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Mitochondrial dysfunction has long been implicated in Type 2 diabetes (T2D). This rela- tionship appears to be bidirectional, with evidence that mitochondrial dysfunction is both caused by and causal of T2D-related phenotypes. A potential causal role in T2D onset would be supported by evidence of a genetic predisposition to mitochondrial dysfunction, since inherited genetic risk factors precede and contribute to disease onset. Here, a genetic study design is used to investigate the potential role of T2D-associated genetic risk loci (T2D loci) in disrupting mitochondrial function through the altered expression of nuclear- encoded mitochondrial genes (NEMGs). The mitochondria are targeted by multiple T2D drugs and therefore such loci may be informative for effective treatment and prevention measures. The functional cis–genes regulated by T2D loci were identified based on the co-location of T2D loci with adipose tissue expression quantitative trait (eQTL) within a genetic distance of 1 LDU. T2D loci and eQTL were previously mapped using LDU- based gene mapping, which is compared and contrasted in this thesis to other popular tests of association. 50 of the identified T2D cis–genes were NEMGs and implicated a number of pathways in the inherited risk of T2D, including the relevant pathway of branched-chain amino acid catabolism. These same 50 genes were enriched for decreased expression in T2D cases compared to controls in independent gene expression datasets. Compared to the total known NEMGs, the 50 cis-NEMGs showed further enrichment for decreased expression, suggesting that T2D-eQTL co-location may identify specific subsets of causal genes. Finally, a candidate T2D locus associated with the cis–NEMG ACAD11 was fine-mapped using targeted sequence data for 94 T2D cases and 94 controls. Sev- eral candidate causal variants were identified, including two low-frequency haplotypes, one of which contained both an ACAD11 splicing mutation and a mutation predicted to disrupt the observed binding of HNF4A and COUP-TFII within the ACAD11 promoter region. Open Access

The brain is as an important regulator of appetite and metabolism. Glucokinase (GK) is a key component of glucose sensing, and is expressed in many glucose sensitive neurones (Rosario et al., 2016, Pocai et al., 2005b). Two regions where glucokinase is expressed are the arcuate nucleus and the paraventricular nucleus of the hypothalamus (Jetton et al., 1994b). Using viral techniques to specifically alter glucokinase expression, I show that upregulation of glucokinase in these nuclei individually, significantly improves glucose tolerance and increases insulin secretion. Conversely down regulation significantly worsens glucose tolerance and reduces insulin secretion. Interestingly the incretin effect is negligible within the arcuate nucleus but within the paraventricular nucleus, glucagon like peptide-1 (GLP-1) secretion is blunted when glucokinase expression is reduced. GLP-1 secretion has been believed to be entirely regulated by the enteroendocrine L-cells of the small intestine however these results suggest that the brain may play a role. The pathway of action of both the arcuate and paraventricular GK appears to be via ATP-sensitive potassium channels (KATP) channels (Maekawa et al., 2000, Hussain et al., 2015b, Ma et al., 2018a). These results suggest an important role for arcuate and paraventricular nuclei glucokinase in glucose homeostasis via KATP channels. Open Access

The main pathological hallmarks of Alzheimer’s disease (AD) are amyloid β (Aβ) plaques and neurofibrillary tangles in the brain. Neuroinflammation is strongly implicated in the progression of the disease, supported by the presence of clusters of activated glial cells surrounding Aβ plaques in AD patients and in animal models of AD. An increase in inflammatory mediators released by these glial cells is thought to contribute to the toxicity of Aβ in AD, leading to neuronal dysfunction and cell death. In addition, neurovascular dysfunction and blood-brain barrier (BBB) deficits have been described in AD, potentially as a consequence of both Aβ deposition and neuroinflammation. Annexin A1 (ANXA1) is an anti-inflammatory mediator, which has recently been shown to suppress microglial activation by acting on the formyl peptide receptor 2 (FPR2), and has also been shown to reduce BBB permeability. We hypothesised that ANXA1 has neuroprotective properties in AD, and our aim was to investigate the effects of ANXA1 treatment on Aβ deposition, neuroinflammation, BBB permeability, and memory using in vitro and in vivo models of AD. We show that ANXA1 reduces Aβ load in models of AD by increasing the expression of Aβ degrading enzymes and by stimulating microglial phagocytosis of Aβ. We also show that these effects are mediated by the activation of FPR2. In addition, ANXA1 had anti-inflammatory properties by reversing the Aβ-mediated increase of pro-inflammatory cytokines in vitro and in vivo in the 5xFAD mouse model of amyloidosis. We also found that ANXA1 treatment increases microglial clustering around Aβ plaques and induces the secretion of anti-inflammatory markers, including transforming growth factor β and arginase. These effects were paralleled by increased synaptic density in the hippocampus and reduced memory deficits. Furthermore, we show that administration of ANXA1 is able to reverse BBB leakage and vascular disruption in 5xFAD mice at 12 weeks of age. These data suggest that ANXA1 plays a pivotal role in Aβ clearance, modulating inflammation, and maintaining BBB integrity in AD, and support the further investigation of ANXA1 as a potential pharmacological tool for AD therapeutics. Open Access

The development and survival of preantral follicles within the mammalian ovary must be tightly regulated in order to ensure a healthy supply of oocytes for the entirety of a woman’s reproductive lifespan. A complex regulatory network of both stimulatory and inhibitory factors exists to control activation of primordial follicles and preantral follicle growth. This thesis aimed to investigate interactions between local intraovarian growth factor and steroid signalling pathways. The epidermal growth factor (EGF) family, previously known to be essential during ovulation, was shown to play a role earlier during preantral follicle development. ErbB receptor subtypes EGFR and ErbB2 as well as EGF-like ligands were expressed in isolated mouse preantral follicles. Cultured preantral follicles exposed to exogenous EGF ligand grew significantly larger than controls and displayed widespread alterations in the expression of genes and proteins essential to follicular development. Receptor inhibitor studies showed that EGF signalling between and within preantral follicles likely activates both EGFR homodimers and EGFR-ErbB2 heterodimers that converge on the MAPK cascade. Androgens were also shown to stimulate preantral follicle growth and alter expression of proteins key to follicular development in culture. Androgens signal primarily through the androgen receptor (AR) that acts classically as a ligand activated transcription factor, however it was shown that AR can also induce rapid non-genomic phosphorylation events in the MAPK cascade in mouse granulosa cells. Interactions between androgen and EGF signalling were uncovered in preantral follicles. Androgens regulate expression of ErbB receptors and EGF-like ligands in cultured preantral follicles. Interestingly, selective inhibition of either EGFR or ErbB2 attenuated the effect of androgens on follicle growth, indicating that ErbB activity is required for maximal androgenic stimulation. The mechanisms of androgen-EGF crosstalk remain unclear, however this novel interaction provides insights into the complex and interconnected regulatory networks within the ovary that control preantral follicle development. Open Access

The neonatal ovary encompasses a finite pool of primordial follicles which serve the need of the entire reproductive lifespan. Follicle activation is therefore a tightly controlled process, the rate of which has a profound influence on fertility. Despite this the exact mechanisms responsible for maintaining the primordial follicle pool or, conversely, for activating the initiation of follicle recruitment remain unknown. Many of the extracellular signalling molecules implicated in early preantral follicle development are members of the transforming growth factor beta (TGFB) superfamily. The aim of this thesis was to examine the role of TGFB signalling in primordial follicle activation and subsequent early preantral follicle development. TGFB signalling is classically facilitated by the SMAD intracellular transcription factors. The SMAD2/3 cascade is the major TGFB signalling pathway expressed in the early preantral follicle. Pharmacological inhibition of the upstream receptors of the SMAD2/3 signalling cascade ALK4/5/7 with small molecule inhibitor SB505124 accelerated primordial follicle activation in a neonatal ovary culture while in a paradoxical manner inhibited development of the small multilayered preantral follicle in an isolated follicle culture. A screen of ligands that converge preferentially to the SMAD2/3 pathway highlighted TGFB2 as the most abundantly expressed in the early preantral follicle. TGFB2 inhibited primordial follicle activation in the neonatal ovary culture while in a differential manner promoted the growth of isolated small multilayered preantral follicles suggesting the action of TGFB2 is dependent on the developmental stage of the follicle. Pharmacological inhibition of ALK4/5/7 (SB505124) and the MAPK/ERK (U0126) signalling cascade ablated TGFB2 induced preantral follicle growth suggesting the coordinated action of SMAD and non-SMAD signalling underlies the context dependent action of TGFB2 in preantral follicle development. In conclusion these novel data suggest that TGFB signalling plays an important role in both maintenance of the primordial follicle pool and early stage preantral follicle development. Open Access

The pancreatic islets of Langerhans play a fundamental role in the stabilisation of blood glucose levels. Pancreatic islets are spherical structures composed of multiple cell types, with each individual cell type secreting a peptide hormone, such as insulin and glucagon, which regulates whole body glucose homoeostasis. Defective hormone secretion from islet cells is a hallmark of certain metabolic diseases, including type 2 diabetes mellitus (T2D). The most abundant islet cell type is the pancreatic β-cell, a specialised cell type that secretes the hypoglycaemic hormone insulin in response to raised glucose levels. Alterations in both β-cell mass and function are the causative factor for the development of T2D. Both genetic and environmental factors are known to underlie the decline in β-cell function typical of T2D. Specifically, genome wide association studies (GWAS) have identified over 100 genomic loci that are associated with T2D risk. Among these loci, variants that lie within/near ADCY5, SLC30A8 and PAX6 show associations with both T2D and abnormal glycaemic parameters typical of a diabetic phenotype. Therefore, the aims of this thesis were to understand how variants associated with T2D manifest at the level of the pancreatic islet. The expression of these genes was therefore manipulated through the generation of tissue-specific transgenic and knockout mice and by RNA interference in human tissue. Reducing the expression of ADCY5, encoding adenylate cyclase five, in human islet tissue reduced glucose-stimulated insulin secretion. This was accompanied by impairments in the metabolic and non-metabolic parameters that govern the secretory response of islets to glucose and other secretagogue. Deleting ZnT8, a β-cell zinc transporter and the gene product of the Slc30a8 gene, in the mouse β-cell significantly impaired the ability of these animals to mount effective responses to glucose. Interestingly, the reverse phenotype, i.e. improved glucose tolerance, was seen in animal models that overexpress ZnT8 in the β-cell. Finally, deletion of Pax6 in the adult mouse resulted in a drastic diabetic phenotype accompanied with changes in the cellular architecture of the islet and alterations in β-cell glucose signalling. Therefore, ADCY5, SLC30A8/ZnT8 and PAX6 gene variants likely negatively impact upon β-cell mass and function leading to a diabetic phenotype. Furthermore, these genes highlight distinct pathways, intrinsic to the pancreatic β-cell, which could be therapeutically targeted in the treatment of T2D. Open Access

Germ cells represent the link between generations. At the time of fertilisation, sperm and egg come together to form a single cell, called the zygote, from which an entire organism is ultimately constructed. In mouse, two unique properties underlie the exceptional potential of gametes: 1) the ability to execute meiosis; and 2) the correct establishment of complementary epigenetic signatures (genomic imprints) in sperm and oocyte that enable post-implantation embryonic development in the next generation. At the molecular level, both the progression through meiosis and the establishment of sex-specific methylation imprints are critically dependent on a wave of global DNA demethylation that is initiated in primordial germ cells (PGCs), the embryonic precursors of the gametes, following their colonisation of the embryonic gonads. The work I present in this thesis focuses on the molecular mechanisms regulating this wave of DNA demethylation. Specifically, I conclusively show that DNA demethylation proceeds independent of the 5-methylcytosine oxygenase Tet1, previously proposed to act as the putative DNA demethylase. Rather, I show that, with respect to DNA demethylation, Tet1 appears to protect the hypo-methylated state in PGCs following the major loss of 5-methylcytosine. Most strikingly, however, with respect to germ cell development, Tet1 appears to play an important role in regulating the timing of the major developmental and transcriptional transitions that occurs in the mouse foetal germ line between embryonic day (E) 10.5 and E14.5, with PGCs derived from Tet1-knockout embryos showing increased stability of the demethylated pre-meiotic germ cell state. Beyond understanding the role of Tet1 in gonadal PGCs, I also present results focusing on the potential relationship between cell signalling, DNA demethylation and licensing for gametogenesis, with preliminary data suggesting a potential role for Akt-mTORC1 signalling in activating the expression of the key meiotic regulator Dazl. Open Access

Osteoporosis is characterised by low bone mass, reduced bone mineral density and a deterioration of bone microarchitecture, resulting in increased susceptibility to fragility fractures. Oestrogen deficiency and thyrotoxicosis are established risk factors for osteoporosis. Oestrogen and thyroid hormone have opposing actions on adult bone and I hypothesise that accelerated bone loss at the menopause is due to unopposed actions of thyroid hormone on the skeleton. To test this hypothesis, I investigated the effect of altered thyroid status on the skeleton in adult sham-operated and ovariectomised wild type and thyroid hormone receptor (TR) α and β knockout mice (TRα0/0 and TRβ-/-). Skeletal phenotype analysis included determination of structural, densitometric, histomorphometric and biomechanical parameters. Skeletal phenotypes of euthyroid wild type mice were compared to hypothyroid and thyrotoxic wild type as well as TRα0/0and TRβ-/- mice. Bone mass was elevated in TRα0/0and reduced in TRβ-/- mice despite similar bone formation rates. These data suggest that the differences in bone mass are due to differing osteoclast activity. The skeletal phenotypes of TRα0/0 and TRβ-/- mice following manipulation of thyroid status were then described in detail. TRβ-/- and wild type mice rendered hypothyroid, had significantly lower bone formation than hypothyroid TRα0/0 mice suggesting that, in the absence of thyroid hormone, TRα may inhibit bone formation. Finally, the effect of oestrogen withdrawal in each of these groups of mice was also investigated. These studies demonstrate oestrogen deficiency bone loss was greater in hypothyroid compared to hyperthyroid mice, indicating that accelerated bone loss following oestrogen withdrawal cannot result from unopposed actions of thyroid hormones. Nevertheless, the studies contained in this thesis provide new insight regarding the roles of TRα and TRβ in bone maintenance and the effects of interactions between oestrogen and thyroid hormones on the regulation of bone mass. Open Access