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The successful development of the human fetus during pregnancy depends on a tight regulation of the uteroplacental vasculature in order to allow sufficient provision of oxygen and nutrients from the mother to the placenta and fetus. To accomplish this, the fetoplacental circulation develops de novo, whilst the uterine vasculature undergoes dynamic remodelling. Such distinct features of these circulations suggest that regulation of their vascular function may differ. If so, then in vitro examination of these blood vessels may serve as a useful model for tissue-specific mechanisms of human vascular tone regulation. A reduction in intracellular Ca2+ ([Ca2+]i) in vascular smooth muscle cells leads to an increase of myosin light chain phosphatase (MLCP) activity, which induces myosin light chain dephosphorylation and vasodilation. Further molecular signal transduction mechanisms may serve to desensitise the myofilaments to changes in [Ca2+]i. There is surprisingly little information on the nature of desensitisation to Ca2+ in human myometrial (MA) and placental arteries (PA). Therefore, this study aimed to primarily assess Ca2+-desensitisation mechanisms in MA and PA. Here, we showed (i) that the phenomenon of Ca2+-desensitisation occurred in MA and PA. (ii) That it was promoted by the activation of protein kinase G (PKG) and dependent upon an active MLCP. (iii) That MA displayed greater PKG-mediated Ca2+-desensitisation capabilities compared to PA. In addition, this sensitivity remained greater in MA and PA over the soluble guanylate cylase/PKG axis, but was, surprisingly, reversed at the NO level. Moreover, these differences were not the result of differing expression levels of PKG and PKG-interacting proteins involved in the vasodilatory pathway, as these were similar between the two artery types. These results highlight a variation in vasodilatory responses between MA and PA, which may illustrate a difference in the regulation of vascular tone between the two circulations. Further investigation of the mechanisms involved may prove useful in the development of more tissue-specific therapies.

In this double-blind, placebo-controlled study, 274 frail elderly men, mean age, 74 years with total testosterone (T)≤12 nmol/L or free T≤250 pmol/L were randomized to transdermal T (50 mg/d) or placebo gel for 6 months. Outcome measures, included muscle strength (dynamometry), lean and fat mass (dual energy x ray absorptiometry), physical function and quality of life (Aging Males' Symptom rating scale, AMS and Short Form 36, SF 36). Testosterone concentrations at baseline were 11.0±3.2 and 10.9±3.1 nmol/L (mean±SD) in the active and placebo groups respectively. Testosterone concentrations increased to 22.9±10 and 18.4±9.2 nmol/L in the T-treated group at 3 and 6 months respectively, while remaining unchanged in the placebo group. Isometric knee extension peak torque in the T group by, mean (SD) 4.7 (31.0) Newton meters (Nm) and decreased in the placebo group by 4.7 (25.5) Nm. The adjusted difference between the groups was significant (P=0.02). The other muscle strength endpoints (isometric knee flexion and isokinetic knee extension and knee flexion peak torques) improved in both the groups, but the adjusted difference between the groups was not significant. Lean body mass increased and fat mass decreased significantly in the T group by 1.08±1.8 and 0.9±1.6 kg respectively. Physical function tests improved in the older (≥75 years) and frailer (≥2 frailty criteria) in men, not in the entire cohort. Somatic and sexual symptoms (AMS) and physical summary score (SF 36) improved in the testosterone group compared to placebo. There was no increase in prostate-related adverse events in the testosterone group compared to placebo. It is concluded that short-term testosterone treatment of frail elderly men with low to borderline-low testosterone levels may preserve age-associated loss of muscle strength. In addition, treatment improved body composition and quality of life. Physical function improved among older and frailer men. These encouraging results need to be replicated in studies of a longer duration.

FOXE1, a forkhead transcription factor, plays an essential role in the development of the thyroid and hair follicle. Loss-of-function FOXE1 mutations cause aberrant morphogenesis of both tissues, apparently due to a defect in cell migration. In the mature thyroid, FOXE1 plays an essential role in the control of thyroid hormone synthesis, modulating both thyroglobulin and thyroid peroxidase gene expression. Several studies have shown there is considerable inter-individual variability in thyroid hormone levels, and this is due at least in part to genetic factors. One of the main features of the FOXE1 protein is a highly polymorphic polyalanine tract that can range in size from 11 to 19-Ala residues (wild-type being 14-Ala residues). The aims of this thesis were: 1) To determine the precise location of FOXE1 expression within the human hair follicle. 2) To identify genes involved in cell migration that could be potential FOXE1 target genes. 3) To investigate the possible role of FOXE1 polyalanine polymorphisms in thyroid dysfunction. 1) Immunohistochemical analysis confirmed previous observations that FOXE1 was expressed in human epidermis and hair follicle outer root sheath. However, technical difficulties prevented more detailed analyses (e.g. colocalisation with hair-specific proteins). 2) Bioinformatics yielded several potential FOXE1 target genes. Subsequent QPCR-based expression profiling and siRNA-based gene knockdown experiments suggested TIMP3 as a likely direct downstream target. Thus, FOXE1 may have a direct role in the modulation of ECM architecture by migratory cells. 3) The genotyping of a small cohort of subclinical hypothyroid subjects, showed there to be an increased incidence of the 16-Ala allele compared with the control group. Luciferase assays revealed the transcriptional activity of the 16-Ala allele to be significantly reduced compared with wild-type. This provides the first evidence that polymorphic variation can affect the transcriptional activity of FOXE1, and be a possible contributing factor in the sub-optimal thyroid function.

Subcellular fractionation revealed that pp97, pp96 and pp64 are head protein whereas pp90 and pp55 are tail proteins. Advanced proteomic analysis (GeLC-MS) identified 37 proteins, including AKAP4, AKAP3, CALI, HSPAlL and HSP70 as candidates for the dephosphorylated proteins. AKAP4 was excluded because it was localised to the tail. Two AKAP3 antibodies showed non-specific binding and a better quality antibody will be needed for further investigations. CALI was excluded because it was localised to the tail fraction. HSP70/72 and HSPA1L were strong candidates for pp64. However, immunoprecipitation of dephosphorylated proteins using phospho (S/T) PKA substrate Ab and HSPA1L Ab was unsuccessful and further work is now required to address this.

This thesis investigates the effects of testosterone and its metabolites on the electrical activity of single corticomedial amygdala neurones in the male rat. Experiments concentrate, in particular, on those corticomedial amygdala neurones which project directly to the medial preoptic/anterior hypothalamic junction. An attempt to relate the observed neuronal effects of testosterone to sexual behaviour has also been made. The first Chapter reviews the electrophysiological experiments on the effects of sex steroids on single neurones in the central and peripheral nervous system. The second Chapter describes experiments which show that long term castration lengthens the absolute refractory periods of corticomedial amygdala neurones which project to the medial preoptlc/anterlor hypothalamic junction. Adjacent corticomedial amygdala neurones which project to the capsule of the ventromedial nucleus of the hypothalamus did not show this effect. Chapter 3 describes an experiment which shows that long term testosterone treatment reduces the absolute refractory periods of corticomedial amygdala neurones which project to the medial preoptlc/anterlor hypothalamic junction, In castrated rats. Results show a direct effect of testosterone In the central nervous system. Chapter k Investigates the effects of two major metabolites of testosterone, oestradlol and dihydrotestosterone, on the absolute refractory periods of these corticomedial amygdala neurones. Oestradlol, but not dihydrotestosterone produces the same reduction effect as testosterone. Results provide direct evidence that oestradlol has the same effect as testosterone In the central nervous system. Chapter 5 describes two similar experiments which show that the testosterone reduction of the absolute refractory periods of these corticomedial amygdala neurones Is correlated with the time at which the hormone stimulates full sexual behaviour. Chapter 6 discusses the significance of the testosterone effect on corticomedial amygdala neurone absolute refractory periods.

Endocannabinoids are endogenous ligands for cannabinoid receptors that play a pivotal role in fertilisation, embryo development, transport, implantation and pregnancy maintenance. The endocannabinoid system (ECS) consists of ligands (e.g. anandamide, AEA), receptors (e.g. CB1, CB2 and TRPV1) and ligand modulating enzymes (e.g. NAPEPLD and FAAH). The ECS interacts with sex steroid hormones and cytokines to regulate reproduction. Progesterone (P4), essential for pregnancy maintenance, increases FAAH activity in human lymphocytes, keeping AEA levels low. An elevated plasma AEA level is detrimental for implantation and pregnancy maintenance and any ECS dysregulation adversely affects pregnancy outcome. The hypothesis therefore was that manipulating the ECS could be an effective way of interrupting implantation. (a) The effect of RU486, a P4 antagonist used to initiate medical termination of pregnancy (MTOP), on plasma AEA levels and levels of AEA and the ECS in trophoblast were investigated. These were examined using UHPLC-MS/MS, immunohistochemistry, qRT-PCR and Western Blotting. (b) The effect of exogenously administered AEA to female rats during the implantation window was studied. The results show that ethnicity and BMI can affect the ECS, increasing AEA levels. RU486 administration causes a rise in plasma (p=0.005) and trophoblast (p=0.0062) AEA levels. Trophoblast NAPE-PLD (p=0.0006), FAAH (p=0.021), TRPVR1 (p=0.042) and CB1 (p=0.03) are significantly elevated at the mRNA level but not at the protein level though protein levels were generally higher. Exogenous administration of AEA to rats, around the day of implantation causes a reduction in viable implantation sites and an increase in resorbed units. At gestation day 14 there was a significant correlation between number of viable embryos and plasma AEA levels (p=0.0091). The complete ECS was detected in implantation, interimplantation and resorbed units at day 14. These studies have shown that manipulating the ECS can interrupt implantation showing the importance of this system in pregnancy maintenance.

The initial aim of this research was to establish the distribution of the protein caveolin-1 in the human term placenta, using an indirect immunofluorescence technique. The findings of this survey showed that caveolin-1 was expressed in all the predicted places (vascular endothelium, smooth muscle and fibroblasts), but also by cells lining the basal plate. These cells were investigated further, firstly by transmission and scanning electron microscopy, then by immuno-gold electron microscopy. The findings of the immuno-gold study suggested a vectorial nature of caveolae trafficking in both foetal endothelial cells and in the endothelial-like cells lining the basal plate. This study also showed that some leucocytes express caveolin-1. The cells lining the maternal blood space above the basal plate of the placenta had been thought to be trophoblastic (foetal) but the initial results obtained using immunofluorescence indicated that the lining consisted rather of a mosaic of two types of cells. Some of these were trophoblastic but others were seen to be more similar to endothelial cells. To determine whether these latter cells were of maternal derivation, placentae from neonates of known gender were used in a cytogenetic analysis, to establish their provenance unequivocally. The probe used in these in situ hybridisation experiments was complementary to a portion of the human Y-chromosome (only found in males). Cells to which this probe hybridised must therefore be male; a failure to hybridise meant that they must be maternal. Some cells resident in the basal plate were shown to have more than one Y-chromosome in a single interphase nucleus and were taken to be polyploid trophoblast ‘giant’ cells. Further collaborative experiments showed that the area fraction occupied by the lining endothelial cells was greater in pre-eclamptic placentae than in placentae from normotensive births; the significance of this in the aetiopathology of this disease is discussed. Cells that play a key role at the maternal-foetal interface have been carefully characterised in this study and have been shown to form a continuous endothelial/epithelial layer of both maternal and foetal origin.

It has been hypothesised that patients with long-term treated HIV infection may exhibit features of accelerated physiological ageing. Given previously established links between, a) anti-retroviral therapy and mitochondrial DNA (mtDNA), and b) mtDNA and ageing, I hypothesised that anti-retroviral therapy may lead to the accumulation of mtDNA mutations, in an acceleration of the molecular process seen in normal human ageing. Using a combination of single cell molecular analyses and ultra-deep sequencing (UDS), I demonstrated that HIV-infected patients with prior exposure to polymerase (pol) γ inhibiting NRTI (nucleoside analogue reverse transcriptase inhibitor) therapy show increased accumulation of somatic mtDNA mutations within cells, in a pattern similar to that seen much later in life due to normal ageing. Empirical data and in silico modelling suggested this is likely to be mediated by the accelerated clonal expansion of pre-existing (age-associated) mtDNA mutations, rather than by increased mutagenesis. I went on to further develop the UDS methodology, to explore more fundamental questions about the characteristics of mtDNA mutations in ageing, health and disease. In so doing, I showed that low-level mtDNA heteroplasmic mutation appears to be universal, and that many ostensibly somatic mutations may in fact have been maternally transmitted at very low levels. I explored the utility of serum FGF-21 (fibroblast growth factor 21) and phosphorus magnetic resonance spectroscopy (31P-MRS) as non-invasive measures of muscle mitochondrial dysfunction in anti-retroviral treated patients. Both showed significant abnormalities, although neither proved sensitive or specific in my patient group. Finally I explored the frequency and severity of fatigue in contemporary HIV-infected patients, showing that half of all patients have excessive fatigue despite good immune function and suppressed HIV viraemia. Patients with prior exposure to pol γ inhibiting NRTIs were almost universally fatigued, suggesting that persistent mitochondrial dysfunction due to accumulated mtDNA mutations may be important in driving fatigue in this patient group.

The model organism Caenorhabditis elegans has been extensively studied in biogerontology. A putative mechanism of ageing is oxidative damage, but this has been challenged in recent studies. One piece of supporting evidence for the role of molecular damage in C. elegans ageing is the presence of blue fluorescent material within intestinal lysosome related organelles, believed to be lipofuscin. This heterogenous, cross-linked aggregate of oxidatively damaged lipids and proteins that accumulates in an age-dependent fashion in post-mitotic mammalian cells. Lipofuscin can be identified by its fluorescent properties, fluorescing blue under UV light. C. elegans intestinal fluorescence has been equated with lipofuscin based on its similar spectral properties, and its increase in ageing populations. This investigation initially set out to verify this assumption. Individual animals were followed in situ on agar pads and their in vivo fluorescence measured from early adulthood until after death. Intestinal blue fluorescence proved to increase not with age, but instead in a striking burst at death. Such death fluorescence also appeared in young worms when killed, irrespective of age or cause of death. Using NMR-based comparative metabolomics the blue fluorophore was identified as anthranilic acid, derived from tryptophan by kynurenine pathway activity. Death fluorescence is generated within intestinal cells, typically in an anterior to posterior wave, by action of the conserved calpain-cathepsin necrotic cell death pathway, a type of cell death previously only observed during neurodegeneration. Using mutational analysis and in vivo fluorescent reporters, we demonstrate that this wave is propagated by calcium influx. Strikingly, inhibition of systemic necrosis delays stress-induced mortality, demonstrating its role as a driver of organismal death. This first description of the biology of death in C. elegans implies that the passage from life to death entails a regulated, programmed transition that is amenable to analysis.