Oestrogen receptors (ER) have classically been described as ligand-inducible nuclear transcription factors. The pleiotrophic effects of ER function have a predominant role in the direct regulation of the growth, differentiation and development of tissues of the human reproductive system. There are two ER subtypes, ER and ER which differ in their specificity for ligand and the consequent actions they orchestrate. Moreover, the latter exists in multiple splice variants of which ER is the only fully functional homologue. Research into the underlying differences in subtype responses to ligand has involved examination of the intranuclear dynamics of individual receptor subtypes. Studies into the mobility of ER in response to ligand have exclusively focused on studies of full length ER and ER independently in transfected cell lines. The studies described in this thesis have investigated the kinetics of ER using Fluorescence Recovery After Photobleaching (FRAP) in infected cell lines which lends itself to more precise expression of the subtype of interest. The morphological impact of natural oestrogenic and synthetic ligands on ERs was examined and the influence on the intranuclear dynamics assessed. Further to this, the effect of co-expression of different ER subtype combinations was examined. Studies on the intranuclear mobility of ER have confirmed and extended the findings of others. Previous work on the development of ER agonists and antagonists has been to target specific overexpressing ER subtypes in a physiological setting. In this study, we demonstrated for the first time an overwhelming ER -selective effect in slowing the rate of mobility within the nucleus, suggesting the study of intranuclear dynamics is an important parameter for the examination of efficacy of a compound. Differential responses to ligand based on co-infected partnerships indicate that heterodimerisation has a profound effect in augmenting ligand-dependent regulation and activity.

This project focussed on a protein called tear lipocalin that is believed to interact with the lipid layer of the tears and to promote tear film stability. By modulating this protein, it was proposed that tear film stability could be increased, hence the modulation of lipocalin was suggested as a goal for dry eye therapy. The effects on tear lipocalin expression of two potential therapies for dry eye, pharmacological stimulation with phosphodiesterase inhibitors and hormonal regulation with aldosterone and oestrogen HRT were investigated. Initially a rabbit model was employed and the rabbit tear protein profile was characterised. Stimulation of rabbit secretion using phosphodiesterase compounds did not alter rabbit tear lipocalin secretion. Clinical trial of phosphodiesterase compounds in humans did not alter tear lipocalin expression but variation in tear lipocalin isoform expression was noted and new isoforms of tear lipocalin were identified. The steroid hormone aldosterone appeared to modulate rabbit lipocalin and low MW protein expression. A clinical trial to examine the effect of gender, menopause and oestrogen hormone replacement therapy (HRT) on lipocalin secretion demonstrated that oestrogen HRT down regulated lipocalin secretion, increased the thickness of the lipid layer and improved symptomatology compared to the untreated menopause group. High levels of tear lipcalin in the menopause group were associated with symptoms of ocular burning, suggesting that increasing tear lipocalin levels might aggravate the ocular symptoms associated with dry eye. In this thesis it was demonstrated that tear lipocalin secretion was modulated by steroid hormones, but did not appear to be modulated by phosphodiesterase inhibitors. Variation in tear lipocalin concentration did not correlate with measures of tear film stability and indeed, high levels of tear lipocalin may be deleterious for dry eye symptoms. Further analysis of tear a stimulants and hormones will lead to better understanding of tear regulation and will open avenues for dry eye therapy. The application of proteomic tools to tear film will advance our understanding of tear film composition and the role each component in tear film stability. / Doctor of Philosophy (PhD)

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Effects of oestrogen on prolactin transcription patterns in living pituitary tissue Amanda Louise Patist PhD in the Faculty of Medical and Human Sciences, September 2013, The University of ManchesterOestrogen is a well-known modulator of the transcription and secretion of prolactin as well as having a role in the physiological proliferation and possibly also in pathological hyperplasia of lactotrophs. Our group has studied prolactin gene promoter regulation in single living pituitary cells in Fischer 344 (d2eGFP-hPRL 455) rats, that express a destabilised eGFP reporter gene under the control of a human prolactin genomic fragment, and identified prolactin transcription cycles that occur in a non-circadian and non-synchronised fashion. Pulsatile transcription has been identified in fetal tissue, stabilising during development. Here we assess the effects of physiological and supraphysiological oestrogen exposure in vivo on prolactin transcription in the adult rat. We have established and validated models of both states by evaluating the expression of the hPRL-d2eGFP transgene during the oestrous cycle and in males with long-term oestradiol-releasing implants, respectively. The oestrous cycles of adult female rats were synchronised by IP LHRH injection. A 1.8-fold increase in the number of cells expressing the prolactin transgene at oestrus (n=7) as compared to diestrus (n=5) and a 10.6 fold increase in mean fluorescence per cell was identified by flow cytometry. In males, chronic oestrogen stimulation induced a 2.5-fold increase in pituitary weight, a 5.2-fold increase in number of cells expressing the transgene and 4.4-fold increase in fluorescence per cell, as indicated by flow cytometry (n=3). Immunofluorescence, qPCR and serum analysis confirmed the high-production state of lactotroph cells in both female and male models. 250µm pituitary slices were imaged for 48 hours using time-lapse confocal microscopy and pulsatile fluorescent reporter activity was evident in both female and male models. Interestingly, no significant difference was seen between transcription cycle patterns (timing or amplitude of transcriptional pulses) in individual cells between high and low oestrogen states. Using a novel mathematical model, that calculates transcription rate from fluorescence data, we have been able to study the transcription rates displayed by single cells and the estimated points at which a cell switches from one rate to another. Patterns in switches in transcription rates were similar between high and low oestrogen states, suggesting that individual pituitary cells within the context of tissue, continue to display cyclical patterns of gene expression, in states of both high and low prolactin production. This implies that cyclical transcription is a fundamental property of prolactin gene expression that persists in different physiological states, and that modulation of cycle characteristics is not the mechanism for increased prolactin synthesis in these circumstances.

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Background: Reproductive and menstrual factors have been evaluated as surrogates for long-term hormonal exposures in several prospective studies of colorectal cancer, yet findings have been conflicting. Methods: The relation of reproductive and menstrual factors (self-reported via a reproductive history questionnaire) with incident colorectal cancer was investigated among women enrolled in the Women's Health Initiative Observational Study (WHI-OS), a longitudinal cohort of 93 676 postmenopausal women (aged 50-79 years at enrolment) in which 1149 incident cases of colorectal cancer occurred over a median follow-up of 11.9 years. Multivariable Cox proportional hazards models that included established colorectal cancer risk factors were constructed to examine the association of colorectal cancer incidence with reproductive and menstrual factors. Results: Having had two children (vs nulliparous: hazard ratio (HR) = 0.80, 95% confidence interval (CD: 0.64-0.99) was inversely associated with colorectal cancer risk. Compared with never users, ever use of oral contraceptives was associated with lower colorectal cancer risk (HR = 0.74, 95% CI: 0.63-0.86); however, no relationship was observed for duration of oral contraceptives use (4 years vs 1 year: HR = 0.94, 95% CI: 0.67-1.32). None of the remaining reproductive and menstrual factors was associated with colorectal cancer incidence. Conclusions: Parity and prior use of oral contraceptives were associated with lower colorectal cancer risk in this cohort of postmenopausal women.

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