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Our group have previously shown that the period around mammalian conception is sensitive to environmental factors that can affect the early embryo before implantation with enduring consequences over the lifetime. Our past work in mice has shown maternal low protein diet exclusively during preimplantation development with normal nutrition thereafter (Emb-LPD diet) leads to increased endocytosis in trophectoderm (TE) of blastocysts followed by growth, cardiovascular and metabolic dysfunctions. The maternal nutrient depletion associates with a reduction in mammalian target of rapamycin complex 1 (mTORC1) signalling (pathway regulating cellular growth) in the early embryos, which is responsive to insulin and BCAAs, both reduced in the Emb-LPD maternal serum and uterine luminal fluid, respectively. We reasoned that the reduced uterine metabolite composition is sensed by embryos through mTOR, activating endocytosis as an early biomarker of adversely programmed embryos, to increase nutrient uptake to counter the poor quality of maternal diet, yet associating with later health complications. The current mouse study determined the role of individual BCAA and insulin depletion conditions on endocytosis in TE after establishing an improved in vitro embryo culture and endocytosis model. Thus, 2-cell embryos were cultured to the blastocyst stage in KSOM medium supplemented with BCAA and insulin at normal (N, NBCAA/N-INS; 100% of concentrations found in control-fed mothers) or with a combination or individual BCAA and/or insulin (INS) depleted by 50% (L-): low valine (L-VAL), low isoleucine (L-ISO), low leucine (L-LEU). After culture, blastocyst fluorescent markers for lysosomes (LysoTracker) and endocytosed and processed bovine serum albumin (BSA-BODIPY) were used with confocal microscopy and VOLOCITY imaging software for quantitative analysis. From many combinations of media with individually varying BCAAs and insulin, my 48 h cultures showed: (i) Culture with L-ISO was sufficient with control concentrations of other BCAAs and insulin to induce enhanced endocytosis in blastocysts; (ii) L-LEU alone had a weaker effect on endocytosis than L-ISO but L-VAL had no effect; (iii) L-INS with control BCAAs had no effect on endocytosis; (iv) Combinations of L-ISO and L-LEU enhanced endocytosis, also when combined with L-INS; (v) Control and experimental culture conditions had no effect on embryo development rate or blastocyst cell number. Additional studies on the enhanced endocytosis phenotype revealed that induction with L-ISO/N-INS also stimulated expression of the structural endocytosis vesicle protein, clathrin, and the main multi-ligand endocytic receptor, megalin, in blastocysts. Induction also increased active lysosomes containing the hydrolase cathepsin B. The timing of the change in endocytosis phenotype, assessed by screening earlier stage morulae, showed that the response only occurred from the early blastocyst stage onwards when the endocytosis system had fully matured. I also explored whether deficiency in mTORC1 signalling in blastocysts could activate the enhanced endocytosis phenotype and, in particular, if the mTORC1-associated transcription factor for lysosome expression, TFEB, was involved. Experimental analysis showed that embryos cultured in control medium with NBCAA/N-INS with increasing concentrations of the mTORC1 inhibitor, rapamycin, led to a dramatic relocation of TFEB from mainly cytoplasmic (inactive) to nuclear (active) localisation, where it acts to promote transcription for lysosomal biosynthesis. Furthermore, treatment with the L-ISO/N-INS protocol in the absence of rapamycin also stimulated nuclear localisation of TFEB and subsequently, increased lysosomes. Collectively, these mouse model experiments indicate L-ISO, and to lesser extents L-LEU and L-INS, act as environmental cues to stimulate compensatory blastocyst endocytosis in low nutrient conditions, mediated through altered TFEB activation shown to be responsive to reduced mTORC1 signalling. In separate experiments, the same endocytosis assay markers were used (BSA-BODIPY and LysoTracker Red) on human embryos donated for research, and compared outcomes based upon maternal BMI and conventional IVF versus intracytoplasmic injection (ICSI). In the first experiment, patients were separated based on normal (18-23) or high (28-33) BMI as a measure of normal or dysfunctional physiological/nutritional states. Whilst no difference was found between blastocysts in terms of embryo survival and cell number, those from the high BMI group exhibited the enhanced endocytosis phenotype using LysoTracker Red. Moreover, the range of scores varied across the high BMI group showing individual embryo variation. Lastly, examination of human embryos for TFEB localisation revealed increased nuclear versus cytoplasmic location for the high BMI group, but not quite at significance level. ICSI procedure showed no effect on endocytosis phenotype, although ICSI embryo scores showed high variation in the high BMI group. Collectively, this preliminary data supports the concept that maternal nutrition/metabolism affects embryo endocytosis phenotype in the human as shown in the mouse model, with the potential to be a biomarker for embryo long-term health.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible disease of the lung, characterised by excessive extracellular matrix (ECM) deposition and loss of pulmonary function. Fibroblasts are the main cell type responsible for ECM deposition and peptides from the renin-angiotensin system (RAS) have been shown to modulate fibroblast function. Angiotensin (ANG)-II, has been shown to exert profibrotic effects on human cardiac fibroblasts, such as increased collagen deposition. ANGII is known to bind to two receptors (AT1R and AT2R), with profibrotic effects attributed to AT1R. ANGII is cleaved to form ANG(1-7). This peptide has been shown to exert antifibrotic effects on fibroblasts, such as decreased cell proliferation, via binding to the Mas receptor. However, most investigations have been performed in rat fibroblasts. Another related peptide called ANG(1-9), has also been shown to exert antifibrotic effects on fibroblasts, via binding to AT2R, again this work has only been performed in rat fibroblasts. The main aims of this thesis were to investigate the effects of ANGII and related peptides on signalling pathways in both IPF derived and healthy human lung fibroblasts (HLFs) and to explore the effects of ANG peptides on HLF function. A second aim was to characterise Mas receptor expression on HLFs. The effects of ANGII, ANG(1-7) and six related peptides on intracellular Ca2+ release were explored and the ability of ANGII to phosphorylate ERK was also investigated. Fibroblast functions in response to both ANGII and ANG(1-7) were also examined, including proliferation, collagen deposition, matrix metalloproteinase (MMP) and cytokine secretion. Receptor involvement was investigated with telmisartan (AT1R antagonist), PD-123319 (AT2R antagonist) and A-779 (Mas receptor antagonist). Mas receptor expression on HLFs was also investigated with immunocytochemistry and radioligand binding studies. ANGI ANGII and ANGIII caused intracellular Ca2+ release via AT1R. ANGII also induced ERK phosphorylation, although this did not translate to fibroblast function. No effects of ANGII were observed on cell proliferation, collagen deposition, MMP secretion or IL-6 production. In the Ca2+ assay, ANG(1-7) and ANG(1-9) partially inhibited the responses to both ANGII and ANGIII. These effects did not appear to be modulated by AT2R or the Mas receptor, indicating a novel binding site for these two peptides. The effects of ANG(1-7) did not appear to correlate with functional activity, with no reduction in cell proliferation or collagen deposition observed with this peptide. Immunocytochemistry showed clear nuclear expression of the Mas receptor in HLFs, with very low cell surface expression, providing an explanation for the lack of functional response. Together these results indicate that although many ANG peptides can affect intracellular signalling pathways, peptides from the RAS do not affect functional responses of HLFs.

The first 4 days of the mouse pregnancy is the preimplantation period. Cells multiply and the first differentiation occurs, establishing distinct stem cell populations; the trophectoderm (TE) which gives rise to the placenta and the inner cell mass (ICM) which gives rise to the embryo proper and yolk sac. Adaptation has been shown to occur at this early stage in response to diet and shows prolonged effects on offspring health (Wakins et al, 2008). Mice were fed a high fat diet (HF) or high protein (HPD) diet during the preimplantation period to determine if adaptation at this stage was specific in its response to poor diet or whether default adaptation occurred to non-optimal diet. It was found that adaptation was in fact specific to dietary challenge. Maternal causes for these adaptations were investigated, with uterine growth factor expression being found to be altered. HF embryos were further investigated with pregnancies maintained to a few days prior to birth (E17.5), with HF diet being fed throughout gestation or only for the preimplantation period (emb-HF) and compared to control fed mice. It was found that both the HF and emb-HF fetuses were proportionally larger than their placenta compared to controls, reflecting the proportions of TE and ICM cells observed in the preimplantation embryo. Bone development and glucose tolerance were also affected in these fetuses. These results suggest that a HF environment experienced during the preimplantation period programmes the growth trajectory of the fetus and placenta. These adaptations may occur in order to establish optimal nutrient supply during gestation in anticipation of nutrient availability. Inappropriate adaptation has been linked with later life diseases such as diabetes and hypertension.

Stress incontinence (SI) is more common in white women (61%) than in black African women (27%). Previous studies have demonstrated that collagen XVII is increased and the collagen I:III ratio is decreased in continent black women compared to white suggesting better adhesiveness and elasticity in the tissue of black women. We decided to examine this concept further and analyse the elastin content in paraurethral vaginal tissue of black and white women with or without stress incontinence. A further study was set up to examine if topical oestrogen could increase elastin and change associated components of white women with pelvic organ prolapse (POP). Oestrogen treatment has been shown to increase pro-mmp2 and new collagen formation in SI women, therefore it seemed possible that such treatment could affect the collagen and elastin components of POP favourably. The clinical symptoms of POP and surgical outcome may also be improved. The first study examined the elastin content by histochemistry in paraurethral vaginal tissue while the second study assessed how locally applied oestrogen (Vagifem) given over three months influenced the mRNA expression of MMP2, elastin and collagen XVII and also elastin, collagen I and collagen III protein content of vaginal skin from women with POP. In the racial comparison study black controls showed a highly significant increase in elastin content compared with white controls (p<0.01). For SI to occur in black women a severe insult that reduces elastin production appears to be necessary as black women with SI showed significantly lower elastin content compared with black controls (p<0.05). After application of topical oestrogen to white women with POP it was shown that mRNA for MMP2 is up-regulated (p<0.01) while that for ER alpha receptor is not (p=NS). The message for collagen XVII was down-regulated (p<0.01) while collagen I and III protein were increased significantly (p<0.001 in both cases). The mRNA for elastin was significantly increased (p<0.05) after treatment but the increase in elastin protein staining did not quite reach significance. In conclusion, black women have higher elastin content in vaginal tissue compared to white and this may contribute to the lower incidence of SI in black women. Topical oestrogen over a short period has remodelling effects on key factors of the extracellular matrix of vaginal tissue. Although significant rises in elastin mRNA were shown but not protein, this treatment over longer term application could enhance some of the changes seen. This research provides evidence that black women have beneficial characteristics of vaginal skin that could resist SI and POP. Oestrogen treatment with refinements for white women could mimic black characteristics and alleviate POP symptoms.