The mammalian Target of Rapamycin (mTOR)kinase is a central regulator of cellular growth and metabolism and plays an important role in ageing and age- related diseases. The increase of invitro data collected to extend our knowledge on its regulation, and consequently improve drug intervention,has highlighted the complexity of the mTOR network. This complexity is also aggravated by the intrinsic time-dependent nature of cellular regulatory network cross-talks and feedbacks. Systems biology constitutes a powerful tool for mathematically for- malising biological networks and investigating such dynamical properties. The present work discusses the development of three dynamical models of the mTOR network. The first aimed at the analysis of the current literature-based hypotheses of mTOR Complex2(mTORC2)regulation. For each hypothesis, the model predicted specific differential dynamics which were systematically tested by invitro experiments. Surprisingly, nocurrent hypothesis could explain the data and a new hypothesis of mTORC2 activation was proposed. The second model extended the previous one with an AMPK module. In this study AMPK was reported to be activated by insulin. Using a hypothesis ranking approach based on model goodness-of-fit, AMPK activity was insilico predicted and in vitro tested to be activated by the insulin receptor substrate(IRS).Finally,the last model linked mTOR with the oxidative stress response, mitochondrial reg- ulation, DNA damage and FoxO transcription factors. This work provided the characterisation of a dynamical mechanism to explain the state transition from normal to senescent cells and their reversibility of the senescentphenotype.

Telomerase is best known for its nuclear telomere maintenance function via its enzymatic activity requiring the two major components TERT (protein) and TERC (RNA). However, telomere and TERC-independent functions for TERT have been described recently. Our group and others have previously shown that telomerase shuttles to mitochondria improving their function and decreasing cellular oxidative stress. Oxidative stress and mitochondrial dysfunction are well known to increase during ageing and have been implicated as a cause for age-related neurodegenerative diseases. I report that TERT protein localises in mammalian brain mitochondria specifically in neurons. Moreover, although telomerase activity is negligible in brain, I found considerable amounts of the telomerase protein TERT in mouse and human brain by independent techniques. Dietary restriction (DR) is known as a condition that improves mitochondrial function, delays or prevents age related diseases and improves cognitive function. In this study mTert was detected in brain mitochondria under basic conditions, and it becomes elevated in brain mitochondria of animals in three independent experiments of short term (3-6 months) DR. Decreased signalling through mTOR has been described as a major mechanism of the DR response. Accordingly, I found that mTOR phosphorylation was down regulated in brains from DR animals. Moreover, to analyse whether decreased mTOR signalling causes mitochondrial redistribution of mTert, mTert wild type and mTert knock-out mice were treated with rapamycin what resulted in increased mTert protein in brain mitochondria and correlated with decreased mitochondrial ROS production. The conclusion is that down-regulation of mTOR is a possible mechanism to increase mTert protein levels within mitochondria under DR. Together, this data shows that mitochondrial mTert might be an important new player in the protection of neurons and improve brain function during ageing.

A group of women, who were not using oral contraceptives, kept daily records of basal body temperature, mood and energy for periods varying from six months to two years. The women reported to the laboratory weekly at the same time to be tested on concept formation, digit symbol substitution, choice reaction time and rotor pursuit tasks. The mood, energy and performance data were normalised to a standard 28 day cycle firmly anchored around menstruation and ovulation, which was pinpointed by using the temperature curves. Mood and energy were both found to be high around ovulation but low paramenstrually. No significant differences associated with different phases of the menstrual cycle, were found in performance on the digit symbol substitution and rotor pursuit tasks but performance on concept formation and choice reaction time was found to be good premenstrually and preovulatory but deteriorated following ovulation and during menstruation. It is hypothesised that changes in estrogen levels may be responsible for the mood and energy levels observed; that the changes in performance are a function of the information load of the task and that this may be due to fluctuations in arousal across the cycle or to the direct effect of gonadotrophins upon information processing. The generalis-ability of the data obtained from the sample studied was investigated using a menstrual questionnaire which was administered to 300 students.

The Myt1l gene, which encodes a neuron-specific transcription factor, is highly expressed in the developing brain, suggesting a crucial role in the processes of neurodevelopment. Although the expression of this gene decreases after birth, it continues to have a good level of expression throughout adulthood in localized areas. Nevertheless, the role of this gene in neural development and resultant behaviours has not been firmly established yet. In an effort to elucidate its function, lentiviral vectors containing synthetic microRNA-adapted short hairpin (shRNAmir) targeting Myt1l were produced to significantly decrease its expression. The knockdown efficiency of these vectors was corroborated in-vitro in the human neural stem cell line (SPC04) and the mouse cell line Neuro-2A. The effects of downregulating MYT1L at gene expression level during differentiation of SPC04 were assessed at two time points: pre-differentiation and day 7 differentiation. The results from this experiment identified five genes being co-expressed with MYT1L during stem cell differentiation: BCL11B, MYT1, SYN1, SNAP25 and JPH3. Further in-silico analysis localized a MYT1L binding site in the promoter region of each one of those genes, suggesting that they might be direct targets of MYT1L. In order to expand our understanding of the possible behavioural effects of this gene, an invivo study was also performed in which adult mice received microinjections of lentiviruses expressing Myt1l shRNAmir in the dorsal hippocampus. These mice manifested a very subtle and transient increase in anxiety-like behaviour, which normalised with time. Even though the dorsal hippocampus has been only weakly linked to anxiety, our results seem to support this association when the expression of Myt1l is reduced. The findings of this thesis have laid the foundations of understanding the function of MYT1L pathway and the impact it has on behaviour.

Fat/Dachsous signalling regulates planar cell polarity (PCP), which is the polarisation of tissue structures perpendicular to the apical-basal axis, and interacts with the Hippo pathway to suppress overgrowth of tissues in Drosophila. Recent studies in vertebrates have implicated Fat4 and Dchs1, the receptor-ligand pair and the vertebrate homologues of Drosophila Ft and Ds, respectively, in regulating PCP in the kidney and cochlea. However, the role of Fat signalling is largely undetermined in vertebrate development. To determine the role of Fat signalling in craniofacial development, a basic characterisation was carried out using histology, OPT scanning, skeletal preparations, immunohistochemistry and in situ hybridisations. This revealed that loss of Fat4 and/or Dchs1 results in arrested growth of salivary glands, disruption of hair polarity of the utricle, delayed differentiation of osteoblasts of the cranial bones and arrested lateral tangential migration of the Facial Branchiomotor neurons (FBNs). In Drosophila and vertebrates, PCP is controlled by two pathways; the Frizzled-PCP and the Fat-PCP pathway. During development, FBNs undergo tangential caudal and lateral tangential migrations within the plane of the neuroepithelium and are a model system to study PCP. Previous studies have shown a critical role for Fz-PCP during caudal migration. The role of Fat-PCP signalling during FBN migration was analysed by using mouse mutants for Fat4 and Dchs1 and by expression analysis. Loss of Fat4/Dchs1 results in an arrest of lateral migration of the FBNs and a loss of polarity as revealed by cell shape and Golgi orientation analysis with no effect on FBN specification. Fat4 and Dchs1 are expressed as complementary gradients in the hindbrain. Generation of chimeric tissue revealed that the gradient of Dchs1 is necessary for polarised FBN migration suggesting that the role and mechanisms of Fat-PCP signalling are conserved between vertebrates and Drosophila. The Islet-1cre and Hoxa3cre conditional knockouts revealed that there is a requirement for Dchs1 both cell autonomously within the FBNs as well as non-cell autonomously in the neuroepithelium whereas Fat4 is largely required non-cell autonomously. Analysis of double Fat4-/-Vangl2Lp/Lp mutants revealed that Fz-PCP exclusively regulates caudal FBN migration whilst Fat-PCP is necessary for the lateral polarised migration of the FBNs even in the absence of Fz-PCP. The two pathways work on orthogonal axes to regulate FBN migration. This study establishes that Fat signalling is largely required during craniofacial morphogenesis, provides further evidence of Fat signalling in regulating PCP in vertebrates and the first evidence that gradients of Fat4/Dchs1 may establish PCP in vertebrates.

Neural tube defects (NTD) are congenital malformations caused by abnormalities in the developmental process of neurulation. Folate metabolism appears to be a determinant of risk of NTD since periconceptional supplementation with folic acid has been shown to reduce the frequency of NTD in humans while sub-optimal folate levels are a risk factor. The mechanisms underlying prevention of NTD by folic acid or susceptibility owing to reduced levels are not known. The aims of this thesis were to understand the role of the folate and, the closely linked, methylation cycle in the cause and prevention of NTD. The effect of methylation cycle intermediates, homocysteine and methionine, on cranial neural tube closure were investigated in cultured mouse embryos. Homocysteine exposure was embryotoxic but did not increase the incidence of NTD, which suggests that increased levels of homocysteine are not a direct cause of cranial NTD. Embryos cultured with high levels of methionine or methylation cycle inhibitors specifically developed cranial NTD in the absence of other developmental defects. These results suggest that the integrity of the methylation cycle is essential for cranial neural tube closure to occur. Mouse embryos that are homozygous for the Splotch211 mutation exhibit NTD that are preventable by folic acid. In Splotch mice, increased apoptosis has been suggested to be responsible for the production of NTD in homozygous embryos. However, in this study immunohistochemical measurement of apoptosis and proliferation in the neuroepithelium in the cranial region of neurulation-stage embryos suggest that the Splotch mutation does not result in increased apoptosis. Finally, in order to test whether there is an underlying defect in folate metabolism in human NTD fetuses, a series of human embryonic cell lines were analysed. The deoxyuridine monophosphate (dUMP) suppression test was modified for use with mammalian fibroblast cell lines and the efficiency and sensitivity of the modified test were analysed by the use of inhibitors of one-carbon metabolism. The test was then applied to human NTD and control cell lines and the results indicate that a subset of the NTD cases have a diminished response, suggestive of an abnormality of folate metabolism.

Ageing describes the progressive functional decline of an organism over time, leading to an increase in susceptibility to age-related diseases and eventually to death, and it is a phenomenon observed across a wide range of organisms. Despite a vast repertoire of ageing studies performed over the past century, the exact causes of ageing remain unknown. For over 50 years it has been speculated that mitochondria play a key role in the ageing process, due mainly to correlative data showing an increase in mitochondrial dysfunction, mitochondrial DNA (mtDNA) damage, and reactive oxygen species (ROS) with age. Therefore, a major aim of the current project was to assess mitochondrial dysfunction, in the form of complex II activity, in the skin cells of differently aged humans. Mitochondrial complex II of the electron transport chain (ETC) was chosen to be examined, as it has recently been implicated in the generation of ROS, as well as in the ageing process of lower organisms, and is the least studied complex of the ETC. Complex II activity was found in the present study to decline in an age-dependant manner in human skin fibroblast cells, which may have been partially related to an observed decrease in the expression of specific nuclear-encoded complex II subunits with age. Further investigations into the cause of the decrease in complex II activity with age revealed that the decline was specific to senescent cells, and was not present in non-senescent cells, which was determined following sorting into subpopulations via fluorescence-activated cell sorting (FACS). The decrease in activity with age was not reflected in another mitochondrial complex examined, complex IV, for which there was no alteration in activity with age for either unsorted, senescent, or non-senescent cells. This finding could suggest the specific targeting of complex II in senescent cells only for future age-related therapeutics. Interestingly, an age-dependant decrease in complex II activity was not observed for human skin keratinocytes, despite being observed in human skin fibroblasts. In the present study, it was also observed that different cell types undergo differing rates of maximal complex II activity, which could have important consequences in terms of the rate of ageing of specific cell types. In addition to the observed decrease in mitochondrial complex II activity with age, it was demonstrated in the present study that mtDNA damage is increased with age in the skin of both humans and a taxonomic group for which age- 3 related changes have not been previously studied, the whales. It was confirmed that the T414G mutation, which is a general biomarker for mtDNA mutations, was higher in human skin fibroblasts from older individuals when compared to younger individuals. Furthermore, an increase in mtDNA damage with age was also found in multiple whale species, for which mtDNA damage was measured in the form of strand breaks within a large region of the mitochondrial genome, using novel primers designed and optimised through the present study. Whales from three distinct species were chosen to be examined based on their differing levels of UV exposure, as a model for different ages. It was found that the level of mtDNA damage increased with both natural age and increased UV exposure. The three whale species studied appeared to have developed alternative mechanisms of coping with UV-induced damage. MtDNA damage was found to be lowest in those whales with the highest expression of heat shock protein 70 (Hsp70), suggesting that this UV-defensive mechanism may be useful in future studies for the prevention of age-related phenotypes. Overall, the present study provides important new insights into the potential role of mitochondria in ageing.

Introduction: There is a nationwide shortage of sperm donors and over the last few years this has been evident at Newcastle Fertility Centre (NFC). As the most common cause for rejection of sperm donors is suboptimal semen quality, external factors that may influence semen quality (i.e. season and vitamin D) were studied to investigate their impact (if any) to improve donor recruitment. Methods: A retrospective review of donor sperm treatments at NFC between Jan 2000 and Dec 2010 was performed to investigate sperm donor shortage. A retrospective review (Dec 2006 to Nov 2009) and a longitudinal study (32 sperm donors) of the semen analyses were conducted to investigate seasonal variation in semen parameters. We performed a retrospective review of donor insemination treatments over 6 years to investigate seasonal variation in donor conceptions. The correlation between semen parameters and serum vitamin D was investigated in a cross sectional study (125 participants) and a cohort study, to examine the change in semen parameters with a rise in serum vitamin D level (with vitamin D supplementation and seasonal rise). Results: A significant reduction in the number of sperm donors recruited and a smaller pool of available donors was seen, which lead to fewer patients receiving treatment and a longer wait for treatment. Seasonal variation with improved semen parameters in winter / spring was noted, but was more prevalent in sperm donors than in patients attending NFC. However, there were no variation donor conceptions by the season of original sperm production. In the cross sectional association study there was no significant difference in the semen parameters between men with different serum vitamin D levels, however in the cohort study, semen parameters deteriorated significantly with increased serum vitamin D levels secondary to vitamin D supplementation and also seasonal rise. Conclusions: A significant local problem of sperm donor shortage is confirmed. Despite significant seasonal differences in donor semen parameters (but not donor conceptions), we do not recommend restricting recruitment of sperm donors to winter / iii spring. A negative association between vitamin D and semen parameters is noted; therefore vitamin D supplementation should not be recommended to improve to semen parameters.

Neural induction is the process that occurs during embryogenesis whereby ectoderm is converted into neural tissue. However, the cellular and molecular mechanisms that govern this process are still not completely understood. For instance, in Xenopus, calcium signalling is known to play a pivotal role in neural induction but whether a similar mechanism occurs in mammals is unknown. As part of a wider study on genes regulating neural stem cell (NSC) development, we have discovered a novel role for Neuronatin (Nnaf), a regulator of intracellular Ca2+ concentration. In my thesis, I describe the use of a mouse embryonic stem cell (ESC)-derived neural differentiation model to dissect the function of Nnat in neural development. Using gain- and loss-of function experiments I show that ESCs in which Nnat expression had been knocked-down have a dramatically decreased ability to generate NSCs, and subsequently neurons, while ESCs that over-expressed Nnat generate an excess of NSCs and neurons. I reveal that Nnat interacts directly with the sarcoendoplasmic reticulum Ca2+-ATPase 2 (Apt2a2/SERCA2), and that Apt2a/SERCA inhibitors cause an increase in cytosolic calcium levels rescuing the ability of Nnat knockdown cells to generate neural cell-types. From these experiments I conclude that Apt2a/SERCA inhibition mimics the function of Nnat and, via the increase of cytosolic calcium, causes neural induction in Nnat knocked-down ESCs. I go on to show that this increase in cytosolic Ca2+, caused by inhibiting Apt2a/SERCA, activates Erk signalling and that inhibition of Erk signalling dramatically reduces the ability of ESCs to generate NSC. Together these results suggest that Nnat functions by increasing cytosolic Ca2+ levels, which in turn causes activation of Erk signalling leading to neural induction. In line with this, the expression of BMP4, which is an inhibitor of neural induction, was found to be up-regulated by Nnat knocked-down.

Osteopontin (OPN), a glycosylated phosphoprotein found in brain, is reduced in the surviving dopamine-containing neurons in the SN (SN) in Parkinson’s disease (PD). OPN prevents the death of dopaminergic neurons in cell culture and animal models of PD. This effect may be mediated through interactions between the RGD binding domain of OPN and integrin receptors resulting in an attenuation of reactive gliosis and the production glial-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). Therefore, the aim of these studies was to determine the involvement of OPN and an RGD containing peptide in the regulation of glia and neurotrophic factors in the neuroprotective properties of OPN in both in vitro and in vivo models of PD. Treatment of rat El 4 ventral mesencephalic cultures with full-length rat OPN or a peptide fragment of OPN containing the RGD binding domain protected cells from MPP+-induced toxicity. This was accompanied by an attenuation of microglia and macrophage numbers, and an increase in GDNF and BDNF levels. However, blocking GDNF function with a neutralising antibody did not inhibit OPN’s neuroprotective action. These results in vitro prompted the investigation of whether similar therapeutic effects could be found in adult rat brain. It was therefore determined whether the effects of exogenously administered OPN on gliosis and neurotrophic factor levels in rat brain could confirm the trans ferability of the in vitro findings to an in vivo model of dopamine neuron degeneration. OPN and its integrin-binding fragment protected dopaminergic neurons against 6-OHDA toxicity and attenuated 6-OHDA-induced microglial number in vivo, although neither treatment with OPN or the peptide fragment altered levels of GDNF or BDNF in the SN or striatum up to 8 days after the injection. In summary, both OPN and the RGD-containing peptide were neuroprotective and reduced reactive gliosis in vitro and in vivo and increased neurotrophic factors in embryonic primary cultures. OPN and a smaller RGD containing peptide could thus be a potential treatment for PD.