Corticosterone (CORT), the main glucocorticoid in birds, plays a fundamental role in maintaining homeostasis and energy-balance, and is therefore tightly linked to an individual’s energetic state and the prevalent environmental conditions. CORT also has pleiotropic effects, ranging from reproductive function, the regulation of behaviour, morphology and immune function. Thus, inter-individual variation in CORT can potentially underpin a range of life-history traits, and through its pleiotropic effects act as a physiological mediator of reproductive decisions, causing birds to direct resources towards reproduction or self-maintenance dependent upon energetic condition and/or environmental quality. In turn, the role of CORT as a mediator of life-history traits has lead to the suggestion that inter-individual variation in CORT may be associated with individual differences in fitness. Despite this, the causes and consequences of large inter-individual variation in baseline CORT, specifically during reproduction, remain relatively unknown. The main aim of this thesis was to address these knowledge gaps by monitoring a nest-box population of blue tits, Cyanistes caeruleus, breeding on the east banks of Loch Lomond, UK over three years (2008-2010), and measuring baseline CORT concentrations in both adult and nestling birds at a standard stage of breeding in each year. Although environmental quality is often linked to variation in baseline CORT in breeding birds, this has rarely been investigated at the individual level. Chapter 2 focuses on the relationship between foraging conditions measured at the territory-scale and baseline CORT in adult and nestlings in 2008-2010. Synchrony with the peak in caterpillar abundance was the only factor to influence nestling CORT, and only in 2008. However, I found that synchrony between breeding and the peak in caterpillar abundance, weather variables and the density of oak trees influenced baseline CORT in adult birds. Importantly, the relationships between adult baseline CORT and these foraging conditions were only evident in some years; when conditions were most demanding. In addition, the effects of the foraging conditions measured upon adult baseline CORT appear to be synergetic and/or additive in nature. As inclement environmental conditions are often associated with elevated baseline CORT and reduced fitness in birds, it has been suggested that elevated baseline CORT should also be associated with reduced fitness (the ‘CORT-Fitness’ Hypothesis). However, this may not be the case, as modulation of CORT in the face of environmental challenges can adaptively influence physiology and behaviour to improve breeding performance and/or survival. In Chapter 3, I tested these assumptions and my results indicate that the foraging conditions linked to maternal baseline CORT differ to those associated with a proxy of fitness i.e. reproductive success. Specifically, maternal baseline CORT appears to be linked with factors that affect energetic demand, i.e. movement between trees, rather than reproductive success, i.e. total number of prey provided to offspring. In addition, in 2009 only, maternal baseline CORT was positively correlated with fledging number. In Chapter 4, I investigate whether there is a link between maternal baseline CORT and brood sex ratio adjustment over three years. I discovered that maternal baseline CORT was not correlated with brood sex ratio in any year. Maternal body condition, however, was linked to brood sex ratio adjustment in one year. Furthermore, experimental manipulation of maternal CORT during egg laying did not result in brood sex ratio adjustment or affect maternal condition, hatching success or chick development. Chapter 5 investigates the role of maternal baseline CORT in reproductive trade-offs. I reduced the costs of egg laying through supplemental feeding and compared maternal baseline CORT, brood care and maternal return rates between manipulated and control mothers. Reducing costs negated the physiological stress associated with provisioning effort in manipulated mothers and improved their return rates the following year compared with controls. Therefore, maternal CORT may mediate reproductive trade-offs in this species. As baseline CORT is often linked with energetic status and environmental conditions, and there is some evidence that CORT affects feather growth, I hypothesised that it may be linked to the expression of UV colouration in the crown feathers of female birds (Chapter 6). The results show that baseline CORT was indeed negatively correlated with UV colouration, and that UV colouration was positively correlated with reproductive success consistently over the three years, thus suggesting this trait signals maternal quality. Finally, Chapter 7 summarizes the main findings and considers how my results add to our knowledge base and discusses pertinent avenues of future research. This thesis presents compelling evidence that inter-individual variation in baseline CORT is significant, as the results show that it is associated with foraging conditions, reproductive success and may also influence reproductive trade-offs and UV plumage colouration. However, the results do not support a role for baseline CORT in brood sex ratio adjustments in blue tits. The results also reveal the complexity of the relationships between inter-individual variation in baseline CORT, environmental conditions and reproductive success. Specifically, both foraging conditions and proxies of fitness, i.e. reproductive success were linked to baseline CORT differently between years, most likely due to the contrasting conditions experienced in those years. Therefore, although inter-individual variation in CORT is linked to life-history traits in breeding birds, relating this variation to individual fitness is challenging. Furthermore, there remains a lack of knowledge concerning the repeatability of baseline CORT concentrations in blue tits. Ultimately, my thesis suggests that in order to achieve a full understanding of how inter-individual variation in baseline CORT is linked to fitness, single year or short-term studies are inadequate; instead, researchers must relate individual differences to long-term measures of fitness.

Animals may adopt a range of energy strategies to persist and perhaps prosper under various prevailing environmental conditions. For example, they may achieve similar growth by investing heavily in maintaining a high capacity food capture and processing system or by using a lower capacity system that is cheap to run but which is less effective at accruing further resources. The way that individuals within species allocate energy resources is an intriguing issue that has implications for understanding competition, population structuring and the response of populations to environmental change. Energy budgets account quantitatively for the pathways by which food energy results in variation in somatic resources and constitute an important basis for evaluating links between behaviour and aspects of performance. Atlantic salmon have been an important model for exploring relationships between growth performance, metabolic strategies and individual behaviour. Juvenile salmonid fish have been a particularly useful subject for exploring individual variation in metabolism because it has been possible to relate standard metabolic rate (SMR) to behavioural traits and lifestyle within a species. SMR correlates with dominance status, which is reflected in the ability of fish to access high value food patches and may promote faster growth under some conditions. This thesis focuses on the within- and among-individual variation in SMR and looks at how a fish’s external environment and social interactions can influence its SMR, and whether this variation in SMR can affect other aspects of metabolism such as feeding. Experiments were carried out to determine: (A) whether the presence of a shelter reduces SMR; this may provide an additional reason for the extent to which these fish will compete aggressively for shelters when these are limiting (Chapter 2); (B) the extent of intraspecific variation in SDA (i.e. differences in the measured parameters that are used to define SDA), and to test whether the SDA for a given meal varies as a function of individual traits such as SMR. If SMR is directly related to scope for growth, then the speed and size of the SDA response should also correlate with SMR (Chapter 3); (C) whether ventilation rate is sufficiently accurately related to metabolic rate (MR) at a range of temperatures and activities so as to allow energy expenditure to be predicted outside a respirometer (Chapter 4); and (D) how visual isolation and the presence of a conspecific can affect SMR, using the method developed in the previous chapter (Chapter 5). Access to shelter was shown to have a significant impact on SMR, producing on average a 30% increase in metabolic costs in the absence of shelter. Therefore, the presence of appropriate shelter not only reduces the risk of predation but also provides a metabolic benefit to fish that is likely to have implications for growth performance and activity budgets. Variation in SMR was shown to affect the profile of energy consumption during digestion of a meal. The peak in post-prandial oxygen consumption, the duration of elevated metabolism and the overall magnitude of SDA all increased with the size of meal consumed. However, for a given meal size, fish with a higher SMR also had a higher peak and greater magnitude of SDA, yet experienced a shorter duration over which metabolism was elevated following the meal. Intraspecific variation in SMR is thus linked to variation in digestive strategies, although the costs and benefits of a given SMR are likely to vary with resource availability and predictability. Ventilation frequency was shown to be a good correlate of MR, since MR was found to correlate strongly with VR in all fish tested, at all temperatures and the relationship was independent of causal factor. The relationship was linear, and both the slope and corresponding intercept of the regression equation were strongly dependent on the fish’s body weight and the test temperature. Visual measurements of VR may therefore provide a highly accurate, cheap and non-invasive method of measuring the energy consumption of fish engaged in natural behaviours. Comparing SMR of individual fish when alone with response to presence and absence of physical shelters showed that all fish exhibited a reduction in SMR when provided with an appropriate shelter. However, when grouped, there were both positive and negative group effects on SMR depending on the relative size of fish. The consequences are that, although the group effect on SMR may be small on average, at the population level it is large, of variable sign and profoundly important in terms of the overall energy budget at the individual level. This thesis therefore shows that metabolic rates can vary extensively within as well as between individuals, depending on the context, and this variation will have profound effects on overall energy budgets.

In a variety of vertebrate species, early life environmental cues are important drivers of an individual’s phenotypic trajectories, priming physiological pathways, with consequences for growth, reproductive-related traits and lifespan. These phenotypic responses are believed to be adaptive in the short-term, but may impinge on health and survival over the long-term. Much of the work in this field has focused on the potential constraints imposed on animals after exposure to early life adversities, including nutritional deficit, sibling competition, and high predator pressure. Such stressful experiences can result in direct, but also indirect (via the maternal route) increases in the exposure to glucocorticoid stress hormones in the developing individuals. Glucocorticoids, whose production and secretion is regulated by the Hypothalamic-Pituitary-Adrenal axis (HPA axis), have been hypothesised to be the main candidates mediating the programming effects of developmental stress. Earlier predictions based on this assumption came from studies conducted in mammals. In mammals it is particularly difficult to manipulate exposure to circulating hormones in developing individuals because of the physiological intimacy between mother and offspring via the placenta and lactation. Here, I circumvent this complicating factor by using the precocial Japanese quail as a study species. In chapter 2 I measure corticosterone (B, the main avian glucocorticoid) stress responses to a standardised environmental stressor in growing quail aged 8- and 16-days-old. The results are consistent with those previously reported in other precocial birds, showing that the magnitude of the stress response (i.e. peak B within 30 min period) is higher in the 8- than the 16-day-old hatchlings. I find no differences in baseline B concentrations between the two groups. I then describe the main experiment in which I elevate B concentrations in ovo and/or in the endogenous circulation of the hatchlings (oral B administration from day 5 to day 19 post-hatching) in order to obtain four distinct phenotypes: pre-hatching B-treated birds, post-hatching B-treated birds, both pre- and post-hatching B-treated birds, and controls. I examine the specific and combined effects of pre- and post-hatching B on (1) growth trajectories and physiological stress responses before sexual maturity (post-hatch day 22) and upon adulthood (post-hatch day 64); (2) adult gene expression patterns within the hippocampus and hypothalamus, and (3) oxidative stress in the blood and the brain in the adults. The main results of Chapter 3 show that post-hatching B, regardless of pre-hatching experiences, decrease HPA axis responsiveness in the juveniles, but only in the female quail; whilst pre-hatching stress, when not combined with post-hatching B, increase HPA responsiveness in both sexes upon adulthood. I also show that both pre- and post-hatching B induce short-term alterations in triglyceride basal concentrations, which are linked with the sex and basal glucose concentrations of the birds; the effects of pre-hatching B exposure were visible also upon adulthood with sex-specific alterations on basal glucose concentrations. Overall these results suggest that early life stress can trigger both transient and permanent physiological changes, depending on the sex and the quality of both the pre- and post-hatching environment. In Chapter 4 I show that the gene expression responses to pre- and post-hatching B are overall subtle, results similar to those reported in previous genomic studies that have manipulated early life rearing environments. The effects are, however, distinguishable, strongly tissue-specific and involve well characterised key candidate genes in the regulation of the HPA axis. These data also suggest important novel regulatory mechanisms, likely linked with cellular redox state, which may be driving the long-term effects of developmental stress. Finally, in chapter 5, I show that developmental B induces alterations in the basal antioxidant defences upon adulthood. The magnitude of these effects, once more, depends upon the timing of exposure, interactions between the pre- and post-hatching B and the tissue examined. As there are no differences in terminal oxidative damage, these results suggest that the B-treated birds could avoid oxidative stress via altering body oxidative defences. In summary, my findings throughout this thesis, illustrate the complexity of glucocorticoid programming and the importance of integrating analyses at multiple levels, from physiology to genome-wide investigations. The results of this thesis also strengthen the importance of examining the effects of early life stress over differing life stages in order to consider the overall balance of costs and benefits that may ultimately affect Darwinian fitness and survival.

P2X receptors are ATP-gated ion channels with myriad roles in humans and other higher vertebrates. Research over the past decade has described the cloning, pharmacology, and physiological role of this receptor family in a number of invertebrate organisms, as well as in unicellular amoeba and algae. However, questions remain regarding the extant of P2X receptor phylogeny and function in many invertebrates. A greater understanding of invertebrate P2X receptor pharmacology and function may provide insights into structure-function relationships in “higher” homologues, as well as novel roles for this ligand-gated ion channel family. This thesis investigated P2X receptor expression and function within eukaryotic phylogeny, with particular emphasis on invertebrate animals. Homology searching of transcriptomic and genomic datasets identified a number of candidate P2X receptor sequences across a range of phyla. Notably, homologues were bioinformatically identified within orders of the class Insecta, where they had been previously thought to be absent. Homologous sequences were also identified in a multicellular alga of the Charophycea, a class of the Viridiplantae division Charophyta, considered to represent the closest extant taxon to terrestrial plants. Following the identification of a P2X receptor homologue in the cnidarian Hydra vulgaris (AEP) (‘aepP2X’) by bioinformatics approaches, total RNA preparations were used for cDNA synthesis to generate templates for PCR to yield aepP2X clones. These clones encoded proteins that exhibited concentration-dependent ATP-evoked inward currents when expressed heterologously in HEK293 cells (EC50 ca. 120 μM; holding potential of -60 mV). Co-application of the classic P2 receptor antagonists pyridoxalphosphate-6-azophenyl-2', 5'-disulphonic acid (PPADS) and suramin was ineffective, as were phenol red and brilliant blue G (BBG). The synthetic ATP analogues α,β-methylene ATP and β,γ-methylene ATP (1 mM) did not evoke currents at aepP2X. Consistent with most mammalian homologues, aepP2X was found to be a non-selective cation channel with negligible chloride ion permeability. Immunohistochemistry using a custom polyclonal antiserum raised against a C- terminal epitope of aepP2X suggested the expression of this receptor occurs in developing and late-stage nematocysts in whole Hydra polyps. A P2X receptor from the microcrustacean Daphnia pulex (DpuP2XB) was also expressed heterologously in HEK293 cells, and was found to be largely insensitive to extracellular ATP (EC50 > 1 mM), although reduction of total divalent cation concentration of perfusing extracellular solution partially restored ATP sensitivity in DpuP2XB. Whilst a P2X receptor homologue from the placazoan, Trichoplax adhaerens (TadP2XA) did not respond to extracellular ATP in HEK293 cells, a P2X receptor from the lancelet Branchiostoma floridae (BfloP2X) does respond in a concentration-dependent manner to A TP . Currents evoked through BfloP2X displayed rundown with repeated 1 mM ATP applications.

In many taxa there is considerable intraspecific variation in life-history strategies from within a single population, reflecting alternative routes through which organisms can achieve successful reproduction. Brown trout (Salmo trutta) and Atlantic salmon (Salmo salar) show some of the greatest within-population variability in life history strategies amongst vertebrates, with some individuals of a population migrating while other individuals remain resident (the phenomenon of ‘partial migration’). Furthermore within each migratory ecotype, multiple discrete male and female life histories (e.g. time spent in freshwater and saltwater) co-exist and interbreed on many spawning grounds, although the effect of the various combinations of life histories on offspring traits remains unknown. These issues are covered in the General Introduction (chapter 1), which introduces the rationale for the experimental work which forms the core of this thesis. In the first of these empirical investigations, full-sibling groups of offspring from freshwater-resident and anadromous brown trout and half-sibling groups of Atlantic salmon were reared in the laboratory under common garden conditions to examine potential differences in their early development, physiology, behaviour and phenotypic flexibility. I found that freshwater-resident parents produced eggs that were slower to hatch than did anadromous parents, but their offspring were quicker to absorb their yolk and reach the stage of exogenous feeding. Their offspring also had a higher conversion efficiency from the egg stage to the start of exogenous feeding (so were larger by the start of the fry stage) than did offspring from anadromous parents (chapter 2). Using Atlantic salmon I showed that mothers that had spent longer at sea (two versus one year) produced offspring which were heavier, longer and in better condition at the time of first feeding. However, these relationships disappeared shortly after fry had begun feeding exogenously. At this stage, the juvenile rearing environment (i.e. time spent in fresh water) of the mother was a better predictor of offspring traits, with mothers that were faster to develop in fresh water (migrating to sea after two rather than three years of age) producing offspring that had higher maximal metabolic rates and aerobic scopes, and that grew faster. Faster developing fathers tended to produce offspring that grew faster, were in better body condition and had higher maximal metabolic rates (chapter 3). I also found that the parental type in brown trout had a significant effect on dominance status in territorial interactions, with offspring of anadromous fish being dominant over size-matched offspring of freshwater-residents, but only when both had been raised under intermediate levels of food availability (chapter 4). Lastly I found that offspring of anadromous trout were less able to maintain their growth rate when reared on a Daphnia diet than were those of freshwater-residents, and showed a correspondingly greater increase in growth following a switch in diet from Daphnia to Chironomid larvae. Offspring of anadromous parents also showed less morphological flexibility in response to diet than did the offspring of freshwater-residents (chapter 5). The results of these studies are summarised in the General Discussion (chapter 6), which suggests that parental effects (either direct or environmentally-mediated through differences in rearing environments) may be an important mechanism underlying the expression of threshold traits and play a significant role in the perpetuation of non-breeding partial migration and life history strategies within populations.

The main aim of this thesis was to investigate the effects of environmental conditions, particularly unpredictable or potentially negative ones, on the maternal transmission of the primary avian stress hormone, corticosterone, to developing embryos. We currently lack information on the extent to which conditions in the maternal environment are transmitted to the offspring in birds via egg compositional changes. It is possible that maternally derived hormonal signals communicate information about the external environment to developing embryos and directly influence the fitness of their offspring in a negative or positive way. I found, using captive zebra finches, that the experimental stressor of unpredictable food availability (as these birds are used to ad libitum food) experienced by mothers can elevate yolk CORT concentrations, but only when combined with the additional demand of laying a replacement clutch (potentially a buffering system to prevent mild stressors impacting on CORT transmission to the embryo). I then looked at yolk CORT concentrations in two populations of gulls (herring and lesser black-backed gulls) in which the population trajectories differed depending on environmental conditions (potentially a reflection of different exposures to stressful stimuli). The results however did not support this hypothesis, as there were no differences according to habitat type or between species (where they coexist). This would suggest that the different environmental circumstances (harsher for the herring gull) experienced by these two species are not reflected in differences in their eggs (at least in terms of CORT). This could be the result of the eggs being buffered from the maternal CORT environment or it may be that the difficult environmental conditions are not occurring during the breeding season. We also identified that experimental human disturbance during the laying period does not appear to elevate yolk CORT concentrations, although there was a trend for concentrations to be higher following the loss of the first clutch in the herring gull (as seen in the zebra finches). I also measured yolk CORT concentrations in Common Eider eggs and looked for differences according to the degree of nest shelter. I found no relationship between shelter and yolk CORT, but birds that laid in more sheltered sites had, on average, smaller eggs. This may indicate lesser quality birds are nesting in the sheltered sites and that yolk CORT is not affected by maternal condition. Finally, I looked at another mechanism through which information relating to the maternal environment could be transferred to the embryo. I investigated whether there were any links between maternally derived immunity and CORT by comparing the anti-microbial lysozyme and CORT concentrations in the albumen. I found no correlation between CORT and lysozyme, suggesting that CORT may not affect lysozyme production. It may be that other factors such as colony density and ‘cleanliness’ are more important in determining the concentrations of lysozyme deposited in the egg or that lysozyme production is not sufficiently costly to be influenced by the maternal stress state. The overall theme of my findings is that CORT concentrations in eggs do not appear to vary much with maternal environments. I will discuss these findings in their broader ecological and evolutionary context and discuss if stress hormones are indeed being used as adaptive signals for preparing the embryo for its postnatal environment.

A considerable number of abiotic (e.g. temperature) and biotic factors (e.g. intra-interspecific interactions) contribute in shaping species’ distribution and invasiveness but knowledge is still lacking regarding the importance of physiological and behavioural traits in determining the distributions of ectotherms and especially the invasion success of non-native species into novel habitats. With rising temperatures, distribution shifts in many of fish species have been observed. Additionally, changing thermal conditions are facilitating the colonisation of invasive species. It is crucial that we gain an increased understanding of the mechanisms underlying the effects of environmental change on the distribution of aquatic species and the ecological damage caused by invasive species. Physiological traits are likely to present a fundamental constraint on the environments that are habitable to a given species. Whole animal traits associated with energy metabolism and locomotory performance are especially likely to be important in this regard. Nevertheless, the effects of traits such as metabolic rate and aerobic scope (AS) on the distributions of native and invasive species have not been thoroughly studied. In a first step toward understanding these effects, I performed a phylogenetically-informed analysis of links between AS and absolute latitudinal distribution range in 86 fish species. From the results obtained in Chapter 2, I found no evidence for the direct implication of AS in the currently observed distributions of fishes. Moreover, no association between AS and invasion success in 59 freshwater fish species (23 invasive and 36 native species) was found. These results suggested that peak AS is not a constraining or determining factor in the distribution range and invasion success in fish. Following on from these results, there is a possibility that there could be a trade-off between peak and breadth of performance for AS across temperatures in fishes. Species with a higher peak AS might only be able to function normally over a narrow range of temperatures. In Chapter 3, I collected AS data from literature and conducted phylogenetical-informed analysis to test the trade-off theory in AS across 28 fish species. No evidence could be found for a trade-off between peak and breadth performance in AS for fish. Interspecific competition between invasive and native fish species might cause changes in the structure of native fish communities. Furthermore, these interactions can vary over competitive context (e.g. for prey or cover), differ over a range of environmental factors (e.g. in response to temperature variation) and be linked directly or indirectly to species’ metabolic capacity (e.g. aerobic scope). With increasing temperatures, invasive species might gain a competitive advantage over the native species though shifts or changes in competitive behaviour and traits such as AS. After having investigated broad patterns among AS and geographical distributions in fishes, the remainder of my thesis focused on trade-offs in energy allocation and tolerance to environmental stressors in a pair of species to determine the role of aerobic capacity as a factor in competition between these two species. Specifically, I examined interactions between native stone loaches (Barbatula barbatula) and invasive bullheads (Cottus gobio), two species which occupy the same ecological niche and that are believed to compete for similar habitats. Physiological and behavioural traits could play an essential role in the spread of invasive species, particularly the internal underlying mechanisms that modulate an organism’s response to environmental changes. In Chapter 4, I examined physiological and behavioural responses of invasive bullheads and native stone loaches to acute and acclimated temperature shifts (13-21°). I found that invasive bullheads had a lower AS than stone loaches over all temperatures tested. Bullheads were also less active overall and preferred colder temperatures (17.5-19°C) than stone loaches (21-22.4°C). Therefore, changes in AS in response to thermal variation are unlikely to be a contributing factor in invasion success of bullheads in Scottish rivers. In Chapter 5, I investigated the direct competitive interactions between bullheads and stone loaches at three different temperatures (13°C, 17°C and 21°C). Overall, native stone loaches were better competitors for shelter use and in particular at colder temperatures. There was no clear causal effect of temperature or AS on competitive outcomes between these two species. Low competitive ability found in invasive bullheads suggests that bullheads may not be actively displacing stone loaches. It has been suggested that a successful invader should have a wide tolerance range for different environmental factors. For example, invasive species might be more tolerant to hypoxic events as compared to native species. In Chapter 6, I looked at the hypoxia tolerance and avoidance behaviour of bullheads and stone loaches over different dissolved oxygen (DO) concentrations (100%, 80%, 60%, 40%, 30%, 25% and 20% DO levels). Surprisingly, I found that bullheads were less tolerant to hypoxic conditions with a Pcrit value of 4.96 mg O2 l-1 at 14°C. Avoidance behaviour towards progressive hypoxia was similar between bullheads and stone loaches; both species spent most of their time utilising the shelter even in hypoxic conditions. Low tolerance towards hypoxia did not give an advantage to invasive bullheads over native stone loaches in particular during harsh environmental conditions. The results from this thesis suggest that metabolic traits may not play as strong a role in constraining species distributions as previously suspected, particularly in the specific case of interactions between native stone loaches and invasive bullheads in Scotland. Instead, other physiological factors, life history traits, and population demographics may play a primary role in affecting invasion success in this case.

There is now strong epidemiological and animal research showing that undernutrition in gestation and early postnatal life is linked with a higher incidence of cardiovascular disease in adulthood. The physiological processes involved are not yet clear. The aim of this thesis was to investigate how aspects of the cardiovascular system in the adult sheep are affected by early life periods of undernutrition, and to investigate to concept that mismatches in these periods may influence these responses. Welsh Mountain ewes received 100% of global nutritional requirements at all times (C) except from minus 30 to day of conception (B), from minus 15 to 15 days after conception (A), or from day 1 of gestation to 31 days gestation (U) when they received 50% of total nutrient requirements. Offspring of groups C & U were then fed ad libitum (CC & UC) or at a level that reduced body weight to 85% of individual target weight from 12 to 25 weeks postnatal age (CU & UU). The adult sheep cardiovascular function was studied at 2.5 years and 3.3 years. At 2.5 years the UC males showed an increased interventricular wall thickness without loss in function. These effects were not seen if early postnatal restriction was also received. In contrast, females subject in the gestational undernutrition (UC) showed a dampened heart rate response to a stressor, which was not seen when combined with a postnatal challenge (UU). Basal adrenaline was elevated in male and female singletons exposed to the postnatal challenge (CU & UU). The stressor produced an enhanced adrenaline response in the females in the postnatally challenged group (CU). This effect was attenuated when combined with a gestational challenge (UU). Thus early life undernutrition alters adult cardiovascular physiology and may have consequences for cardiovascular function and disease in later life. These effects are sex-specific. The cardiovascular system is affected by the mismatch between gestation and early postnatal nutrition.

It has been suggested that the heat shock response of Xenopus oocytes might provide a good model system to study the regulation of translation in early development: oocytes were thought to contain a store of hsp70 mRNA, which was unmasked reversibly on heat shock. However, more recently it has been reported that the hsp70 apparently synthesized in oocytes is in fact made entirely in the attached follicle cells. A method was developed to reliably remove follicle cells. Two-dimensional gel analysis of radioactively labelled oocytes revealed that, although hsp70 was the major protein synthesized during heat shock if follicle cells were present, it could not be detected if these cells were removed. Oocytes do contain up to 15pg of hsp70 mRNA; it is not clear if this is translated. In some experiments synthesis of a group of proteins was induced in oocytes during heat shock, but none was hsp70. To test whether oocytes can synthesize hsp70, plasmids encoding Xenopus hsp70 genes were injected. These were transcribed efficiently, yielding RNA that was indistinguishable in length and polyadenylation status from heat-induced transcripts in follicle cells. Synthesis of hsp70 was detected in defol1iculated, injected oocytes: it was translated well even during heat shock (when translation of other mRNAs was greatly reduced), suggesting that oocytes regulate translation of exogenous transcripts appropriately. Surprisingly, flu nucleoprotein RNA transcribed in oocytes was also preferentially translated during heat shock in some experiments. The 5' leaders of hsp70 and NP RNAs contain a region of homology. As injection of RNA provides a more direct way of controlling transcript levels in oocytes, polyadenylated RNA encoding hsp70 was transcribed in vitro (from a variety of templates) and injected into oocytes. Products of a hsp70/globin fusion and a truncated hsp70 gene were never detected. Native hsp70 was synthesized, but it was not made preferentially during heat shock. Similarly, when influenza NP mRNA was injected, preferential translation was never observed. It appears therefore that exogenous RNA is regulated appropriately during heat shock when it is introduced by the route of DNA injection, but not when RNA itself is injected.

Glyoxalase 1 (Glo1) of the glyoxalase system catalyses the metabolism of the reactive dicarbonyl metabolite, methylglyoxal, and thereby prevents potentially damaging glycation of protein and DNA. Glo1 is hypothesised to be a potential factor in the development of vascular complication of diabetes, such as diabetic nephropathy. The induction of diabetes in mice deficient in Glo1 provides a pre-clinical in vivo model to test this hypothesis. Glo1 mutant mice with putative Glo1 deficiency produced by the International Mouse Knockout Consortium (IMKC) were acquired from the European Mutant Mouse Archive. The initial aim of this study was to study the exacerbation of diabetic nephropathy by Glo1 deficiency in streptozotocin-induced diabetic mice, with an initial objective to confirm Glo1 deficiency in the IMKC Glo1 mutant mouse and subsequent objectives contingent on this. The preliminary studies were unable to confirm Glo1 deficiency in this mouse model and so a revised aim was to characterise the mechanism of compensatory Glo1 expression in the mutant mouse and explore similar occurrence in similar precursor mouse embryonic stem cells (ESCs) and related clinical application. Genotyping of Glo1 mutant mouse offspring by PCR revealed only heterozygotes and wild-type (WT) littermates, and no homozygotes without Glo1 wild-type alleles. Studies of the Glo1 mutant mouse revealed levels of Glo1 activity, protein and mRNA identical to those of wild-type control siblings. Other components of the glyoxalase system were also analysed – activity of glyoxalase 2, concentrations of methylglyoxal (MG) and D-lactate, and tissue protein content and urinary excretion of MG-derived glycation adduct MG-H1 and found no significant change in Glo1 mutant mice, with respect to WT controls. This suggested a functionally normal Glo1 and glyoxalase system in Glo1 mutant mice. Therefore, Glo1 mutant mice have a mutated Glo1 gene but with compensatory Glo1 expression identical to that of WT control. This provided a possible explanation for the unexpected normal phenotype of Glo1 mutant mice reported in the IMKC project. To explore the mechanism of compensatory Glo1 expression, Glo1 copy number was quantified by Taqman® method, normalizing response to transferrin receptor protein-1 (Tfrc). Glo1 mutant mice had 3 copies of Glo1 in all tissues analysed with amplification extending from 3’-end of exon 1 to the 5’-end of exon 6. Taqman copy number assay was established to detect and quantify mutant Glo1Gt(..)Lex and WT alleles. Most mutant mice contained two copies of Glo1 and one mutant copy of Glo1Gt(..)Lex – Glo1(+/+)Gt(..)1Lex. In some cases, however, 2 copies of both Glo1 and mutated Glo1Gt(..)Lex – Glo1(+/+)Gt(..)2Lex were found. Inheritance studies suggested a simple Mendelian inheritance with a WT allele accompanying the Glo1Gt(..)Lex mutant allele on arms of chromosome 17 such that Glo1 deficiency was prevented. This was indeed observed throughout the all breeding of the Glo1 mutant mice. I hypothesised that Glo1 copy number increase may have arisen in the mutant mice during gene trapping by copy number alteration (CNA) induced by increased methylglyoxal concentration, or dicarbonyl stress, in mouse ESCs. To explore and model this, mouse ESCs were cultured with exogenous 200 μM MG under atmospheres containing 20% oxygen - typical of most cell culture conditions, and 3% oxygen - typical of ESCs oxygen exposure in vivo. Incubation of ESCs for 12 days with MG induced CNV increase of Glo1 by up to 16% in both 20% and 3% oxygen atmospheres. Increase in Glo1 CNV at day 12 with MG treatment was associated with an increase in Glo1 protein. Therefore, functional low level CNA of Glo1 was induced by exposure to high levels of exogenous MG. No evidence was found for Glo1 CNA with dicarbonyl stress induced by Glo1 silencing or cell permeable Glo1 inhibitor. Finally, I hypothesised that GLO1 CNA may occur in clinical dicarbonyl stress, a severe example of which is patients with renal failure receiving haemodialysis - associated with ca. 5-fold increase in plasma MG concentration. DNA of peripheral mononuclear cells from healthy subjects and patients with renal failure receiving hemodialysis renal replacement therapy were examined. Human GLO1 copy number was not significantly different between the patients and the control subjects. This requires further investigation in this case and other examples of clinical dicarbonyl stress. From these studies I conclude that the IMKC Glo1 mutant mouse does not exhibit the Glo1 deficiency; rather, it maintains wild-type levels of Glo1 expression through Glo1 copy increase likely induced during gene trapping. Dicarbonyl stress in mouse ESCs in vitro induced low level Glo1 copy number increase – a model of Glo1 CNA in putative gene trapping associated dicarbonyl stress. It is unclear if GLO1 CNA occurs clinically. These findings reveal that focussed copy number alternation of GLO1 may provide a protective response to dicarbonyl stress in some circumstances.