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Grey seal pups are weaned abruptly after a short, intensive nursing period, during which they lay down fat and protein. Fat stores are crucial for insulation when they first go to sea and protein is essential for muscle and oxygen store development. Both components also provide them with energy to sustain them during an extended postweaning fast and their first days and weeks at sea until they can forage effectively. There is thus a trade off between the requirements for fat and protein in metabolism and for other functions. It is crucial for the survival of grey seal pups that they begin to feed before these reserves become critically depleted. The ability to do so depends on the size of their fat and protein stores at weaning, energy partitioning during the postweaning fast and the development of adequate diving and foraging capabilities. The management of fat and protein stores through energy partitioning and appropriate timing of departure are key to survival but the mechanism by which this management is achieved is unknown. To investigate the mechanism that controls energy partitioning and timing of departure from the colony, changes in body mass and composition and levels of hormones that are involved in energy balance in other mammals were measured during the postweaning fast of wild grey seal pups. The impact of initial energy reserves and hormone levels on energy use and departure was investigated directly, by manipulation of energy reserves and hormone levels, using supplementary feeding and hormone treatment. This is the first time such intervention studies have been performed in wild fasting pups. The impact of the stress of repeated handling on hormone levels and energy utilisation was also examined. The movement patterns and development of maximum diving capabilities of pups after departure were investigated using satellite telemetry. Leptin was present in grey seal serum but could not be measured accurately using two currently available immunoassays. Prolactin was not detected in postweaned pup serum and was eliminated as a potential candidate for control of fuel use and timing of departure from the colony. Cortisol and thyroid hormones (TH) were measured in serum but Cortisol could not be measured in grey seal saliva. Cortisol and TH are potentially involved in energy expenditure and fuel partitioning during fasting. Cortisol and total thyroxine levels decreased from weaning to midway through the fast. Both hormones increased in response to both natural and artificially induced changes in fuel availability. Dexamethasone, a potent Cortisol analogue, caused a short-term increase in mass loss, and induced reversible and short-lived changes in Cortisol levels and immune function, but its impact on energy partitioning was unclear. The effects of the drug were not mediated through TH. There were no changes in TH or Cortisol consistently associated with the timing of departure and dexamethasone did not induce departure. Neither hormone therefore seemed likely to be involved directly in the cue to leave the breeding colony. Handling regime did not impact significantly on Cortisol secretion, TH levels or long-term fuel use in grey seal pups. Fatter animals, or those provided with additional food, relied more heavily on fat to meet energetic requirements than leaner pups. There was no difference in energy partitioning between the sexes. Grey seal pups had a greater tolerance to protein depletion than terrestrial mammals but may have reached critical protein levels soon after departure from the colony. They were therefore under considerable pressure to begin to feed very soon after they have gone to sea, despite their extensive fat reserves. Pups showed marked inter-individual differences in movement patterns, which were very different from those of adults. Initially they undertook coastal movement, but showed wide dispersal from the colony within their first few months at sea. They began to exhibit adult-like repeated trips from known haul-outs to discrete offshore areas 4-5 months after departure. They were able to reach almost all areas and depths available in the North Sea, but were constrained in their ability to remain submerged, in terms of dive duration, post-dive surface interval and percentage of time spent diving over extended periods, compared with adults. Their maximum ability to remain submerged when they first went to sea was related to the duration of the postweaning fast and thus the degree of development on land. In contrast, maximum diving capabilities achieved during the first three months at sea did not vary substantially between animals and increased with time since departure, and were thus likely to be a product of diving-induced development of oxygen stores and cardiovascular control. Overall, larger body size and longer fast duration conferred increased diving capabilities, which may present one mechanism for increased survivorship in bigger animals.

Phocid seals go through dramatic seasonal changes in body mass and composition because of the spatial and temporal separation of foraging, reproduction and moulting. The regulation of fat mobilisation during these processes is a key factor for their survival. Unlike in humans, reproductive fitness and first-year survival are positively linked to adiposity in seals. Therefore, the rapid fluctuations in body mass and metabolic profile suggest that seals could be a good study model for understanding how fat expansion and loss can be achieved in a mammalian system without significant comorbidities. In humans, excess fat accumulation leads to tissue remodelling, potential hypoxia, chronic inflammation and oxidative stress. Although these processes are strongly interrelated, evidence suggest hypoxia, as result of limited vascularity or impaired diffusion, is the underlying trigger. How seals avoid the potential detrimental effects of large, fluctuating adiposity is unknown. Given that grey seals (Halichoerus grypus) have a similar fat percentage to obese humans, I hypothesised that oxygen availability in blubber will be affected by increasing tissue depth and adiposity. In chapter 2, I measured partial pressure of oxygen (pO₂) and temperature in vivo at different blubber depths in healthy juvenile grey seals. Adipose tissue pO₂ was similar to other species. Blubber pO₂ dropped significantly with increased fatness, but not with blubber depth, which is consistent with other animal models of rapid fat deposition and most human studies. Grey seal blubber therefore not only undergoes oxygen restriction during diving as previously reported, but also during fattening. In chapter 3 I investigated if fatness changes were reflected in tissue morphologic modifications and if adipocyte area and vascularity could impair oxygen diffusion in adult females and their pups during the breeding season, during which mothers reduced their mass by 23% while pups mass increased by 72%. Adipocyte area did not significantly shrink during the mothers' fast v but adipocytes did significantly enlarge during suckling in pups. Moreover, the rate of adipocyte growth was faster in inner than in outer blubber. Adipocyte diameter in mothers and weaned pups was close to the maximum O₂ diffusion distance, suggesting that the bigger cells could be susceptible to hypoxia. Vascularity did not reduce in mothers during mass loss, but angiogenesis was associated with adipocyte enlargement in inner blubber in pups. Whether vascularity growth is quick enough during adipogenesis to provide sufficient oxygen is unknown. Collagen area per adipocyte was associated with adipocyte area in females, but not in pups, suggesting pups' adipocytes are not mechanically constrained while expanding. In chapter 4, I investigated how hypoxia related molecular pathways were associated with adipocyte area alterations. Despite the expected reduced oxygenation described in chapter 2 and 3, no significant changes in hypoxia inducible factor (HIF-1α) were detected in mothers, which is consistent with the limited changes in adipocyte area. However, Hif-1α did significantly increase in pups at late suckling, and was reduced at the post-weaned fast. These changes were paralleled by alterations in adipogenic markers and thus the role of HIF during blubber development warrants further attention. Blubber collagen showed a unique distribution pattern of parallel bundles. This repeated orientation was not observed in human adipose tissue. Moreover, collagen area was similar between both species despite adipocyte area being significantly bigger in seals. Collagen content and distribution might allow blubber to expand further than subcutaneous adipose tissue in humans, which could help prevent ectopic fat accumulation that is a key trigger for comorbidities. Downstream effects of HIF-1α accumulation were investigated in chapter 6, with a novel explant in vitro approach. The HIF-1α downstream metabolic pathways seem to be regulated conversely to what is reported in the literature. We suggest that the metabolic pathways are more susceptible to nutritional state than oxygen availability. This thesis contributes to the understanding of oxygen management during blubber development and contraction in seals, the molecular shifts induced by hypoxia and key insights into the comparative anatomy and physiology of adipose tissue structure and function.

The neuromuscular junction (NMJ) defines the functional interface between nerve and muscle. Reactive oxygen species (ROS) are linked to pathological denervation in multiple neurodegenerative diseases, but whether and how ROS causes denervation is unclear. Our main hypothesis was that ROS recapitulates the signalling pathways of developmental denervation to cause pathological denervation in multiple diseases. To address our hypothesis, we made the local consequences of NMJ detectable as motor deficits by using disparate exogenous and endogenous models to induce synaptic inactivity in developing Xenopus laevis tadpoles. We resolved whether: (1) synaptic inactivity increases mitochondrial ROS; (2) chemically heterogeneous antioxidants rescue synaptic inactivity induced motor deficits; (3) ROS and mitochondrial calcium (mtCa2+) crosstalk exists; and (4) denervation is achieved by activating non apoptotic caspase 3. Regardless of whether it was achieved with muscle (α-Bungarotoxin), nerve (α-Latrotoxin) targeted neurotoxins or an endogenous pruning cue (SPARC), synaptic inactivity increased mitochondrial and cytosolic ROS in vivo. In addition, selectively inducing mitochondrial ROS—using mitochondria-targeted Paraquat (MitoPQ) - recapitulated synaptic inactivity induced motor deficits. In α—Bungarotoxin, α-Latrotoxin and SPARC, but not MitoPQ denervation models Ru360, specific mtCa2+ uptake inhibitor, blocked Ca2+ uptake in mitochondria and regulated ROS. In addition, Z-DEVD-FMK and AZ-10417808, specific caspase inhibitors, blocked caspase 3 activation, prevented denervation and mtCa2+ increase. Most importantly, the manganese porphyrins, MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+ two novel antioxidants, blocked ROS and mtCa2+ increase, prevented caspase 3 activation and abolished nerve, muscle and environmental toxin-induced pathological denervation. We conclude that ROS are a shared biochemical feature of pathological denervation and recapitulate an endogenous redox-regulated developmental denervation pathway.