The insect Malpighian tubule is involved in osmoregulation, detoxification and immune function, physiological processes which are essential for insect development and survival. As the Malpighian tubules contain many ion channels and transporters, they could be an effective tissue for targeting with novel pesticides to control populations of Diptera. Many of the insecticide compounds used to control insect pest species are no longer suited to their task, and so new means of control must be found. The malarial mosquito, Anopheles gambiae, spreads the Plasmodium parasite which is responsible for over one million deaths each year, and is one of the species on which many current insecticides are no longer effective. Anopheles is notoriously difficult to study due to a lack of natural mutation stocks and transgenic capabilities, as well as the difficulties involved with maintaining a colony. The fruit-fly Drosophila melanogaster is a useful model organism for Anopheles, and previous studies suggest that the mechanisms of Malpighian tubule function are well conserved between the two species. Following microarray investigations to identify genes which were highly enriched in both the Anopheles and Drosophila Malpighian tubules, four homologous genepairs were selected, AGAP097752 and CG15406, AGAP012251 and Picot, AGAP009005 and ZnT35C, and AGAP002587 and CG8028. Analysis of the Anopheles Malpighian tubule microarray data-set showed ion channels and transporters to be highly expressed in the tubules, although similarly to Drosophila, very few of the renal up-regulated genes have been characterised. The gene-pairs chosen were all novel, but putatively predicted to be involved in sugar transport, phosphate transport, zinc transport and monocarboxylate transport respectively. These are functions which are likely to be essential, but so far remain unstudied in the insect renal system. The gene-pairs were chosen with two main purposes; to determine how closely expression of the genes was conserved between Anopheles and Drosophila, and also to determine which of the genes were essential, and could therefore be effective insecticide targets. The homologous gene-pair AGAP007752 and CG15406 have well-conserved expression in the Malpighian tubules, suggesting that they are functionally important genes. This was shown in Drosophila, where knockdown of CG15406 4 expression was lethal to the fly. A direct role in tubule fluid secretion was not found, and experiments to determine the sugars transported by CG15406 were inconclusive, possibly due to an abundance of highly-expressed sugar transporters in the tubules. The inorganic phosphate co-transporters AGAP012251 and Picot also show conservation of expression in the Malpighian tubules, and are likely to be involved in the transport of inorganic phosphate into the tubules for incorporation into metallo-organic concretions. In the Anopheles tubules the concretions are found in the main segment, in the Drosophila tubules they are located in the distal initial and transitional segments, where AGAP012251 and Picot are expressed. Picot is essential for Drosophila development through to adulthood, and for survival as an adult, although the transporter does not appear to be directly involved in fluid secretion. Expression of neither AGAP012251 nor Picot is confined to the tubules. The putative zinc transporters AGAP009005 and ZnT35C show a highly conserved expression pattern, and appear to be involved in the secretion of excess zinc from the Malpighian tubules. ZnT35C is essential early-on in Drosophila development, and for survival in the adult fly. Similarly to Picot and CG15406, there is no direct role for ZnT35C in fluid secretion from the tubules under normal zinc conditions. The putative monocarboxylate transporters AGAP002587 and CG8028 are not as well conserved, as AGAP002587 is highly upregulated in the tubules of female mosquitoes both before and after a bloodfeed, whereas CG8028 has no sex-specific up-regulation. CG8028 is not essential for Drosophila development or survival, and plays no discernable role in fluid secretion. The data collected during this investigation suggests that in general there is a high level of conservation of expression between homologous transport genes in the Anopheles and Drosophila Malpighian tubules. The three gene-pairs which show the greatest conservation of expression are also essential for development and survival in Drosophila. This suggests that cross-species studies are an effective way of finding essential and important genes. The data collected also suggests that Drosophila is a reliable model for Anopheles, and could be used as a high-throughput system of finding genes which could be effective insecticide targets in Diptera.

Wound healing and recovery from injury were investigated in eggs and larvae of herring (Clupea harengus L.), plaice (Pleuronectes platessa L.) and salmon (Salmo salar L.). The resistance of herring eggs to mecha.nical damage was first examined. The chorion of eggs before and just after fertilisation could be burst by loads of 4-30 g but eggs 5 h post-fertilisation could withstand over 1000 g without bursting. Resistance remained high until just before hatching when it decreased to 20-680 g. The vitelline membrane, however, showed a lower resistance at all stages. Early herring and plaice larvae were caught and eaten by medusae (Aurelia aurita, Tiaropsis multiserrata, Bougainvillea sp.), hydroids (Sarsia sp.), megalopa larvae of the prawn Nephrops norvegicus and adult mysids. Early herring larvae survived minor stings from an Aurelia ephyra, and also experimentally inflicted lesions such as superficial scratches, suction wounds and amputation of up to 2 mm of the tail in sea water. The caudal region of the primordial fin regenerated within a month when less than 1 mm was cut off. Yoll, sac and first feeding herring also survived an incision of 0.3 mm long through the body ventral to the notochord and dorsal to the gut; in starving larvae survival was poorer in the later stages of starvation. When skin was removed in larvae of all 3 species the mortality depended on the area of the lesion, thp maximum area tolerated increasing with larval size. In sea water the threshold area was 0.1-0.2 mm2 dO.r 6-8 mm long plaice, <: 0.3 mm for 10-13 mm long herring and 0.3-0.4 mm for 14-17 mm long herring. In river water the threshold was 1~2mm for 19':1l2m1m long saImon and 6.5-8 mm for 26-28 mm long salmon. The thresholds w ore about 1-3% of the total body surface area. Tolerance was increased in isosmotic salinities, the threshold area being as high as 10-14% of the body surface in 24-28 mm long salmon in 8%0. Healing of skin lesions was observed in vivo and by histology, the main response being a mass migration of epidermal cells from the periphery of the lesion. Wound areas of 0.1-7 mm closed in 4-12 h, the mean rates of cell migration being 40-110JUm/h at 10_11°C. The rate of migration was temperature dependent. The normal skin structure was restored with regeneration of a new basement membrane and dermis within 3 weeks. Older stage larvae showed an inflammatory response similar to adult fish.

Energy is the fundamental currency of life that drives organismal growth and development. Energy requirements vary greatly between species but also within species due to differences in physiology, behaviour and life history. The consequence of this variation is of great interest to ecologists, as it is potentially a trait upon which natural selection can act. One of the main components of an organism’s energy budget is its baseline level of metabolism, hereafter termed its standard metabolic rate (SMR). It has been shown in several species of salmonid fish that a high standard metabolic rate correlates with dominance, aggression and boldness. This competitive advantage has been shown to result in higher growth over conspecifics in simple lab environments, but the ecological consequences are less clear. This thesis examined the performance of contrasting metabolic strategies across a range of environmental conditions to ascertain the ecological consequences of SMR variation. Experiments also investigated the relationships between SMR, food intake and absorption efficiency to help relate energetic strategies to performance. The effects of environment on the outcome of different energetic strategies were profound. Higher population densities increased intraspecific competition for preferable feeding territories, but fish with a higher SMR tended to be the best competitors and so were most likely to get a preferred territory (Chapter 2). However, for a given quality of feeding territory, whether relatively good or poor, lower SMR individuals grew best due to their lower energy requirements. The benefit to high SMR fish of being able to secure better territories was diminished under less predictable feeding conditions, and disappeared under a structurally complex habitat, resulting in these fish having no performance advantage over fish with a lower SMR (Chapter 3). These high SMR individuals performed poorly in the presence of low densities of a heterospecific competitor, being subject to a disproportionate proportion of the aggression from a more dominant species (brown trout, Chapter 4). At higher densities of trout, intraspecific interactions appeared much more important for both species, resulting in the salmon with the highest SMR exhibiting the fastest growth. These three chapters demonstrate that environmental conditions, both abiotic and biotic, have great consequences for the success of different energetic strategies. The consequences of metabolic strategy on physiology proved just as interesting. High SMR individuals expended more energy when digesting a given size of meal but reduced the duration of this specific dynamic action (SDA, the rise in metabolism associated with processing and digesting a meal) response (Chapter 5). This suggested that their digestion was more rapid than that of low SMR fish, but this did not lead to a higher rate of food consumption (Chapter 5) nor did they sacrifice absorption efficiency (Chapter 6). This thesis demonstrates that the performance of fish with alternative energetic strategies is dependent on the prevailing environmental conditions, which helps explain the persistence of variation in SMR within populations.

Freshwater planarian Schmidtea mediterranea is an emerging model for studying in vivo gene functions and regulation in native cell niches. The obligate asexual strain of this species reproduces by fission, in which succession of soma occurs without passing through the germline. To achieve this somatic immortality the somatic stem cells need to overcome the end replication problem. Therefore it can be hypothesised that somatic telomere maintenance in asexual S. mediterranea must possess a germ-like property, with which age-related erosions can be adequately repaired. In this PhD project, the telomere repeat unit in S. mediterranea was confirmed to be the vertebrate-like TTAGGG. Attrition of whole body telomere length was found in ageing sexual worms and also in asexual worms which had not gone through recent fission events. Opposite telomere length dynamics were observed in regenerated samples of the two strains, with erosion in the sexuals and reset in the asexuals. The telomere maintenance was found to increase during regeneration in both strains, with a higher level of increase in asexual worms. A homolog of the telomerase reverse transcriptase subunit, Smed_Tert, was identified and characterised in this organism. High level of Smed_Tert expression was seen in germ cells in mature sexual worms and adult stem cells in asexual worms. Knockdown of Smed_Tert expression by RNA interference caused progressive telomere erosion, however effects on cell proliferation and viability have not been observed in knockdown samples. Four alternate splice isoforms of Smed_Tert were identified. The enhanced telomerase activity during regeneration correlates with a proportional increase in the full-length isoform and a decrease in isoforms with a truncated TRBD domain, suggesting a dominant negative regulation of telomerase by alternative splicing. Significant increase in the expression of the full-length isoform was seen in regenerating asexual samples but not in sexual strains, which correlates with their telomere length dynamics. It is hoped that the comparative studies between the sexual and asexual strains can improve our understanding of how soma can evolve to become an effective inheritable unit.

Zebrafish, in contrast to mammals, are capable of functional regeneration after complete transection of the spinal cord. In this system I asked: (1) Which spinal cell types regenerate in the lesioned spinal cord? (2) To what extent do the dopaminergic and 5-HT systems regenerate and (3) do dopaminergic axons from the brain influence cellular regeneration in the spinal cord? (1) Lost motor neurons are replaced by newly born motor neurons that mature and are integrated into the spinal circuitry after a spinal lesion in adult zebrafish. Using immunohistochemical and transgenic markers in combination with BrdU labeling, we showed that also 5-HT, parvalbuminergic, Pax2+ and Vsx1+ cells are newly born after lesion. Thus, my work shows that diverse cell types are newly generated in the lesioned spinal cord of adult zebrafish. (2) After spinal cord lesion, zebrafish completely recover locomotion within six weeks. Previous work suggested that axonal regeneration is crucial for functional recovery. Here I analyzed changes in the density of 5-HT and dopaminergic axon terminals in the lesioned spinal cord during recovery. Rostral to the lesion site, I observed die-back and sprouting of dopaminergic axons within two weeks post-lesion. Caudal of the lesion, axons are lost indicating Wallerian degeneration. At six weeks post-lesion I tested functional recovery with a behavioral swim test. In recovered fish, a third of the axonal density was restored just caudal of the lesion site, but not at far caudal levels. In contrast, in fish that had non-recovered, only few axons had bridged the lesion site. Thus dopaminergic axon regrowth correlates with functional recovery. Re-transection of the spinal cord in recovered animals abolished re-gained swimming capability, suggesting that behavioral recovery critically depends on axons that crossed the spinal lesion site and not on an intraspinal circuit. 5-HT axon terminals are of both intra- and supraspinal origin. The overall time course of changes in axon terminal density during recovery is similar to that of dopaminergic axon terminals and also correlates with functional recovery. Overall, the organization of the spinal dopaminergic and 5-HT systems, consisting of neuronal somata in the spinal cord and descending axons, differs significantly from their unlesioned organization. I observe sprouting rostral to the lesion site and limited innervation of the caudal spinal cord, as axons do not regrow into the far distal spinal cord. (3) We further hypothesized that signals released by descending axons are involved in cellular regeneration around the lesion site. Dopaminergic axons of supraspinal origin sprout rostral, but are almost completely absent caudal to the lesion site at two weeks post-lesion. Moreover, we observe that expression of the dopamine receptor drd4a is only increased rostral to the lesion site in the ventricular zone of progenitor cells, including olig2 expressing motor neuron progenitor cells. Correlated with these rostro-caudal differences, numbers of regenerating motor neurons are almost two-fold higher rostral than caudal of the lesion site. To functionally test whether dopamine is involved in motor neuron regeneration, we ablated tyrosine hydroxylase positive, mostly dopaminergic axons by injecting the toxin 6-hydroxydopamine. This treatment significantly reduced motor neuron numbers only rostral to the lesion site. As a gain-of-function experiment, we injected the dopamine agonist NPA after spinal lesion, which increased motor neuron numbers only rostral to the lesion site at two weeks post-lesion. These results suggest that dopamine released by descending axons, augments the generation of motor neurons in the lesioned spinal cord of adult zebrafish. In summary, during spinal cord regeneration I observe generation of various cell types and plastic changes of descending axonal projections. Dopamine released by descending axons is able to increase motor neuron regeneration, showing for the first time that signals from descending axons influence cellular regeneration in the spinal cord.

Our initial studies reported on the cloning and characterization of FoxP3 (T regulatory cell specific) for the first time in any teleost species along with T-bet and STAT6 in zebrafish, which are specific to Th1 and Th2 cells respectively and looked at their expression in the zebrafish.  Our next studies focused on CD4 expression by T cells and addresses the question whether teleost Th cells are functionally similar to their mammalian counterpart.  A CD4 specific antibody was developed and using molecular techniques have been able to characterise for the first time CD4+ cells within the zebrafish. In addition, to the CD4L gene, three other novel CD4 related genes have also been discovered which need to be investigated, which could mean in fish different T helper cell populations exist expressing CD4 related molecules.  Another FoxP3 gene was discovered, a factor important in controlling the differentiation and function of T regulatory (Treg) cells, and so we shifted our investigations to studying these FoxP3 molecules within the zebrafish model.  These two orthologs of mammalian FoxP3 were only found in zebrafish and it appears that higher vertebrates including modern fish only have one copy of the FoxP3 gene.  Through the development of an antibody we were also able to look at the expression of the zebrafish genes within cells and determine where they were being expressed.  Lastly, having all the molecular tools that were developed during the course of this thesis, allowed us to use <i>in vivo </i>zebrafish models of <i>Mycobacterium marinum </i>and <i>Salmonella typhimurium </i>infection, and attempt to determine the types of adaptive immune responses that were being developed. Understanding the regulatory mechanisms behind the teleost adaptive immune response will help to establish the zebrafish model for vertebrate immune function.

This thesis focuses on the link between condition, defined as the magnitude of fat reserves, and maturation in two sub-populations of Atlantic herring (Clupea harengus L.). Histological, fatty acid (FA), univariate and multivariate analyses were used in a multi-scale approach to elucidate the relationship between body fat and maturity. Furthermore, the accuracy of commonly used proxies of condition and maturity was tested. No evidence was found to support the hypothesis that a threshold of fat must be exceeded for first maturation to occur; however, a size threshold was observed. During maturation, herring may be capable of both selectively incorporating certain FA into the ovary and also of synthesising FA within the ovary itself. Mesenteric fat was highly dynamic during maturation and likely plays a role in gonad development. Commonly used morphometric condition indices were not related to mesenteric fat and the relationship between morphometric indices and other more direct indices was dependent on maturity stage. Macroscopic maturity staging was unreliable and errors led to an under-estimation of the herring spawning stock biomass of up to 26%. A gonadosomatic index was validated which was able to discern between immature, mature and recovering fish more accurately than macroscopic staging. Few differences were found between North Sea autumn-spawning (NSAS) and Norwegian spring-spawning (NSS) herring in this study. The FA profiles of both sub-populations were similar over the maturity cycle and the effects of length and maturity stage on mesenteric fat were analogous for both populations. A photoperiod cue of first maturation was found for Atlantic herring. However, this cue differed between the subpopulations, with NSAS herring maturation being triggered by the spring equinox and NSS herring maturation being triggered later. A multi-scale approach was successfully employed to demonstrate that there is an intrinsic link between fat reserves and maturity in herring.

The principal aim of this thesis was to investigate the effects of altering the energy density of the diet (kJ/g) on post-restriction weight regain in domestic cats and laboratory mice. Secondly, evidence for body weight regulation was examined in mice. In cats, consumption of the same number of calories of a low energy dense diet (with added water) induced significantly less weight rebound than a more energy dense diet (with no added water). This was accounted for by differences in physical activity, as cats on the low energy dense diet were significantly more active. It was assumed the less energy dense diet held cats in a perceived state of energy restriction and they were actively searching for more food. In mice, cellulose was used to alter the energy density of the diet. Post-restriction body weight rebound was also observed on an energy dense diet (no cellulose). However, body weight not did reach pre-restriction levels when consuming a low energy dense diet (with added cellulose) and digestibility was significantly reduced. It was likely that the fibre-rich induced a gut processing limit on the bulk of digestible food that could be consumed. Overall, the energy density of the diet was shown to modulate post-restriction body weight rebound, but results varied with species and the energy dilutant used. The implications for human weight management have yet to be elucidated. The second principle finding was that post-restriction hyperphagia was induced to replenish food deficiencies incurred during caloric restriction, rather than to replenish body mass loss in mice. This provided preliminary evidence for a ‘calorie-counting’ body weight regulatory system in mammals.

The molecular mechanisms that control growth appear to be conserved across the animal kingdom, with nitric oxide regulation of cell proliferation and growth being found to be very significant. Indeed, in Drosophila larval development and mammalian systems NO has been shown to be particularly important in these processes, and previous work in our laboratory has identified the Drosophila forkhead transcription factor dFOXO as a critical target through which NO signalling exerts its regulatory effects on growth, although little is currently known concerning the precise mechanisms involved. Accordingly, in this thesis, we investigate the processes through which NO may modulate growth and demonstrate that targeted expression of a constitutively active NO Synthase to whole larval salivary glands or clones of cells within the glands, results in reduced endoreplication and growth as measured by nuclear size. Targeted over expression of dFOXO itself is shown to result in similar phenotypes, and subsequent molecular analysis of potential signalling targets required for this inhibition of growth reveals that dFOXO, Thor and Myc expression are regulated in vivo by NO. To elucidate if NO acts directly on dFOXO, the genetic interaction of components of the insulin signalling pathway is analysed, exploiting RNA interference to assay what components are necessary for the NO signal to be effectively transduced, and it is demonstrated that NO control of growth is not through sGC, one of the most significant known targets for NOmediated regulation in other organisms. We subsequently investigated the roles of Thor, a Drosophila 4E-binding protein, and the kinase, Lk6, homologues of which are known to be important in growth regulation in other organisms, and thus potential effectors of NO and dFOXO. However our data demonstrated that neither Thor nor Lk6 are required for the inhibition of growth by NO. Interestingly a potential anti-oncogenic effect of NO signalling was also revealed following analysis of interactions between NO and Ras or Myc induced growth in which NO was able reduce the overgrowth produce by both these oncogenes. Overall this research confirms dFOXO as an essential target for NO induced inhibition of growth. The work also eliminates two dFOXO transcription targets, Thor and Lk6, as necessary for NO to regulate growth.

Mechanisms associated with growth regulation have been shown to be highly conserved in mammals and Drosophila, especially when examining the insulin signalling pathway. Previous studies suggest that nitric oxide (NO) signalling can inhibit growth and cell proliferation in a Drosophila forkhead box O (dFOXO)- dependent manner. dFOXO is a component of the insulin signalling pathway and has also been demonstrated to inhibit growth via its interactions with components in this pathway; however, the mechanism by which dFOXO and NO interact is unclear. Since inhibition of growth by NO is dependent on dFOXO, this thesis examines the effect of co-expressing nitric oxide synthase II (NOS2) with three dFOXO alleles (dFOXO25, dFOXO21 and dFOXOBG01018) in Drosophila salivary glands taken from third instar larvae. It concludes that the dFOXO25 null allele appeared to be the strongest deletion of dFOXO given that salivary gland nuclei appear most similar in size to the wild type. This indicates that NO-induced growth inhibition only occurred to a very small degree as a result of a powerful loss-of-function of dFOXO exhibited in dFOXO25 homozygotes. This thesis also investigates the effects of NO on salivary glands taken from the same developmental stage when co-expressed with overexpressed oncogenes, dMyc and RasV12. Nuclei measurements were larger than the NOS2-only expressing line and smaller than the lines expressing only each of the oncogenes. However, TEM analysis revealed that co-expression might induce endoplasmic reticulum (ER) stress in the glands. Research shows that NO and these oncogenes can provide the reactants necessary to generate peroxynitrite, which is associated with the generation of ER stress. When examining the effects of these growth regulators on mitochondria and Golgi, this thesis reports that dFOXO, NOS2 and dMyc can increase mitochondrial biogenesis. Golgi was unaffected by expression of the growth regulators.