In this investigation energy demands from offspring were varied for the MF1 mouse after litter sizes were manipulated and set to 5 pups/litter or 16 pups/litter by cross-fostering to examine the effects on immediate lactation and subsequent reproductive performance. Pups reared in manipulated large litters grew slower and were weaned lighter (half the size of pups reared in small litters) than pups reared in manipulated small litters.  Mortality of pups was high in the large litters but pups did not die in small litters. The results of a subsequent reproduction that followed the first lactation suggested an apparent cost for the female group that previously reared pups in large litters.  This is because in the subsequent reproduction, the group raised litters that were significantly (P<0.05) 12.7g less in mass than litters raised by females that previously reared pups in small litters.  This apparent cost, however, did not appear to have severe negative impact on the general reproductive performance because data showed comparable litter sizes and masses at birth, growth rates of pups, and the number and masses of pups weaned between this group and females that previously reared pups in small litters. At reproductive age, female offspring previously weaned from large litters weighted significantly (P<0.05) 5.8g less than female offspring previously weaned from small litters.  During their first breeding attempt or episode, female offspring previously weaned from large litters did not catch-up in body mass with those previously weaned from small litters.  In spite of this, the two groups ingested similar quantities of food prior to mating and during lactation.  The groups did not differ significantly (P>0.05) in efficiencies of energy utilization, milk energy output and daily energy expenditure.  Consequently, they showed comparable breeding success. Different age groups of male and female offspring previously weaned from manipulated small (5 pups/litter) or large litters (16 pups/litter) and unmanipulated normal litters (10 pups/litter) varied significantly in body mass and did not show compensatory growth at the time they were examined for susceptibility to obesity and glucose intolerance.  A batch of six months old male offspring previously weaned from litters whose mothers were fed a particular macronutrient diet during lactation did not differ significantly in body mass when they were also examined for susceptibility to obesity.  All groups and sexes showed a tendency to develop obesity when they were challenged with a diet that contained up to 45% fat by calories.  Development of obesity did not appear to depend on  a particular litter size in which the mice were previously reared and weaned or whether the mice were previously suckled by mothers that were fed a particular macronutrient diet.  However, the development of obesity exhibited sexual dimorphism with the male offspring showing a higher tendency to develop obesity than female offspring.

The calorie restriction (CR) and insulin/IGF-I-like signalling (IIS) are two pathways regulating the lifespan of C. elegans. Recent studies showed that glucose restriction extends the lifespan of C. elegans while excessive glucose shortens the lifespan of the worms. The first step of the glucose metabolism is the transport of glucose across the plasma membrane by the glucose transporters. The work described in this thesis aims to identify glucose transporters in C. elegans and to provide a primary investigation of the in vitro and in vivo function of the identified glucose transporter. Nine putative transporters have been cloned and expressed. Out of the nice cloned putative transporters in the C. elegans genome, H17B01.1 (H17) only is identified as a fully functional glucose transporter using an oocyte expression system in which glucose transport activity is directly measured. The two transcripts of H17 are both capable of transporting glucose with high affinity, as well as transporting trehalose. Heterologous expression of H17 in mammalian CHO-T cells suggests that the protein is localised both on the plasma membrane and in the cytosol. In vitro studies of H17 show that the protein does not respond to insulin stimulation when expressed in mammalian CHO-T cell and rat primary adipocyte systems. In vivo functional studies using H17 RNAi indicate that the worm’s lifespan is not affected by the H17 knockdown. However, glucose metabolism of C. elegans (as measured by glucose oxidation to CO2 and incorporation into fat reserves) is influenced by the decreased expression of H17, especially in the daf-2 mutant strain, e1370. However, the increase of glucose metabolism caused by H17 knockdown observed in daf-2 mutant is inhibited in the age-1 and akt-1 mutant strains. The findings reported in this thesis suggest that the H17 glucose transporter may play an important role glucose metabolism in C. elegans and that this transport and metabolism is influenced by insulin receptor activity and serine kinase cascades.

Nitric Oxide (NO) is known to alter cell proliferation and growth. This project investigated the molecular targets of NO, using the salivary glands of Drosophila melanogaster third instar larvae. A screen of 75 transgenic Drosophila lines expressing Yellow Fluorescent Protein tagged proteins, for those that showed a rapid alteration in sub-cellular localization after organ culture with an NO donor was undertaken. This screen revealed Mi-2 as a target of NO action. This was confirmed in vivo as localization of Mi-2-YFP was altered in whole salivary glands expressing Nitric Oxide Synthase (NOS2). The localization of the endogenous Mi-2 protein was similarly altered in NOS2 expressing single cells. A quantitative analysis using the image analysis software “Velocity” demonstrated an increase in the nuclear concentration of Mi-2 protein in these cells. Targeted expression of RNAi-Mi-2 as well as analysis of Mi-2 transheterozygous mutant larvae revealed that NO can impart its reduced growth phenotype independently of Mi-2. Dref can associate with Mi-2 and can control cell proliferation and growth. Localisation of Dref did not show any noticeable change in single cells expressing NOS. However when these cells were double labelled both with anti-Dref and anti-Mi-2 antibodies, the anti-Dref staining was altered. This indicates that NO can alter the availability of the Dref antigen by reorganising the Mi-2/Dref complex. Mi-2 is a component of the NuRD complex. The effect of NO in altering the Mi-2/Dref complex was exploited to investigate the effect of simj (a regulatory component of NuRD), on either of the proteins after potential disassociation of the complex. The data demonstrated an independent effect of simj on the localization of each protein. NO and FOXO regulate the expression of a common set of genes and FOXO is required for the anti growth properties of NO. Thus the effect of FOXO expression on Mi-2 and Dref protein distribution was determined. Although no consistent alteration of Mi-2 localization was observed, a quantitative analysis showed a large increase of Dref protein concentration in FOXO expressing cells. Double antibody staining of these cells with both anti-Dref and anti-Mi-2 antibodies showed a novel nuclear localization of the proteins imparted by FOXO. Thus this study has identified the regulation of the Mi-2/Dref protein complex as a possible growth control mechanism by NO and FOXO.

Endogenous nitric oxide (NO) produced by the enzyme NO synthase (NOS) has an important role in a variety of physiological processes. However, NO becomes noxious to cells if produced in excess. Therefore, the production of NO is tightly regulated. A particularly exciting and novel aspect of the regulation of NO signalling is the possibility that the expression of NOS genes is controlled by unconventional mechanisms that depend on the presence of natural antisense transcripts (NATs). In this thesis I investigate the important properties of two distinct NOS-related NATs: Lym-antiNOS2 and Mm-antiNOS1. I show that Lym-antiNOS2 RNA is widely expressed in the CNS of the pond snail Lymnaea stagnalis. Furthermore, I demonstrate that the expression of Lym-antiNOS2 is differentially regulated by training leading to long-term memory formation. Moreover, my results indicate that Lym-antiNOS2 RNA is subjected to peripheral trafficking in neurons. As for Mm-antiNOS1, I find that its expression is restricted to embryonic brain tissue and is almost undetectable in the adult brain of Mus musculus.

1. The free radical gas nitric oxide (NO) is now recognised as a ubiquitous and versatile signalling molecule and the investigation of its biological roles has involved a wide range of scientific disciplines in many different species. Yet despite this, its potential roles in the development of rhythmic motor activities in vertebrates have been largely ignored. 2. Physiological experiments recording extracellular ventral root output suggest that NO is playing an inhibitory role in the swimming system of Xenopus laevis larvae, shortening the duration of swim episodes and slowing swim frequency. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry labelled three populations of neurons in the brainstem, which putatively co-localise NO with the aminergic neuromodulators serotonin (5-HT) and noradranaline (NA), and the fast descending inhibitory neurotransmitter, y-aminobutyric acid (GABA). This suggests that the inhibitory role is supraspinal in origin. 3. Intracellular recordings from neurons presumed to be spinal motor neurons provide further evidence for the inhibitory influence of NO. My experiments suggest that NO potentiates both glycinergic and y-aminobutyric acid (GABA)-ergic inhibition onto spinal motor neurons. The facilitation of the release of these inhibitory transmitters is consistent with the observed effects on swim frequency and swim episode duration, respectively. Additionally, NO appears to affect membrane properties, causing a pronounced membrane potential depolarisation and a decrease in membrane conductance. This suggests that NO shuts off a resting membrane conductance. 4. NADPH-diaphorase histochemistry was subsequently applied to determine the four dimensional expression of putative nitrergic neurons in the central nervous system and related structures. The developmental sequence of staining identifies groups and subgroups of interconnected intemeurons, and provides further clues to their identity. NADPH-diaphorase labelling was also located in the eyes, skin and blood vessels, further confirming the validity of this staining technique for identirying nitric oxide synthase. 5. In the related anuran species, Rana temporaria nitric oxide donor drugs appear to have no affect on swimming, but instead reliably initiates a non-rhythmic "lashing" motor pattern similar to that elicited by dimming of the illumination. Interestingly the NADPH-diaphorase technique labelled three clusters of apparently homologous interneurons in the brainstem and additionally the inner layer of the skin was intensely stained, implicating a species-specific role for NO released from brainstem neurons.

This thesis describes a study into two post capture conditions, idiopathic muscle necrosis (IMN) and a post capture opportunistic bacterial infection, affecting the Norway lobster, Nephrops norvegicus, and a comprehensive analysis of the stressors involved in the capture and post capture periods of the fishery process. A multivariate approach was used to study progression in the post capture condition of trawl-caught N. norvegicus for the live transport market. Prolonged periods of aerial exposure resulted in large disruptions to the carbohydrate profile, increases in haemolymph L-lactate and crustacean hyperglycaemic hormone concentrations, and corresponding fluctuations in haemolymph pH. These disruptions increased with the increasing temperature of aerial exposure, which impacted the immunocompetence of the lobsters: circulating haemocytes and phenoloxidase levels were significantly reduced and the degree of bacteraemia increased. The health status of N. norvegicus captured during trials in spring and autumn by means of short trawl (1h duration), long trawl (5h duration) and creeling was assessed using a range of physiological (Haemolymph L-lactate, crustacean hyperglycaemic hormone (CHH), abdominal muscle glycogen concentrations), immunological (total haemocyte counts (THC)) and physical (mortality, damage indices) parameters. Increased duration of trawl did not significantly alter physiological parameters, while time of landing did have a significant effect on L-lactate, where animals landed in the morning exhibited higher concentrations. Seasonal variations in abdominal muscle glycogen concentrations were also apparent. Irrespective of season, individuals captured by short trawls in the morning suffered lowest mortalities. Damage assessment data revealed that a greater proportion of individuals were categorised as heavily damaged following longer trawls conducted in spring. The carbohydrate profile and immunocompetence of N. norvegicus was significantly affected following trawl capture and subsequent handling and aerial exposure post capture. The recovery period was investigated through a range of parameters (L-lactate, glycogen, glucose, THC) and the data suggested that animals should be submerged and unstressed for at least 24h following capture and handling to avoid further alterations to the carbohydrate profile and reduce the window of opportunity for invading bacteria to cause meat spoilage. The influence of air temperature on the condition of N. norvegicus caught for the live export market was assessed by correlating meteorological data with the percentage of catch accepted for live transport. Results illustrated a large degree of variability in the conditions of animals on arrival at the processing plant. Air temperature was the only meteorological factor that had an impact on the morbidity and mortality of the catch. In one instance in particular, mean air temperature on the day of capture had a significant negative impact on the health of the catch.

The bloodstream form of Trypanosoma brucei differentiates into the stumpy form in the mammalian bloodstream, completing differentiation into the procyclic form on uptake by the tsetse fly. The underlying genetic events occurring during this differentiation process in pleomorphic cell lines were investigated through whole-genome microarray studies of key time points during differentiation from stumpy form cells to the procyclic form found in the insect midgut. The microarray was extensively validated and bioinformatic experiments conducted to detect motifs over represented in stumpy form or slender form cells. A positional-dependent motif was identified that was over represented in stumpy form cells, possibly representing a regulatory domain. The transcripts found to be enriched in stumpy form cells included a chloride channel, although RNAi directed against this gene showed no phenotype, suggesting the protein is redundant, as three other homologous proteins exist in the genome and showed similar mRNA profiles on the microarray. Stumpy form cells are G0 arrested and two proteins implicated in G0/G1 regulation in other organisms, Target of Rapamycin (Tor) and Cdh1, were investigated in T. brucei to determine whether these proteins were involved in differentiation. The result of depletion of either protein was rapid cell death in bloodstream form cells, although treatment with the drug rapamycin did not have any effect on the cells in contrast to other eukaryotes where this drug causes G1 arrest. A method for synchronisation of bloodstream form cells was also designed using a supravital dye and flow cytometry to allow investigation into cell cycle-dependent processes. This method was particularly suitable for harvesting populations enriched in G0/G1 stage cells, however differentiation of the isolated G0/G1 and G2/M populations did not show significantly different differentiation kinetics.

upd3 in the haemocytes also caused a reduction to signalling in the JNK pathway. The results here show that the haemocytes relay signal(s) to the fat body through the use of cytokines, a process surprisingly similar to the mammalian system.

For human and animal vision, the perception of local visual features can depend on the spatial arrangement of the surrounding visual stimuli. In the earliest stages of visual processing this phenomenon is called surround modulation, where the response of visually selective neurons is influenced by the response of neighboring neurons. Surround modulation has been implicated in numerous important perceptual phenomena, such as contour integration and figure-ground segregation. In cats, one of the major potential neural substrates for surround modulation are lateral connections between cortical neurons in layer 2/3, which typically contains ”complex” cells that appear to combine responses from ”simple” cells in layer 4C. Interestingly, these lateral connections have also been implicated in the development of functional maps in primary visual cortex, such as smooth, well-organized maps for the preference of oriented lines. Together, this evidence suggests a common underlying substrate the lateral interactions in layer 2/3—as the driving force behind development of orientation maps for both simple and complex cells, and at the same time expression of surround modulation in adult animals. However, previously these phenomena have been studied largely in isolation, and we are not aware of a computational model that can account for all of them simultaneously and show how they are related. In this thesis we resolve this problem by building a single, unified computational model that can explain the development of orientation maps, the development of simple and complex cells, and surround modulation. First we build a simple, single-layer model of orientation map development based on ALISSOM, which has more realistic single cell properties (such as contrast gain control and contrast invariant orientation tuning) than its predecessor. Then we extend this model by adding layer 2/3, and show how the model can explain development of orientation maps of both simple and complex cells. As the last step towards a developmental model of surround modulation, we replace Mexican-hat-like lateral connectivity in layer 2/3 of the model with a more realistic configuration based on long-range excitation and short-range inhibitory cells, extending a simpler model by Judith Law. The resulting unified model of V1 explains how orientation maps of simple and complex cells can develop, while individual neurons in the developed model express realistic orientation tuning and various surround modulation properties. In doing so, we not only offer a consistent explanation behind all these phenomena, but also create a very rich model of V1 in which the interactions between various V1 properties can be studied. The model allows us to formulate several novel predictions that relate the variation of single cell properties to their location in the orientation preference maps in V1, and we show how these predictions can be tested experimentally. Overall, this model represents a synthesis of a wide body of experimental evidence, forming a compact hypothesis for much of the development and behavior of neurons in the visual cortex.

Poly is a novel, essential protein in Drosophila melanogaster, loss of function of which results in late larval lethality. Importantly, Poly is evolutionarily conserved with a human homologue. poly mutation was isolated in a P-element mutagenesis screen that aimed to generate a larger collection of single P-element induced mutants. Mutant poly larvae are characterized by extreme larval longevity without pupation, formation of melanotic masses, smaller imaginal discs and brains, and abnormal nuclear morphology in neuroblasts. During the course of my project, I attempted to identify cellular processes and pathways that Poly might be involved in. Interestingly, my data suggest that Poly is a novel interactor and regulator of Insulin receptor/target of rapamycin (InR/TOR) signalling in Drosophila. Linking environmental cues to cell growth and metabolism is an essential process that multicellular organisms need to accomplish successfully for normal development. InR/TOR signalling is a highly conserved pathway that mediates the link between the environment and cellular processes such as growth, metabolism and ageing. My analysis in Drosophila suggests that Poly interacts physically with the InR and mutation of Poly leads to an overall down-regulation of InR/TOR signalling in Drosophila as revealed by decreases in the phosphorylation levels of Akt, S6K and 4E-BP - all downstream effectors of this pathway. In addition, loss of poly results in constitutive activation of autophagy in Drosophila fat body and a decrease in stored triglyceride levels. Furthermore, I show that localisation and levels of Poly protein are dependent on insulin action in both Drosophila and human cells. Together, these data suggest that Poly is a novel mediator of InR signalling that promotes an increase in cell growth and metabolism. Taking into consideration the observed poly mutant phenotype, I also investigated the potential involvement of Poly during cell cycle progression and the Drosophila innate immune response. While my analysis suggests that poly loss of function does not have a direct effect on cell cycle progression, alteration of Poly has consequences on various aspects of the Drosophila innate immune response. Therefore, I conclude that the Drosophila innate immune response is a cellular process in which Poly plays a crucial role.