Cephalopods, including cuttlefish, have a reputation for ‘living fast and dying young’. The aim of the work described in this thesis was to perform a metabolic assessment of the European cuttlefish (<i>Sepia officinalis), </i>so as to determine if cuttlefish live up to the cephalopod reputation as a high energy consumer. To begin the assessment in Chapter 2, the standard metabolic rate (SMR) of <i>Sepia officinalis</i> was investigated over a water temperature range of 11-21°C. The SMR of cuttlefish was compared to 17 other marine ectotherms at a similar body mass and in corresponding water temperatures. Cuttlefish had an SMR 1.7 times higher than the teleost fish species examined. In Chapter 3, cuttlefish cellular energy budgets were calculated to identify cellular drivers of whole-animal SMR. The purpose of Chapter 4 was to discover the primary catabolic fuel used by adult <i>Sepia officinalis.</i> Respiratory quotients (RQ) and O:N ratios were calculated at 16 and 21°C. Carbohydrates were the primary metabolic substrate in both fasting and fed states. Temperature significantly affected ammonium and phosphate excretion, and the excretion of each substance was tightly correlated with the other. In Chapter 5, experiments were taken outside the laboratory and into a tidal pond. Large (1800 g) cuttlefish were tagged with acoustic jet pressure transmitters and released into a marais in L’Houmeau, France. Cuttlefish were found to be nocturnal, with night time activity significantly influenced by moonlight levels. The respiratory component ‘R’ of the cuttlefish energy budget is calculated in the thesis conclusions of Chapter 6. After deriving estimates of the daily energy expended in activity for <i>Sepia officinalis</i> in the marais, the European cuttlefish may indeed be ‘living faster’ than fish.

No description available.

No description available.

The <i>Drosophila </i>Toll transmembrane receptor functions in both embryonic development and the innate immune response against Gram-positive bacteria and fungi. Proteolytic cleavage of Spätzle, its pro-protein ligand, results in an active cysteine knot-containing dimer (C-106) that binds to and activates Toll. This pathway is considered to be the evolutionary precursor of the Toll-like Receptor (TLR) family, which serve as sentinels of the mammalian innate immune system. This study describes how the pro-domain masks the putative receptor-binding site, located at the ‘head-like’ region of C-106. Spectroscopic and binding analysis reveals that the sole Trp²⁹ residue, located within the Trp-loop in the same region, is not only increasingly exposed as the pro-domain is reoriented prior to receptor interaction, but is also involved in pro-domain binding. mutational analysis of Spätzle indicates that a combination of electrostatic and hydrophobic forces govern the structure of C-106 as well as its interaction with the pro-domain and Toll. Furthermore, low resolution structural analysis of the Toll ectodomain, combined with homology modelling and biochemical data, shows that C-106 binds to the N-terminal tip of Toll, inducing the formation of a 2:2 complex with two sites of interaction between the ectodomain chains; one near the N-terminus and the other at the juxtamembrane position. This data therefore suggests that Toll activation is an allosterically controlled mechanism induced by the end-on binding of C-106. Supplementary analysis of TLR2 activation by agonists of non-microbial origin, specifically the lectins KM+ and Galectin-3, provided a limited insight into the non-specific activation of Toll-like Receptors, thereby implying a more flexible model of receptor activation may exist.

When neurons exit the cell cycle after division at the apical surface of the proliferating neuroepithelium, they sever their attachments to the apical surface while migrating to their correct laminar positions. Although apical retraction is an essential step in neuronal differentiation, its underlying molecular mechanisms remain unknown. The accessibility of the retina, together with the ease of genetic manipulation and <i>in vivo </i>imaging capabilities of the vertebrate zebrafish, make zebrafish retinal ganglion cells (RGCs) an ideal model system to study the molecular basis of apical retraction in the CNS. I was able to show that the axonal guidance molecule, Slit1b, is necessary for apical retraction. <i>Slit1b </i>mRNA is expressed during RGC differentiation. Time-lapse imaging in <i>slit1b </i>morphants revealed a delayed retraction of apical processes in RGCs. Other developmental events, such as axon extension, progresses normally, thus ruling out a general developmental delay. Transcripts of the Slit receptors, <i>robo2 </i>and <i>robo3</i>, were detected in the retina during RGC differentiation. Morpholino knockdown experiments revealed that <i>robo3</i>, but not <i>robo2</i>, was required for normal apical retraction of RGCs. In addition, expression of dominant-negative N-cadherin caused RGCs to lose apical attachments prematurely. Finally, dominant-negative N-cadherin expression in <i>slit1b </i>morphants resulted in early retraction even in <i>slit1b </i>morphants, suggesting that N-cadherin functions downstream of Slit/Robo signalling during apical retraction. This study helped shed light on molecular events underlying apical retraction in RGCs. It now needs to be shown if these mechanisms are more general during neuronal differentiation throughout the CNS.

I have investigated the role of Epidermal Growth Factor Receptor (EGFR) signalling in the control of ommatidial orientation. Both over- and under-activation of the pathway lead to significant defects in ommatidial orientation, while leaving the polarity of ommatidial unaffected. This previously unrecognised function of EGFR signalling further adds to the multiple functions of the pathway in <i>Drosophila</i> eye development. I have shown that the initial rotational event proceeds normally in EGFR pathway mutants, but that orientation becomes disrupted later in development, leading to the severe adult phenotypes seen. It is unclear which of the EGFR ligands is responsible for mediating its function in rotation: both Keren and Spitz may be important. At present, no mutant exist in <i>keren</i>. I have conducted a P element excision screen in an attempt to generate a <i>keren</i> mutant; characterisation of potential mutants is still ongoing. Hopefully, this will allow resolution of the question as to which ligand activates EGFR signalling to control orientation, and will also allow analysis of other potential functions of Keren. Very little is known about the cell biological processes underlying ommatidial rotation. As a first step towards gaining some insight into the mechanism by which ommatidial orientation is achieved, I have investigated a possible role for E-cadherin in this process. E-cadherin interacts genetically with EGFR signalling in rotation; however, it remains unclear whether cadherin-based adhesion has a primary function in the control of ommatidial orientation. I propose a model for the role of EGFR signalling in ommatidial orientation, in which the pathway acts in the 3^rd instar disc, to lock ommatidial in place once they have reached their appropriate positions; in the absence of such a lock, ommatidial orientation can become disrupted during the remodelling of the larval disc into an adult eye.

I examine whether it is possible for the temporal resolution and reliability of a photoreceptor to vary across the eye. This possibility has not been addressed before in any animal and yet it is important for our understanding of how visual systems may be designed to register patterns of optic flow or to track moving targets. In the male blowfly, <I>Calliphora vicina, </I>I show that both spatial and temporal acuity are higher at the front of the eye and fall off with retinal eccentricity. The particular pattern of tuning observed emphasises the importance of tracking to the male fly, a behaviour commonly observed in flies prior to mating. To investigate this possibility further, in Chapter 3 I compare the responses of male and female photoreceptors to simulated target stimuli. For this purpose I use the housefly, <I>Musca domestica</I>, a species whose anatomical and behavioural sex-differences are well documented. The male photoreceptor responds much more powerfully to small moving targets than the female and response amplitude greatly exceeds that predicted from conventional models of photoreceptors dynamics. In particular, the male photoreceptor boosts the signals generated by targets moving within the behavioural regime of distances and speeds. These results allow the limits of male pursuit vision to be determined and demonstrate the impact of behaviour on retinal function. Adaptation of photoreceptor sensitivity and temporal resolution to ambient illumination is a well-known phenomenon. This process is usually considered to be complete within seconds. However, I show in <I>M. domestica</I> that significant improvements in temporal resolution and reliability can occur over a much longer period. These improvements are derived from a more consistent registration of the timing of photon absorption events and appear to be associated with a reduction in rhabdomeral surface area.

No description available.

No description available.

No description available.