A detailed understanding of the mechanisms by which plants sense and respond to major environmental stress factors will significantly contribute towards the prospects of developing crops capable of yielding well over a wider geographical range, including marginalised lands. One of the important stress response mechanisms in eukaryotes is mediated through phosphorylation of the eIF2α-subunit (serine 51/56) by specific kinases, namely double stranded RNA activated protein kinase (PKR), General Control Non-repressible 2 protein kinase (GCN2), Pancreatic eIF2α kinase (PERK) and Heme-regulated inhibitor protein kinase (HRI). This mechanism is a highly conserved phenomenon in eukaryotes and occurs in response to various stress conditions. Unlike in yeast and mammals, the mechanism is however not well established in higher plants, although its components such as eIF2α and GCN2 kinase have been identified in plants. The objective of the study reported herein was therefore to elucidate this mechanism in Arabidopsis, a model plant species. Initially the presence of yeast GCN2 kinase (ScGCN2), human PKR (HsPKR), human HRI (HsHRI) and human PERK (HsPERK) kinase homologues in Arabidopsis and Viridiplantae (green plants and algae) was evaluated through homology and phylogenetic analysis using TAIR10 and NCBI protein sequences, respectively. Arabidopsis lacked homologues of HsPKR, HsHRI and HsPERK however the presence of ScGCN2 homologue, herein referred to as AtGCN2 (Arabidopsis GCN2 kinase), was confirmed. Further evaluation of translation control mechanism through phosphorylation of AteIF2α (Arabidopsis eukaryotic initiation factor 2 α-sub-unit) was conducted using Atgcn2-1 null mutant plants (plants expressing a copy of truncated non-functional AtGCN2 kinase). Unlike WT Col-0, the Atgcn2-1 seedlings failed to induce phosphorylation of AteIF2α after exposure to amino acid starvation (150 µM glyphosate), NaCl (50 and 100 mM), heat (37oC) and cold (4oC) acclimation. On the other hand no strong phenotype of Atgcn2-1 was observed under optimal growth conditions and NaCl stress, except seedlings had relatively shorter roots compared with WT Col-0 seedlings. Failure of Atgcn2-1 seedlings to induce phosphorylation of AteIF2α Ser 56, after exposure to various stress confirmed that Arabidopsis possesses only one GCN2 kinase, as is in the case of yeast, and unlike mammalian systems. Further characterisation was conducted by exposing WT Col-0, Atgcn2-1, jar-1 and NahG seedling to biotic stress; Cauliflower Mosaic Virus (CaMV) and Pseudomonas syringae DC3000 (P. syringae) and positive control treatment using 150 µM glyphosate. The jar-1 and NahG seedlings are mutants defective in jasmonate and salicylic pathways, respectively. Inoculation with CaMV and P. syringae failed to induce phosphorylation of AteIF2α, unlike glyphosate. These results suggested that activation of the AtGCN2 kinase may be independent of jasmonate and salicylic pathways. Due to lack of a strong Atgcn2-1 phenotype, two mutants expressing AtGCN2 under the control of a 35S promoter, namely p35S:AtGCN2 and p35S:GFP:AtGCN2 were generated for further characterisation and localisation of AtGCN2 kinase, respectively. For characterization experiments the p35S:AtGCN2 seedlings were subjected to salinity stress, osmotic stress and temperature shock. In localisation experiments, GFP activities were assessed in non-stressed 7-day old p35S:GFP:AtGCN2 seedlings. During characterisation, higher germination rates were generally obtained with p35S:AtGCN2 and Atgcn2-1 compared with WT Col-0 seeds on ½ MS media containing NaCl, KCl and mannitol. On media infused with PEG6000 however p35S:AtGCN2 had the lowest germination rates. There were also no strong p35S:AtGCN2 phenotypes observed, except for increased root growth compared with WT Col-0 and Atgcn2-1 seedlings. In contrast, Atgcn2-1 seedlings subjected to PEG6000 osmotic stress had the highest increase in root growth compared with both WT Col-0 and p35S:AtGCN2 seedlings. On the other hand localisation of the GFP:AtGCN2 fusion protein was observed in the root and shoot tip tissues of p35S:GFP:AtGCN2 seedlings. The results obtained with Atgcn2-1 and p35S:AtGCN2 seedlings suggested that mutation of Atgcn2 produced root phenotypes. There were no significant differences in the survival of all the three genotypes when seedlings were subjected to heat shock stress. In cold shock experiments however Atgcn2-1 survival was significantly (p<0.05) lower than that of WT Col-0 and p35S:AtGCN2 seedlings, thus suggesting that null mutation of Atgcn2 increased susceptibility of seedlings to cold shock. The homologues of the yeast General Control Non-repressible 4 (ScGCN4) and human Activating Transcriptional Factor 4 (HsATF4), that are activated, when yeast and mammalian eIF2α is phosphorylated, respectively are yet to be identified in plants. To identify putative Arabidopsis ScGCN4 and HsATF4 homologues both in vitro and in silico approaches were explored. In vitro translation experiments using Wheat Germ Lysate (WG) mimicking plant translation under stress (WGeIF2α-P) and non-stress (WGeIF2α) conditions were conducted. To mimic stress conditions mPKR kinase was added into the translation reaction and significantly inhibited protein synthesis compared to control treatment. However, due to technical difficulties it was not possible to identify all translated transcripts under stress conditions (WGeIF2α-P) thereby identifying potential Arabidopsis ScGCN4 and HsATF4 homologues. This prompted the use of in silico tools to identify putative Arabidopsis homologues of ScGCN4 and HsATF4 using the FivePrime Viewer programme (Webb, 2008). A total of 99 TAIR10 transcripts with 5′ upstream Open Reading Frames (uORFs) were identified and only two transcripts, AT4G31590.1 and AT1G58120.1, were identified as putative homologues of ScGCN4 and non for HsATF4. The AT4G31590.1 and AT1G58120.1 transcripts encode for proteins involved in cellulose synthase/ gylcosyl transferase and methyl transferase activities, respectively. Although these genes are involved in key plant growth and developmental activities, there is need to assess translation control of their main open reading frame (mORF) by uORFs through phosphorylation of AteIF2α. Overall the data presented in this study suggest that stress response translation regulation mechanism mediated by phosphorylation of eIF2α is present in Arabidopsis. Plants are known, however, to carry out unique biological processes such as photosynthesis and cellulose biosynthesis that other eukaryotes lack. It would therefore not be surprising for them to have translation regulation mechanisms like other eukaryotes but with unique differences.

This project focuses on the characterisation of two separate aspects of Trypanosoma brucei cell biology; the degradative process of autophagy and a specific cysteine peptidase from the metacaspase family. Autophagy is a widely conserved intracellular mechanism for the degradation of long lived proteins and organelles, that requires the formation of an autophagosome (double membrane bound vesicle) around cargo destined for the lysosome. The molecular machinery involved in autophagy has been well characterised in yeast and bioinformatic screens have identified many of the core components in T. brucei. However, beyond bioinformatics there is limited experimental evidence to support the presence of functional autophagy in T. brucei. A key component of the autophagic pathway is ATG8, a ubiquitin-like protein that is incorporated into the autophagosome membrane. To investigate autophagy in T. brucei the three candidate ATG8 genes (ATG8.1 Tb927.7.5900, ATG8.2 Tb927.7.5910 and ATG8.3 Tb927.7.3320) were fused with yellow fluorescent protein (YFP) and expressed in bloodstream form and procyclic form T. brucei cultured parasites. Fluorescent microscopy was used to monitor the formation of YFP-labelled autophagosomes, which enabled the evaluation of the autophagic response towards a variety of different stimuli. We provide the first direct experimental evidence confirming a functional autophagy pathway in T. brucei and show that it is induced in response to nutrient starvation in the procyclic form and neuropeptide treatment in the bloodstream form and can be blocked by the classical autophagy inhibitor wortmannin. Characterisation of the T. brucei ATG8 family revealed that ATG8.1 and ATG8.2 appear to operate as ‘ATG8-like’ proteins, whereas ATG8.3 behaves atypically, possibly functioning as an ATG12 protein. Furthermore, targeted RNAi downregulation of the predicted T. brucei ATG3 (Tb927.2.1890) caused a reduction in cell growth. The vital role of ATG3 in the autophagy pathway suggested that the process was required for normal procyclic form growth. The second focus of the project was metacaspase 4 (Tb927.10.2440) which belongs to the metacaspases (MCAs), cysteine peptidases of the caspase family found in plants, fungi and protozoa, but absent from mammals. Of the five MCAs possessed by T. brucei, only MCA2, MCA3 and MCA5 contain the conserved histidine cysteine catalytic dyad. MCA1 and MCA4 are predicted to contain key substitutions within their active sites, raising interesting questions regarding potential peptidase activity and functions. The exact role of the T. brucei metacaspases remains largely unknown, with MCA2 , MCA3 and MCA5 appearing to function in association with RAB11 positive endosomes, although independently of the known recycling functions of these endosomes (Helms et al. 2006). To develop our understanding of the MCA family in T. brucei a study into the function of MCA4 was undertaken. An antibody was raised against MCA4 and western blotting of cell lysate revealed that MCA4 expression occurred only in bloodstream form T. brucei. Interestingly MCA4 localised to the flagellar membrane, appearing in a linear array of punctate structures. Dual acylation is known to mediate flagellar membrane association in T. brucei and was implicated in MCA4 targeting following bioinformatic predictions and subsequent experimental confirmation of MCA4 palmitoylation using an acyl-biotin exchange reaction. MCA4 contains a non-canonical active site residue (serine-219) in the position of the predicted conserved active site cysteine. Activity assays using purified recombinant protein revealed that full length MCA4 was unable to autoprocess and was inactive. However, MCA4 peptidase activity could be detected following proteolytic activation with MCA2. Interestingly, removal of the MCA4 active site serine by mutagenesis (MCA4S219G) did not abolish activity of the processed enzyme, revealing an alternative nucleophile was capable of contributing to activity. Furthermore, mutation of the active site serine to cysteine, produced a constitutively active peptidase capable of autolytic processing in a calcium dependent manner. Following these key findings the role of MCA4 in the T. brucei lifecycle was investigated by RNAi and genetic knockout. Rapid depletion of MCA4 by RNAi caused a block in cytokinesis followed by cell death, nevertheless the generation of MCA4 null mutant parasites (∆mca4) was possible. A role for MCA4 in mammalian infection was revealed by monitoring infection progression in mice. Deletion of MCA4 increased host survival and parasite virulence could be restored by the ectopic re-expression of MCA4 in ∆mca4 parasites.

Although sessile organisms, unable to run away from danger, plants are well adapted to the potential harmful effects of sunlight’s high energy photons within the UV-B wavelength range (280-315 nm). For instance they are able to, among other things; produce their own sunscreen to counter any damage to their proteins, lipids and DNA. Plants of course depend on light as a source of energy for photosynthesis but also use specific wavelengths within the electromagnetic spectrum in a number of ways to act as an informational signal, including UV-B wavelengths, which can induce photomorphogenic responses that allow adaptation and survival for plants in the ever-changing environmental conditions they inhabit. It is now well established in plants that there are more than two pathways operating in response to different wavelengths and fluence rates of UV-B. In response to high, potentially damaging UV-B levels plants utilize a non-specific pathway which overlaps with other stress pathways such as pathogen attack and wounding by, for example, herbivores. And in response to low non-damaging UV-B levels plants utilize the UV-B specific photomorphogenic pathways which bring about acclimation, preparing the plant for potential higher doses and actively promoting plant survival (Jenkins and Brown, 2007). A number of photoreceptors have been identified in plants which act throughout the electromagnetic spectrum, but only in the last year has one been discovered operating at UV-B wavelengths. In fact until then no UV-B- specific photoreceptor had been found in any organism and it was not known how plants perceive UV-B light to initiate photomorphogenic responses. Over the last decade evidence was mounting in favour of the most upstream component of the UV-B photomorphogenic pathway and the only UV-B specific component, UVR8 (UV-RESISTANCE LOCUS 8) as being a UV-B photoreceptor. Now it has been demonstrated in plants to be a bona fide UV-B photoreceptor and to perceive UV-B by a novel mechanism (Rizzini et al., 2011, Christie et al., 2012, Wu et al., 2012). It has been demonstrated upon UV-B irradiation that UVR8 can dissociate from a homodimer to a monomer in vivo and in vitro. And unlike other conventional photoreceptors, which use a chromophore to detect specific wavelengths of light, UVR8 uses tryptophan residues found within its protein structure to carry out photoperception. When UV-B is detected via specific tryptophan residues found within the dimeric UVR8 protein, the energy is captured and used to cause disruption and breakage of several salt bridges between adjacent homodimers causing monomerization and subsequently leading to interaction with COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1), nuclear accumulation and signal transduction (Christie et al., 2012; Wu et al., 2012; Favory et al 2009; Kaiserli and Jenkins 2007; Brown et al., 2005). Once UVR8 is in its active form it can then regulate the transcription of a number of UV-B responsive photomorphogenic genes allowing the plant to acclimate to counteract any future potential damage, which in turn promotes the plant’s survival and reproduction (Brown et al., 2005; Oravecz et al., 2006; Favory et al., 2009). When I first started my studies UVR8 was implicated in UV-B responses but it was unknown if it functioned as a photoreceptor. The purpose of my Ph.D was to determine if UVR8 was a UV-B photoreceptor and if so how it perceives UV-B. And more specifically, to address the question: can tryptophan residues within its structure act as an intrinsic chromophore? To investigate this aim I firstly used site directed mutagenesis to mutate specific and multiple tryptophan residues of the 14 found within UVR8’s structure to alanine, phenylalanine and tyrosine. Then I carried out transient expression studies in Nicotiana benthamiana to determine if the mutant protein tagged to GFP was stable and to determine if its subcellular localisation was affected. These UVR8 Trp mutant variants were further analyzed using yeast 2-hybrid assays (Y2H) to test for interaction with COP1, RUP1/RUP2 (REPRESSOR OF UV-B PHOTOMORPHOGENESIS) and also homodimerization. This allowed me to identify Trp mutant candidates to introduce transgenically into Arabidopsis and test further for their ability to complement the null mutant uvr8-1. The mutants were tested using a number of assays to check for monomer/dimer status, subcellular localisation, protein stability, COP1 interaction, photomorphogenic gene expression, hypocotyl inhibition and chromatin binding. Herein I present in vivo data in yeast and plants which shows, as reported by Rizzini et al. (2011), Christie et al. (2012) and Wu et al. (2012), that specific Trps, mainly W285 and W233 within the triad W233, W285, W337 have key roles in photoreception. W337 has a lesser role. These triad Trps, which are all in the conserved motif GWRHT, have now been shown in the UVR8 crystal structure to be brought into close proximity (Christie et al., 2012, Wu et al., 2012). The W285A mutant did not complement uvr8-1 and the W233A mutant only partially complemented, whereas W337A substantially complemented uvr8-1. And although all three Trp mutants constitutively interact with COP1 in planta before and after UV-B irradiation, this is not sufficient to rescue the uvr8-1 mutant for W285A and W233A, suggesting that although COP1 interaction is required for UV-B specific photomorphogenic responses it is not sufficient to mediate a response. Furthermore, for each of the triad mutants their dimer/monomer status is affected, and W285A is constitutively monomeric without being functional. Therefore, similar to COP1 interaction, monomerization on its own is not sufficient for UVR8 activation. In addition, I show that of the remaining 11 trps left of the 14 in total found within UVR8, some (W39, W144, W352) are important for structure and hence function, and the others (W92, 94, 196, 198, 250, 300, 302, 400) are not essential for function and/or structure. To further support the intrinsic Trp chromophore model of UVR8 I also present an action spectrum for dimer to monomer conversion for pure UVR8 protein in vitro from samples expressed and purified from E.coli. The spectrum closely resembles the absorption spectra of UVR8 and Trp in solution, with a maximum response at 280 nm. Moreover, the action spectrum partially resembles the in vivo UVR8 dependent HY5 (ELONGATED HYPOCOTYL 5) expression action spectrum published previously (Brown et al., 2009), although the in vivo HY5 study shows a substantial response at 300 nm, which this in vitro study lacks. Overall I show the importance of specific Trps to the UV-B photoreceptor UVR8 in yeast and in planta and demonstrate that W285 and W233 in particular are important in allowing UVR8 to function as a photoreceptor by acting as intrinsic chromophores.

This thesis is concerned with the development of new electrochemical scanning probe techniques and the application of these to biological problems. These techniques allow high resolution quantitative investigations of surface processes through measurements at a precisely placed electrode probe. A new technique, called intermittent contact scanning electrochemical microscopy, which allowed the probe-surface distance to be decisively determined through the physical interaction of the probe with the surface was developed. Separately, a new type of dual electrode probe was developed and characterised, and a new instrument (including both hardware and software) capable of a wide range of electrochemical imaging modes was developed with wide applications. The quantitative analysis of the electrochemical signal, typically measured at the probe, requires understanding the mass transport between the probe and the surface. Finite element modelling was used extensively throughout to solve the mass transport problem and therefore quantitatively analyse experimental results. Intermittent contact scanning electrochemical microscopy was used to quantify the mass transport through a porous biological membrane, dentin, that separates the pulp and enamel in teeth. Oxygen generation and consumption rates during photosynthesis were determined by measuring the local oxygen flux at an electrode placed a precise distance above a monolayer of isolated chloroplasts or thylakoid membranes. Finally, the new dual electrode probe was used to measure the reduction of an artificial electron acceptor by isolated thylakoid membranes.

Cell motility plays an important role throughout biology, the polymerisation of actin being fundamental in producing protrusive force. However, it is increasingly apparent that intracellular pressure, arising from myosin-II contraction, is a co-driver of motility. In its extreme form, pressure manifests itself as hemispherical protrusions, referred to as blebs, where membrane is torn from the underlying cortex. Although many components and signalling pathways have been identified, we lack a complete model of motility, particularly of the regulation and mechanics of blebbing. Advances in microscopy are continually improving the quality of time series image data, but the absence of highthroughput tools for extracting quantitative numbers remains an analysis bottle-neck. We develop the next generation of the successful QuimP software designed for automated analysis of motile cells, producing quantitative spatio-temporal maps of protein distributions and changes in cell morphology. Key to QuimP's new functionality, we present the Electrostatic Contour Migration Method (ECMM) that provides high resolution tracking of local deformation with better uniformity and efficiency than rival methods. Photobleaching experiments are used to give insight into the accuracy and limitations of in silico membrane tracking algorithms. We employ ECMM to build an automated protrusion tracking method (ECMM-APT) sensitive not only to pseudopodia, but also the complex characteristics of high speed blebs. QuimP is applied to characterising the protrusive behaviour of Dictyostelium, induced to bleb by imaging under agar. We show blebs are characterised by distinct speed-displacement distributions, can reach speeds of 4.9μm/sec, and preferentially form at the anks during chemotaxis. Significantly, blebs emerge from at to concave membrane regions suggesting curvature is a major determinant of bleb location, size, and speed. We hypothesise that actin driven pseudopodia at the leading edge induce changes in curvature and therefore membrane tension, positive curvature inhibiting blebbing at the very front, and negative curvature enhancing blebbing at the sides. This possibly provides the necessary space for rear advancement. Furthermore, bleb kymographs reveal a retrograde shift of the cortex at the point of bleb expansion, suggesting inward contractive forces acting on the cortex even at concave regions. Strains defficient in phospholipid signalling show impaired chemotaxis and blebbing. Finally, we present further applications of QuimP, for example, we conclusively show that dishevelled is not polarised during Xenopus gastrulation, contrary to hypotheses in the literature.

The main theme of this thesis is investigating how a cell’s biological function relates to the topology of its Gene Regulatory Network (GRN). In this context, the limits a biological function places on evolution are examined and also whether genetic networks can evolve the capacity to sense internal mutations. GRNs are modelled using Threshold Boolean Networks (TBNs), abstracting away details so that certain computational approaches become viable. For instance in Chapter 3, all possible TBNs that attain a specified functional path (of the form {v(t)}T t=0) through the expression state space are exhaustively found from a possible 3N2 TBNs, where N is the number of genes (nodes) in the network. This allows for the detailed examination of the complete neutral evolutionary space of a given functional path. It is demonstrated that the major quantities of interest, such as the connectivity of this neutral space under point mutations, the mutational and noise robustness of the TBNs in this space and even the number of networks all depend strongly on the duration T of the paths. The neutral space is found to disintegrate rapidly into disconnected components as T is increased. The effect of more exotic functional forms is also investigated. Chapter 4 focuses on evolving networks which are sensitive to deletion mutations. It is found that increased sensitivity is readily evolvable in TBNs, with the networks evolving to be more topologically balanced (they possess a similar number of excitatory and inhibitory interactions). Networks are only found to achieve maximal sensitivity through attaining long limit cycles. The study of sensitivity is extended to static populations of TBNs in Chapter 5 and the question is asked about whether a population of cells can develop the capacity to sense the presence of a mutant among them. The multicellular framework is also used to investigate the effect of intercellular connectivity on the dynamics. It is found that the greater the intercellular connectivity the more uniform the expression patterns are between cells. Chapter 6 applies the general Ergodic Set (ES) [Ribeiro and Kauffman, 2007] concept to stem cell differentiation and cancer. An alternative hypothesis to that in [Serra et al., 2010] is proposed about how to model stem cell differentiation using ESs. Coupled with results from Chapter 4, I suggest that under this new hypothesis, pluripotent stem cells will correspond to more sensitive TBNs and that differentiated stem cells will correspond to more robust TBNs.

Critical to the development of the peripheral nervous system is the regulation of the growth and survival of neurons during the phase of target field innervation. Neurons extend axons towards their target tissues and branch extensively forming complete functional synaptic connections by the end of development. This study has revealed the first known role for the tumor necrosis superfamily (TNF) member a proliferation inducing ligand (APRIL) in the development of the nervous system. Neurons of the mouse superior cervical ganglion (SCG) were chosen for study as an excellent model of peripheral development. These neurons can be easily dissected and cultured to study their growth and survival under controlled experimental conditions. The target fields innervated by this population of neurons are clearly defined, offering the opportunity to study final target field innervation, meaning this population offers excellent experimental potential. This study demonstrated for the first time that the mRNA transcripts for the TNF superfamily member APRIL and its receptors B cell maturation antigen (BCMA) and transmembrane activator and cycophylin interactor (TACI) are expressed in the SCG and target fields during the development of these neurons at a time when they are innervating their peripheral targets. Investigation of protein expression demonstrated that these neurons express this ligand and its receptors, localised to the cells themselves as opposed to projections innervating distal target fields. Functional analysis of the role of APRIL, BCMA and TACI on the growth and survival of SCG neurons in vitro demonstrated that, in the presence of nerve growth factor (NGF) the principal neurotrophin acting on these cells, either treatment with recombinant APRIL or a function blocking antibody to APRIL, enhance the growth of these neurons independent of survival. This effect is clear over a wide developmental window from embryonic day 16 to post natal day three. The ability of recombinant APRIL to enhance this growth requires the presence of a putative heparin sulfate binding domain. In addition to the activity of these factors, function blocking antibodies against the receptors BCMA and TACI also enhance NGF promoted neurite growth as do soluble recombinant forms of the receptors. Interaction with APRIL signalling acts to promote the growth of SCG neurons in culture. This enhancement of growth requires the activity of mitogen-activated protein kinase kinase and Phosphatidylinositide 3-kinase. Neurons cultured from APRIL null mice grow larger than their wild type counterparts in the presence of NGF. Two target fields innervated by these neurons, the submandibular salivary gland and nasal mucosa are hyperinnervated at post natal day five in APRIL null mice. This phenotype is not evident by post natal day 10 and so points to a transient, but significant, increase in target field innervation.

In the research described in this thesis, the trafficking of each CD23 isoform was studied in detail in both wild-type and mutant CD23 proteins, using an identical cell system and the same method of CD23 ligation for both isoforms. Confocal microscopic analysis demonstrated intracellular sorting differences to exist between the two isoforms, with CD23a utilising the endocytic pathway, and CD23b following both the endocytic and phagocytic pathways in a B-cell line. Site-directed mutagenesis was used to investigate a number of potentially key residues present in the unique N-terminal tail of each isoform. The serine groups at positions 7 and 5 in the CD23a and b isoforms, respectively, and the NNP tri-peptide motif in the b isoform were found to be necessary for accurate trafficking of these proteins. The discovery that the CD23 isoforms utilise different trafficking pathways corroborates the hypothesis that CD23a and CD23b may have functionally different roles. Current models of human CD23 signalling link the CD23a isoforms to a cAMP-generating pathway and the CD23b isoform to stimulation of inositol-1,4,5-trisphosphate production and calcium mobilisation. The ability of CD23 to transmit a signal within various cells and the fact it has a very short cytoplasmic tail, of only 23 amino acids and bereft of catalytic motifs, strongly suggests that CD23 may associate with other molecules involved in signal transduction. The divergence in the signalling pathways associated with each CD23 isoform has been attributed to the unique amino acids at the N-terminal cytoplasmic tails, as the remainder of the proteins are identical. However, there is no definitive evidence to link directly CD23 to any of these individual signalling pathways. The research presented in this thesis utilises the yeast two-hybrid assay to investigate binding partners for the N-terminal tail of CD23. Filamin A was identified as a potential interacting protein, and was found to interact with both the CD23a and CD23b isoforms. It is hypothesised that CD23 interacts with filamin A, which functions as an adaptor protein, to enable downstream responses to CD23 through connections to the appropriate signalling pathways in an isoform-specific manner.

The compound eyes of trilobites, which appeared in the Early Cambrian, represent one of the first preserved visual systems. Application of state-of-the-art microscopy techniques in the present study has revealed fine details of the microstructure and chemistry of these unusual calcite eyes that, until now, have been inaccessible and this has facilitated new insights into their growth and function. Six species from three families of trilobite with holochroal eyes, ranging from Early Ordovician to Middle Carboniferous, and 21 species from three families of trilobite with schizochroal eyes, ranging from Early Ordovician to Middle Devonian, were investigated. High-resolution microscopy techniques, including Electron Backscatter Diffraction, Transmission Electron Microscopy and Electron Probe Micro-analysis have made it possible to ‘see’ through the diagenesis of trilobite lenses to reveal the likely original lens microstructure and chemistry. Computer-based optical modelling has further shown how original lens microstructure and chemistry enhanced lens function. The discovery of sub-micron sized crystals that display a gradual and precise change in orientation shows that in many lenses much of the original microstructure has remained intact despite exposure to pore fluids and elevated temperatures and pressures during diagenesis. Although microstructure varies slightly with lens shape, there is often exceptionally precise crystal orientation. In holochroal eyes there is a direct relationship between lens shape and microstructure; where lens surfaces are planar, crystals are of uniform orientation, but where lens surfaces are convex, c axis orientation fans out, away from the lens axis. Several microstructural patterns have been identified in schizochroal lenses. However, a single original microstructural pattern, in which c axis orientation fans out at lens surfaces but remains parallel to the lens axis in the centre of the lens, may be applicable to all schizochroal lenses. The original chemical composition of the lenses, in particular those of schizochroal type, is less commonly preserved. However, the unravelling of a diagenetic pathway of change, through understanding the intricacies of relationships between different minerals in the lenses, has made possible a better understanding of how these lenses were altered during diagenesis. Lenses in holochroal eyes are invariably low-magnesium calcite, like the rest of the exoskeleton, as has been established for some time. The present study clarifies the original structures of schizochroal lenses and in doing so, ends the controversy over lens function; lenses were originally constructed as doublets, as suggested by Clarkson and Levi-Setti, and were not gradient index lenses, as was suggested by Campbell and Bruton and Haas. Lenses in schizochroal eyes, were constructed of high-magnesium calcite, with highest concentrations of magnesium in the lower ‘intralensar bowl’ and central ‘core’ regions of the lens. The degree of partitioning of high levels of magnesium, of up to 8 mole % MgCO3, in the schizochroal eyes is remarkable given the magnesium-poor ‘calcite seas’ in which they were formed. This is perhaps the first example of element partitioning within biominerals for a specific function. Based on the growth sequence of modern arthropod exoskeletons lenses in trilobite eyes are likely to have grown from the outer surface in, one lamella at a time, with microstructure and chemical composition controlled by an organic matrix. Assessment of the trilobite optical structures, using Code V optical modelling software, leads to the conclusions that the trilobite eyes functioned in a similar manner to the apposition eyes of modern animals. Code V modelling of holochroal and schizochroal eyes, and the subsequent determination of their resolution and sensitivity, shows that both eye types probably had a single optically isolated photoreceptor beneath each lens. Using a combination of specific lens size, shape, spacing, microstructure and composition, the schizochroal eye was adapted to low light intensities, similar to the eye of the modern isopod Cirolana. These adaptations would have provided the trilobite with light and dark detection of a resolution sufficient to identify movement, allowing it to detect prey and defend itself against predators. The birefringent properties of the calcite from which these lenses were made could be a hindrance, resulting in double refraction of light rays and the formation of ‘ghost’ images. Fascinatingly however this property provides the lenses with the refractive power required to make full use of the light available to them, vital for an organism with a crystalline lens with a fixed focal length. Study of the calcified lenses of ostracods and brittlestars and comparison to lenses in schizochroal trilobite eyes confirms that these modern organisms do not provide accurate analogues for trilobite eyes. No other organism that shares all characteristics of schizochroal trilobite eyes has yet been found; the eyes of the phacopine trilobites remain unique in the natural world.

Lamina II of the dorsal horn contains numerous small neurons with varying morphologies, most of which have axons that remain within the spinal cord. It can be distinguished from the other laminae by its lack of myelinated fibres and its constituent interneurons that are densely packed. This region is the major termination site for unmyelinated (C) primary afferent fibres, which convey mostly nociceptive information. It also receives inputs from thinly myelinated (Aδ) fibres, some of which are nociceptive. In spite of its importance and several past attempts, little is known of its neuronal circuitry. This is mainly due to the great functional and morphological diversity of lamina II interneurons, which has made characterisation difficult. A comprehensive classification scheme is essential to identify discrete functional populations of lamina II interneurons, and to enable understanding of their roles in the local neuronal circuitry. The present study aims to investigate the physiological, pharmacological and morphological properties of lamina II interneurons recorded in an in vitro slice preparation from adult rat spinal cord. These properties were correlated with the neurotransmitter content of each cell, which was identified by detection of vesicular transporters in axonal boutons, in order to distinguish discrete functional subpopulations of cells in this region. Both inhibitory and excitatory interneurons were identified in lamina II, based on their expression of vesicular GABA transporter (VGAT) or vesicular glutamate transporter (VGLUT2), respectively. None of the cells that had VGAT-immunoreactive axons displayed staining for VGLUT2, and vice-versa. Injection of depolarising current evoked tonic-, transient-, delayed-, gap-, reluctant- and single spike-firing among these cells. Discharge pattern was strongly related to neurotransmitter phenotype, since most excitatory cells, but very few inhibitory cells had firing patterns that could be attributed to A-type potassium (IA) currents (i.e. delayed, gap or reluctant-firing). This suggests that excitatory lamina II interneurons with IA –type firing patterns are involved in plasticity that contributes to pain states. The majority of inhibitory cells displayed tonic-firing pattern in response to depolarisation. There was also an obvious difference in the response of lamina II neurons to hyperpolarisation, since the majority of inhibitory cells showed inward currents while most excitatory cells displayed transient outward currents. Noradrenaline and serotonin hyperpolarised both inhibitory and excitatory neurons, while only inhibitory neurons responded to somatostatin. This is consistent with the findings of a previous study that had shown that the somatostatin 2 receptor (sst2a) is only expressed by inhibitory neurons in lamina II, and suggests that the pro-nociceptive effects of somatostatin are mediated by ‘disinhibition’. The somatodendritic morphology of 61 lamina II interneurons was reconstructed from projected confocal images of Neurobiotin labelling and assessed according to the morphological scheme developed by Grudt and Perl (2002). Although cells in the islet, central, vertical and radial class were identified, a substantial number of cells (19/61) had morphology that was atypical or intermediate between two classes and therefore could not be classified. Certain morphological types were consistently found in the inhibitory or excitatory population: all islet cells were GABAergic, while all radial cells and most vertical cells were glutamatergic. However, the correlation between these properties may be complex, since there was a considerable diversity in the remaining cells. Some glutamatergic interneurons had axons that contained somatostatin and many of these also contained enkephalin. Somatostatin-expressing glutamatergic cells included various morphological types, while enkephalin was detected in the axons of vertical and radial cells. All cells with axons that were somatostatin- and enkephalin-immunoreactive had delayed-firing patterns. Taken together with the pharmacological data from the present study, this suggests that somatostatin released from these glutamatergic neurons would hyperpolarise subsets of inhibitory neurons and causes disinhibition. This could lead to alterations of pain thresholds. The results from this study demonstrate that distinctive populations of inhibitory and excitatory interneurons can be recognised in lamina II, and these cells are most likely to correspond to discrete functional groups. Electrophysiological, neurochemical, morphological and pharmacological properties of neurons can be correlated but this is likely to be very complex. Future investigations that combine various approaches should allow further understanding of the specific roles of lamina II interneurons in nociceptive processing within the spinal cord.