Mouse mammary gland morphogenesis at puberty is a complex developmental process, regulated by systemic hormones, local growth factors and dependent on the epithelial/epithelial and epithelial/stromal interactions. TEBs which invade the fat pad are important in laying down the epithelial framework of the gland at this time point. The objective of this thesis was to use a combination of ‘pathway-‘ and ‘candidate gene analysis’ of the transcriptome of isolated TEBs and ducts and associated stroma, combined with detailed analyses of selected proteins, to further define the key proteins and processes involved at puberty. Using GeneChip® Mouse Exon 1.0 ST Arrays we identified the epithelial-, epithelial-stromal- and stromal transcriptomes of TEBs and ducts and defined the major functional pathways/biological processes in each compartment. By ranking the transcripts according to their expression levels and known functions in other systems, we identified genes of potential importance for pubertal mammary morphogenesis. We focused our study on Upk3a and Fbln2 and their protein products. Upk3a could only be detected at mRNA level and thus further analysis was based on Fbln2. We demonstrated that Fbln2 V1 and Fbln2 protein are predominantly expressed in the epithelium and stroma of TEBs. Using hormone primed mice we demonstrated that Fbln2 expression and localisation in the mouse mammary gland is positively regulated by E2 and P. Furthermore, by a combination of further in silico analysis, in vitro functional assays, IHC and IF we identified Vcan, Lama1, Fbn1, ColVIαIII, ColIVαI, ColXVIIIαI, Eln, Per, Acan, Nid, Itgb3 and Itga5 as potential binding partners of Fbln2 in mammary gland. Finally, we reported lack of an obvious mammary phenotype in Fbln2 KO-/- mice at puberty but demonstrated that this may be attributed to the over-compensation by Fbln1. This thesis demonstrates the benefit of DNA microarray analysis in studying pubertal development of mouse mammary gland. It identifies Fbln2 as a potential pubertal mammary regulator which by interacting with various ECM proteins at different sites of mammary milieu may contribute to an array of structural and migratory functions during mammary morphogenesis. These data substantially add to the understanding of the development of mammary gland at puberty and reveal many potential avenues for further investigations.

The Drosophila innate immune system is one of the most widely characterised of all metozoan defense systems, and shares many similar characteristics to the innate immune systems of higher organisms. As such, Drosophila has become the model organism of choice for many researchers with regards to the study of the general mechanisms and regulatory elements of innate immunity. There are a number of mechanisms that Drosophila employ in order to combat infection, and these include both humoral and cellular responses. However, perhaps the most widely characterised of these mechanisms is the systemic production of anti-micorobial peptides (AMPs) via the activation of two specific immune signalling pathways – Toll and Imd (Lemaitre et al. 1995a; Belvin and Anderson 1996). In Drosophila, a number of recent studies have identified a role for the diffusible second messenger nitric oxide (NO) in the positive regulation of the Imd pathway, a pathway that is fundamental to host defence against Gram-negative bacteria (Lemaitre et al. 1995a; Nappi et al. 2000; Foley and O'Farrell 2003; McGettigan et al. 2005). To date, the exact mechanism by which NO is mediating its effects on the Imd pathway has not yet been determined. However, it can be suggested that this effect is mediated through activation of the cGMP signalling pathway, via interaction with one of its upstream components, soluble guanylate cyclase (sGC), the main intracellular target for NO (Marletta and Spiering 2003). Therefore, the aim of this study was to determine the potential role of the cGMP signalling pathway on regulation of the Drosophila Imd immune pathway. To do this, the Drosophila Malpighian (renal) tubule was used as a model system. The Malpighian tubule is a very well characterised, extensively studied epithelial tissue and for a number of years has comprised the model system of choice with regards to the study of the epithelial roles of signalling and transport genes (Dow and Davies 2001). The suitability of this tissue as a model system for this study is two-fold: Firstly, for many years, the NO/cGMP signalling pathway has been deemed as critical to tubule function (Dow et al. 1994a). Secondly, a recent study has identified the tubule as an important autonomous immune-sensing tissue where, upon immune challenge with Gram-negative bacteria, Imd pathway-associated AMPs are systemically produced in the tubule principle cells. Importantly, it has been demonstrated that activation of the Imd pathway in the principle cells is regulated via the autocrine production of NO (McGettigan et al. 2005). Data obtained from this study has demonstrated a completely novel role for cGMP signalling in the tubule. Expression analysis has revealed that cGMP acts to modulate the expression of Imd pathway-associated AMPs in a dose-dependent manner; whereby low nanomolar concentrations are shown to stimulate diptericin expression and higher micromolar concentrations of cGMP are shown to inhibit it. This effect does not appear to extend to the fat body, the canonical tissue involved in the systemic induction of AMPs, thus suggesting a completely tissue-specific mechanism. Importantly, it is shown here that the cognate cGMP-dependent protein kinases (cGKs), DG1 and DG2 (MacPherson et al. 2004a; 2004b), mediate differential effects on AMP production in the tubule. Targeted modulation of the expression of these kinases to the principle cells of the tubule using the GAL4/UAS system demonstrates that activation of DG1 mediates positive modulation of diptericin expression in the tubule. By contrast, negative modulation of diptericin expression is shown to occur following the activation of the two main isoforms of DG2, DG2P1 and DG2P2. These data therefore describe a completely novel role for each of these kinases. Significantly, the effects of these kinases on diptericin expression in the tubule are sufficient to impact on survival of the whole fly in response to septic infection with Gram-negative bacteria, as well as contribute significantly to bacterial clearance in the gut following natural infection with E.coli. This study has therefore revealed a critical novel role for both the tubule and cGKs in the regulation of defence mechanisms in response to both septic and natural infection in the adult fly. Interestingly, Q-PCR has revealed that DG1 mediates its effects downstream of Imd. Additionally, studies have revealed that both DG1 and DG2 act to regulate the Imd pathway via modulation of Relish activation, the NFκB transcription factor responsible for the induction of AMPs following activation of the Imd pathway (Hedengren et al. 1999). Translocation assays have demonstrated that targeted over-expression of dg1 to the principal cells of the tubule results in enhanced translocation of activated Relish into the nucleus, whereas targeted knock-down of this kinase by RNAi results inhibition of Relish activation. In contrast to DG1, overexpression of either dg2P1 or dg2P2 to the principal cells of the tubule results in inhibition of Relish activation, even in the presence of immune challenge. However, this study has not revealed the exact mechanism by which these kinases mediate their effects on Relish activation, and therefore it is not clear whether DG1 and/or DG2 are acting directly on Relish, or indirectly via phosphorylation of an, as of yet, unidentified substrate(s). Despite this, a completely novel function for each of these kinases is described here for the first time. Importantly, data described in this study also identifies that, with regards to Imd pathway regulation, DG1 and DG2 may be activated via different sources of cGMP within the cell. Data shows that stimulation of the Imd pathway in the tubule is facilitated by the activation of sGC via interaction with NO. Alternatively, inhibition of the Imd pathway in the tubule is shown to be facilitated by the activation of a receptor guanylate cyclase (rGC). Additionally, it is demonstrated by this study that cGMP-mediated inhibition of the Imd pathway in the tubule is regulated by the dual-specificity, tubule-enriched phosphodiesterase (PDE), PDE11 (Day et al. 2005), thus describing a functional role for this regulatory enzyme for the first time in Drosophila. In conclusion, this study further validates the role of the tubule as a critical immune-sensing tissue in Drosophila melanogaster. In addition, a completely novel role for the cGMP signalling pathway, as a differential regulator of Imd pathway activation in the tubule, is described here for the first time. In particular, an important novel functional role for each of the Drosophila cGKs, DG1 and DG2, is revealed. The data shown in this study therefore contributes to fuller understanding of not only Imd pathway regulation in Drosophila, but also provides a significant advance in the understanding of the complexities of cGMP signalling and its regulation of tubule function.

The PDE 11 family of dual specificity phosphodiesterases was first identified in 2000, and has not been well characterised, although mutations in the gene have been linked to multiple disorders, including major depressive disorder, and cancer. DmPDE11 is a dual specificity phosphodiesterase, which shows 96% similarity with the catalytic domain of HsPDE11A, and around 40% similarity along the length of the protein. The focus of this project was to characterise this important enzyme using the model organism Drosophila melanogaster. The resources available to Drosophila researchers are unrivalled, and include a sequenced genome, unparalleled transgenic technology, of which stocks are freely available, and Homophila, a database of human disease genes and their Drosophila orthologues. Drosophila is genetically tractable to an extent not seen in any other multicellular organisms. The genetic dissection of gene function in Drosophila has allowed the identification and characterisation of numerous cell signalling genes. For example, mutations to Dunce were shown to affect olfactory learning. This allowed the identification and cloning of the mammalian dnc homologue PDE4. cAMP (and cGMP) were subsequently shown to modulate learning and memory in mammals. The 5.8 kb expressed sequence tag (EST) SD13096 had previously been shown to contain sequence present in the incomplete PDE11 RA ESTs previously released by Flybase, but also incorporating a 5’ UTR, and an in-frame start codon within two novel 5’ exons. A Northern blot of DmPDE11 RA produced one band of approximately 5.8kb; as this matches the size of the DmPDE11 RA ORF, was accepted that SD13096 encodes the entire PDE11 RA ORF (Day, unpublished). Expression of this EST in S2 cells revealed that the construct produced a protein of the accepted size, and the protein localised to the cytoplasm. However, PDE assays of S2 cell lysate revealed that the enzyme did not appear to encode an enzyme with either cA- or cG-PDE activity. DmPDE11 RA was replaced on Flybase by the new isoforms DmPDE11 RB and DmPDE11 RC, which had two key changes to the RA isoform. Both new isoforms had different N termini, sharing a second exon, with distinct first exons. Furthermore, exon 11 of the RA exon is not present in the newly predicted isoforms. These new isoforms were verified by reverse transcriptase- polymerase chain reaction analysis. In the course of this verification, two further novel isoforms were identified, which shared the novel N termini with the RB and RC isoforms, but include a novel exon/exon boundary within the original exon 19, which results in a truncated isoform. As such the four isoforms were named DmPDE11 RB long, DmPDE11 RB short, DmPDE11 RC long, and DmPDE11 RC short. The open reading frames of these isoforms were cloned from Drosophila cDNA using high-fidelity DNA polymerase and sequenced for fidelity. The open reading frames were tagged with YFP, and this tag was used to verify expression of these isoforms. Each isoform expressed a protein of the predicted size when expressed in Drosophila. DmPDE11 B and C proteins show distinct localisation in the Malpighian tubule, where the long and short isoforms of each isoform display indistinguishable localisations. DmPDE11 B localises to the apical and basolateral membranes, and DmPDE11 C localises to an unknown organelle, or to vesicles. All 4 isoforms were verified as dual specificity cA- and cG- PDEs. The previous finding (Day, unpublished) that DmPDE11 co-immunoprecipitates with cGMP dependent protein kinase activity, and that cGMP dependent protein kinases co-immunoprecipitate with cG-PDE activity, and thus that cG-PDE(s) interact with at least one cGMP dependent protein kinase, directly or indirectly, was investigated. DmPDE11 C long and short were co-transfected in Schneider 2 cells with the cGKs DG1, DG2P1 and DG2P2. Co-immunoprecipitation of these showed that both the long and short isoforms of DmPDE11 C interact with every cGK screened. Time did not permit the application of this protocol to screen DmPDE11 B interaction with the cGKs. Whether this interaction is direct or indirect was screened by peptide array. Peptide arrays were generated representing the sequence of DmPDE11, DG1, and DG2, and proteins were generated fusing fragments of these proteins with HIS6 and Glutathione-S-Transferase tags. These were expressed in E. coli, and verified by western blotting. HIS6 tagged protein expression was shown to be of higher quality, and was thus affinity purified, and used to overlay and probe the peptide arrays for putative direct interactions. When the PDE11 array was overlaid with tagged protein representing the C terminal half of DG1, and the N and C terminal halves of DG2, a putative direct interaction was identified between DG1 and PDE11 on two separate regions of the PDE11 array, which both fell within the sequence of PDE11 represented by the Middle-HIS6 fragment. As such, this was used to probe the PDE11 array. A reciprocal putative interaction was identified on three regions of the DG1 array, representing sequence in both DG1N-HIS6 and DG1C-HIS6 fragments. Unfortunately, although DG1-HIS6 was verified by western blotting at the analytical stage, attempts to affinity purify the protein failed. Time did not permit the probing of the array with DG1N-GST fusion protein, and so further putative interaction sites on PDE11 may remain. The generation of alanine substitution arrays, and subsequent mutagenesis analysis with yeast two hybrid or co-immunoprecipitation would be necessary to confirm this direct protein-protein interaction as bona-fide. The investigation into a putative direct interaction between PDE11 and DG2 did not yield conclusive data, and so further investigation is required. The role of DmPDE11 in immunity was investigated by the use of DmPDE11 RNAi and deletion lines. The DmPDE11 deletion line showed a qualitative reduction in survival in individual survival assays, but when these data were merged a significant decrease in survival compared to controls was seen. However, fly numbers did not permit the inclusion of all of the necessary controls, and so these assays should be repeated with these. However, upon immune challenge, progeny from a DmPDE11 RNAi (line 9) x Act5c (a ubiquitous GAL4 driver line) cross did not show a decrease in survival compared to parental lines. Transgenic Drosophila expressing H. sapiens PDE11A3 were generated. The protein localised to the nucleus at low levels of protein; increased expression led to nuclear exclusion, and localisation to the basolateral and especially apical membranes, with cytosolic localisation also. The work has provided the tools needed to further research PDE11. The implication of this gene as a tumour suppressor gene, and its role in other processes, means that it is of the utmost importance that this enzyme is further characterised.

The debate regarding the contribution of adult stem/progenitor cells during normal growth and beta-cell regeneration is far from being resolved. Therefore, we addressed in two distinct situations the origin of new beta-cells. We exploited a Cre/loxP lineage tracing system to efficiently label beta-cells in double transgenic mice (Z/AP; RIPCreERTAM) to address the origin of new beta-cell during the beta-cell mass expansion in response to one and two pregnancies. Similarly, we examined origin of new beta-cell after diabetic recovery in triple transgenic mouse (Z/AP; RIPCreERTAM; pIns-c-MycERTAM). Finally we evaluated the functionality of regenerated beta-cells after diabetic recovery in the single pIns-c-MycERTAM mouse model by microfluorimetry, in collaboration with Dr P. Squires. We showed that the beta-cell functionality in the pIns-c-MycERTAM line was abnormal. Second, we showed that the human placental alkaline phosphatase label (HPAP) in the double and triple transgenic mice was 1) specific to beta-cells, 2) irreversible and heritable and 3) tamoxifen dose-dependant. Third, the analysis of the proportion of beta-cells labelled for HPAP in one pregnancy, showed that the HPAP labelling index of the non-pregnant animals (0.44±0.05) was greater that in the pregnant group (0.33±0.06),(paired two-tailed t-test, P-value 0.021), indicating a dilution of the label in pregnant animal pancreata. Furthermore the combined results of the mean HPAP labelling index in non-pregnant animals (0.44±0.12) and pregnant animals (0.33±0.09) in one and two pregnancies reinforced our results above by indicating that the difference between the two groups was considered extremely significant (paired, two-sided student t-test, P-value 0.0007). Likewise, we showed that two to three months after the tamoxifen pulse, beta-cells do not fully lose differentiation or transdifferentiate into other lineages of either endocrine or exocrine compartment. In conclusion, we demonstrated for the first time that non-beta-cell progenitors contribute significantly to the increase of the beta-cell mass in response to pregnancy in combination with pre-existing beta-cell self-duplication.

Injection of 4-hydroxytamoxifen (4-OHT) activates the oncogene c-myc in transgenic pIns-c-MycERTAM mice, triggering β-cell proliferation in the short term as well as apoptosis and reduced insulin secretion, leading to hyperglycaemia. This hyperglycaemia is preceded by a short period of hypoglycaemia, which may be caused by: (i) increased insulin secretion or release from dying cells; (ii) rapid β-cell proliferation; and (iii) increased insulin sensitivity. This thesis characterizes the initial stages of the expression of c-MYC in the plns-c-MycERTAM mouse model and attempts to identify the causes of the transient hypoglycaemia using mathematical models. Furthermore, microarray data were analysed to investigate the early hypoglycaemia from the point of view of transcriptomics. The size and mass of β-cells were quantified during the transient period of hypoglycaemia by means of immunohistochemistry. These data were incorporated in a detailed mathematical model of β-cell dynamics.

The surface nature of Acanthamoeba trophozoites and cysts was investigated with respect to cell surface charge, hydrophobicity and surface carbohydrate composition. Particulate microelectrophoresis revealed a marked negative charge for both morphological forms, though less for cyst surfaces. Hydrophobicity was determined by adhesion to n-hexadecane and indicated a relatively low hydrophobic nature of both forms, though less so for cysts. Surface carbohydrate composition was studied by the use of fluorescent lectins and flow cytometry, using a ligand-receptor approach for further in depth analysis of binding of particular lectins. These studies showed trophozoite and cyst surfaces to be rich in N-acetylglucosamine, N-acteylneuraminic acid, mannose and glucose, with the addition of N-acetylgalactosamine on cysts. The importance of such surface properties was investigated with respect to phagocytosis of polystyrene latex microspheres, of different surface types and size. Investigations into the optimum conditions of uptake of beads indicated a preference for a medium devoid of nutrients, such as saline, though temperature was not a factor. An amoebal predilection for beads of lower charge and greater hydrophobicity was demonstrated. Furthermore, a preference for the largest bead size used (2.0 m) was observed. The influence of either Con A or mannose or glucose on bead association was apparently limited. The fate of foreign DNA ingested by Acanthamoeba appeared to indicate that such DNA was destroyed, as it could not be detected following extraction procedures and PCR amplification.

No description available.

No description available.

Haemocytes, the main type of circulating defence cell in invertebrates, functionally resemble mammalian macrophages, and are responsible for phagocytosis, endocytosis and other innate immune functions. In mammalian macrophages, the extracellular-signal regulated kinase (ERK) pathway has been shown to modulate immune responses. It was therefore hypothesised that this pathway may play a similar role in haemocytes of the gastropod mollusc, 'Lymnaea stagnalis', and intermediate host of the schistosome, 'Trichobilharzia ocellata'. Two ERK-like proteins were detected by Western blotting usmg antibodies that recognise non-phosphorylated and phosphorylated forms of mammalian ERK 1/2; these proteins had approximate molecular weights of 44 and 43kDa respectively. In addition, the upstream regulator of ERK, MEK, was detected in haemocytes as well as the transcription factor downstream of activated ERK, Elk-1. By employing a mitogen-activated protein (MAP) kinase assay kit, the identified ERK-like proteins were found to possess kinase activity. When haemocytes were challenged with 'E. coli' lipopolysaccharide (LPS), the activity of the ERK pathway was found to be significantly upregulated after 5 min challenge and perinuclear migration of phosphorylated ERK was observed by immunocytochemistry. Other compounds, including Zymosan A, adrenocorticotropin hormone, noradrenaline and challenge with heat-killed 'E. coli' did not modulate haemocyte ERK pathway activity. Pharmacological inhibitors used to block signalling to the ERK pathway demonstrated that MEK, PKC, PI-3K and Ras activity are vital for phagocytosis by haemocytes. In addition, the integrin subunits [alpha]V[beta]3 and [beta]l were found to be present on the haemocyte surface and integrin engagement was found to be necessary for phagocytosis. Monosaccharides found on the surface coat of 'T. ocellata', fucose and galactose, were applied to haemocytes in the presence and absence of haemolymph. These sugars proved to have important immunomodulatory effects, since ERK, PKC and phagocytic activity were all affected by these sugars, and differences in experiments with and without haemolymph suggest an important role for serum proteins in the molluscan immune response. In conclusion, this study provided an insight into the signalling machinery involved in the molluscan defence response, and the results should stimulate further research in snail defence responses, particularly following challenge by parasites.

Ageing, immunity and stress tolerance are inherent characteristics of all organisms. In animals, these traits are regulated, at least in part, by forkhead transcription factors in response to upstream signals from the Insulin/Insulin–like growth factor signalling (IIS) pathway. In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance. It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signalling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. In this project we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four Caenorhabditis species. We show, using phenotypic analysis of DAF-16 influenced phenotypes, that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (Caenorhabditis elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Quantitative Real-Time PCR, which positively correlate with the observed phenotypes. We also provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis. Finally, we take a closer look at the daf-16 gene and its isoforms in C. elegans and their role in driving specific responses to stress. These findings impact upon our understanding of the diversification of the IIS pathway and the evolution of longevity in general, and illustrate how such differences could explain both inter and intra-species differences in ageing, immunity and stress response.