Mate choice plays an important role in sexual selection and speciation. The evolution of mate choice is intriguing in cases where choosy individuals gain little except for genetic material from the mate and where the trait used as a criterion for the choice is costly to its bearer. The sensory exploitation hypothesis is an interesting idea that applies to such cases because it suggests that sexual preferences may arise as side-effects of preferences that are under selection in other contexts. The role of mate choice in speciation is strong but is debated because the reasons for population divergence in mate preferences and sexual traits are sometimes hard to explain. Also in this context sensory exploitation offers a potential explanation in that a link between natural and sexual selection may result in divergence in sexual selection whenever populations differ in natural selection. In this thesis, I test several aspects of this hypothesis in a species of fish, the swordtail characin (Corynopoma riisei). In this species males display a flag-like ornament that grows from the operculum. Because females respond to this ornament by biting at it, it has been proposed to act as a food-mimic. By manipulating female food type and quantity, and testing the resulting female preference for the male ornament, I find support for the theory that the preference has evolved through sensory exploitation and that females indeed appear to relate the ornament to a food item. Furthermore, I show that sensory exploitation can lead to morphological divergence among natural populations in this species. Apart from the flag-ornament, other courtship signals are also investigated. The results show that the relative importance of different signals may vary depending on receiver motivation. This suggests that various aspects of both male courtship signals and the conditions during which they are being signalled should be considered to gain a full understanding of mate choice and its role in sexual selection and speciation.

T lymphocytes are considered an essential and advanced component of the immune system, since these cells are able to discriminate self from non-self, start up an immune reaction and further develop into memory cells. However, therapies based on the use of patient derived newly generated T cells reinoculated into humans do not exist. This is due to difficulties in replicating the peculiar conditions required for T cell development in vitro. The systems developed so far are based on the use of animal or unrelated human thymic tissue and therefore they would not be adequate to be used in any clinical application. Having conjectured that human skin cells, rearranged in a threedimensional fashion, would be able to support the development of human T lymphocytes from hematopoietic stem cells, we developed a model consisting of human skin keratinocytes and fibroblasts arrayed on a synthetic matrix so to create a prototype suitable to be translated into the clinic. In this way we were able to induce few hundred cord blood CD34⁺ haematopoietic stem cells to entirely develop into mature CD4⁺ or CD8⁺ T lymphocytes in vitro. However, circulating adult peripheral CD34⁺ precursors failed to survive in the same conditions. Finally we were able to explain our success as consequence of strong induction of the Notch delta ligand Dll-4 by the keratinocytes cultured in the construct. In synthesis, we report here for the first time that skin keratinocytes, in the presence of fibroblasts and reconfigured in a three-dimensional arrangement, are able to induce the differentiation of a minimal amount of cord but not adult blood stem cells into fully differentiated T cells by acting through the Dll-4 Notch signaling pathway in vitro.

Obesity is a multi-factorial metabolic disease, resulting in increased adipose tissue acquisition by the host. This disease increases the risk for developing co-morbidities, including Metabolic Syndrome and other disorders such as breast cancer. Obesity, and particularly abdominal obesity, is characterised by metabolic changes, including chronically elevated insulin concentrations and aberrant secretion of cytokines released from fat tissue, called adipokines. Epidemiologically, the risk of developing postmenopausal breast cancer is increased in obese individuals. The molecular link between obesity and breast cancer however is not well understood. The study presented here aimed at identifying the molecular mechanisms involved in this link, by testing the hypothesis that high insulin concentration and certain adipokines may promote breast cancer progression and/or breast cancer aetiology. A cell culture system of breast cancer cells and breast epithelial cells was employed to investigate changes in cell proliferation, activation of cell signalling pathways, cell cycle progression and apoptosis after treatment with insulin, leptin, TNF-α, adiponectin and IL-6. In MDA-MB-231 breast cancer cells, insulin treatment did not affect cell proliferation, cell cycle or apoptosis. Conversely, IR-phosphorylation, AKT-phosphorylation and ERK1/2-phosphorylation were all significantly increased. Microarray analysis indicated several important changes in gene expression with insulin treatment. Leptin treatment increased proliferation by 21%. Additional analyses of the effect of leptin indicated that neither the PI3-kinase pathway nor the MAP-kinase pathway was involved in mediating this effect. Treatment with TNF-α increased apoptosis, but did not affect cell proliferation or activation of cell signalling pathways. In MCF-10A breast epithelial cells, cell proliferation increased after insulin treatment by 180%. IR-phosphorylation, AKT-phosphorylation and ERK1/2 phosphorylation were all significantly increased while early apoptosis decreased after insulin treatment. Analysis of cell cycle however did not indicate a change in progression. Microarray analysis indicated that insulin treatment may increase expression of genes related to cancer growth. Leptin treatment increased cell proliferation and also increased ERK1/2-phosphorylation, while AKT-phosphorylation was not affected. Leptin did not change cell cycle progression. TNF-α treatment increased cell proliferation and also increased ERK1/2 phosphorylation, while AKT-phosphorylation was not changed. TNF-α treatment tended to increase apoptosis, the change however was not statistically significant. In SK-BR-3 breast cancer cells, cell proliferation did not change after insulin treatment. IR-phosphorylation and AKT-phosphorylation increased after insulin treatment, while ERK1/2-phosphorylation decreased. Gene expression of cyclin D and cyclin E increased with insulin treatment, while apoptotic rate and cell cycle profile were also not affected. Cell proliferation increased by 115% after treatment with 100 ng/ml leptin. ERK1/2-phosphorylation however decreased, while AKT-phosphorylation tended to increase, but the change was not statistically significant. Cell cycle profile was not affected by leptin treatment, G1-phase however tended to increase, but the change was again not statistically significant. Cell proliferation increased by 59% after 48 h treatment with 10 ng/ml TNF-α. AKT-phosphorylation and ERK1/2-phosphorylation increased with TNF-α treatment. Cell cycle analysis showed a decrease in S-phase and G2-phase, indicative of a decrease in cell cycle progression. These results indicate that none of the examined obesity-related factors is convincingly identified as the main molecular link between obesity and postmenopausal breast cancer. Conversely, all treatments affected each of the cell lines in, at least, one of the examined aspects. This indicates that many of the obesity-related factors may affect breast cancer and that a single breast tumour may utilise a unique combination of those factors to promote growth. All treatments increased proliferation in MCF-10A breast epithelial cells, with additional analysis generally supporting growth promotion. Insulin treatment particularly increased cell proliferation, while leptin and TNF-α increased MAP-kinase signalling. This may indicate that insulin and adipokines may have a higher impact on breast cancer aetiology than on breast cancer progression.

The grey squirrel (Sciurus carolinensis) has wildtype and melanic (dark) colour morphs. Melanism is associated with variations in the melanocortin-1 receptor (MC1R) gene in a number of species. The MC1R protein is a G-protein coupled receptor, predominantly expressed in melanocytes, where it is a key regulator of pigment production. To investigate the genetic and molecular basis of melanism, the MC1R genes of the wildtype and melanic grey squirrel were sequenced. The wildtype (MC1R-wt) and melanic (MC1RΔ24) variants of the MC1R were then functionally characterised in a cell-based assay. The MC1R gene of the grey squirrel was found to have a 24 base pair (bp) deletion associated with melanism. The MC1R is typically activated by its agonist, the alpha-melanocyte stimulating hormone (α-MSH), which stimulates dark pigment production by raising intracellular cAMP levels. Conversely, the MC1R is inactivated by its inverse agonist, the agouti signalling protein (ASIP), which stops dark pigment production by lowering intracellular cAMP levels. To investigate the effects that the 24 bp deletion have on receptor function, MC1R-wt and MC1RΔ24 genes were transfected into HEK293 cells. Cells expressing either MC1R-wt or MC1RΔ24 were stimulated with α-MSH or ASIP and intracellular cAMP levels were measured. Unstimulated MC1RΔ24 cells showed higher basal activity than the MC1R-wt cells. Both MC1R-wt and MC1RΔ24 cells responded to α-MSH with a concentration-dependent increase in intracellular cAMP. However, while the MC1Rwt cells responded to ASIP with a concentration-dependent decrease in intracellular cAMP, MC1RΔ24 cells responded with an increase in cAMP. Melanism in the grey squirrel is associated with a 24 bp deletion in the MC1R. Cells expressing MC1RΔ24 have higher basal levels of cAMP than MC1R-wt cells. ASIP acts as an inverse agonist to the MC1R-wt but as an agonist to the MC1RΔ24. As MC1RΔ24 cells have higher levels of cAMP, and higher levels of cAMP lead to dark pigment production, the 24 bp deletion is the likely molecular cause of melanism in the grey squirrel.

The Protein kinase C (PKC) superfamily is a key regulator in platelet activation with individual isoforms playing distinct roles. This thesis focuses on the role of the novel PKC isoforms downstream of several agonists using both pharmacological and genetic approaches and human and mouse platelets. Quantification of the protein levels of PKC isoforms identified different levels of the five major PKC isoforms expressed in human platelets and also differences between levels of the same isoform in human and mouse platelets. Use of a selection of broad spectrum and isoform-specific inhibitors, identified both positive and negative novel roles for PKC in the regulation of human and mouse platelets. A net positive role for PKC was found in GPVI, Clec-2, and PAR receptor signalling, with classical isoforms of PKC playing a major role in aggregation and dense granule secretion. A novel negative regulatory role was also identified in the regulation of ADP-induced platelet activation for PKC~, and both PKCE and PKC~ in human and mouse platelets respectively. Gene knock-out mouse models confirmed a positive regulatory role for PKCe in allb~3 outside-in signalling but identified no other regulatory role for PKCe in agonist induced platelet activation. Despite this relatively minor role, functional redundancy was identified between PKCe and PKCE isoforms in haemostasis, as tail bleeding was significantly increased in mice deficient in both novel isoforms. The work presented here identifies key roles for the PKC superfamily in the complex regulation of platelet activation, with different isoforms supporting and limiting the process of thrombus formation and haemostasis.

Many anticancer therapies, including radiotherapy, act by damaging the deoxyribosenucleic acid (DNA) that is fundamental to cell function and proliferation. H2AX is a histone protein associated with DNA that is phosphorylated to produce γH2AX in response to DNA double strand breaks (DSBs), the most lethal lesions caused in cancer cells. This thesis examines the translation of γH2AX detection assays to clinical situations in order to provide biomarkers of response that might help to guide the treatment of cancer patients. γH2AX immunohistochemistry was developed in preclinical xenograft models, and validated over a range of radiation doses and over time after irradiation. The method was prepared for translation to archived clinical biopsy and surgical specimens. The DSB Biomarkers Pilot Study was established in order to develop a method for γH2AX quantification in direct tumour cell specimens obtained using the clinical technique of fine needle aspiration (FNA) cytology. Eleven patients undergoing anticancer therapy were recruited to the study, and the method evaluated. The coefficient of variation of the measure was 49%. Non-invasive imaging for γH2AX would allow DNA damage to be quantified in all tumour sites, and on multiple occasions. An antibody-based nuclear medicine imaging agent was re-engineered using Fab fragments of the antibody. The novel agent demonstrated improved pharmacokinetics when compared to the whole antibody agent, but reduced target specificity. The findings further develop the potential to exploit DNA damage biomarker measurements in clinical oncology.

Recent studies have identified Two-Pore Channels (TPCs) as the channels activated by NAADP. To date, most studies that characterized these channels have employed heterologous expression or overexpression systems. The research reported here has focused principally on endogenous TPC activity by using single and dual gene knockout (KO) in a mouse system and has yielded insights into TPC expression levels, subcellular localisation, NAADP binding, and channel function. Mouse models that had been generated by both the “gene-trapping” and the “genetargeting” techniques were obtained and validated. These included a knock-down strain (“hypomorph”). Surprisingly, all TPC mutant mice showed no gross phenotypes. In addition to the two known isoforms in mouse, TPC1 and TPC2, the expression of a shorter variant of TPC1 was discovered; this has an alternative (truncated) N-terminus, and has been termed (DELTA)N-TPC1. All TPC variants/isoforms were widely expressed in all mouse tissue types tested. Overexpression of mouse TPCs in mouse embryonic fibroblasts showed that (DELTA)N-TPC1 and TPC2 were expressed primarily in late endosomes/lysosomes while TPC1 was expressed in both endosomes and lysosomes. Dileucine sorting motifs target TPCs to late endosomes/lysosomes; it was shown that truncation or mutation of dileucine motifs significantly reduced localization in late endosomes/lysosomes. Furthermore, TPCs were shown not to be the direct binding target of NAADP, as the high affinity NAADP binding was retained in hepatic membranes from TPC double KO (DKO) mice. It is concluded that NAADP binds to an (as yet, unidentified) accessory protein. The functional role of TPCs was studied in depth using mouse pancreatic acinar cells. NAADP is known to release Ca²⁺ from the acidic stores in response to the stimulation by the hormone cholecystokinin (CCK). In all TPC mutant mice, CCK was still able to evoke Ca²⁺ oscillations, but with slower and attenuated oscillations in the TPC1 hypomorph, and with slower oscillations in TPC DKO. In all TPC KOs, oscillations were disrupted by known inhibitors of the NAADP-signalling pathway (Ned-19, GPN and bafilomycin A1), indistinguishable from the responses with wild-type cells. This suggests that TPCs are not involved in CCK signalling, although it is possible that functional compensation masked the phenotype arising from the impaired signalling.

Increases in temperature damage plant cells, and plants react to heat stress by inducing of a number of protective mechanisms. In this study, it has been shown that heat damages cells both directly while the plants are being heated, and indirectly through heat induced oxidative stress during recovery from heating. Different stress response pathways are induced in each case. Evidence implicates salicylic acid, abscisic acid, ACC (a precursor of ethylene), calcium ions and active oxygen species in pathways resulting in thermotolerance (i.e. increased survival at high temperature). Addition of these potential second messengers results in increased survival and decreased oxidative damage after heating (as measured using the TBARS assay), while silencing the pathways through use of mutants, inhibitors or transgenes results in decreased thermotolerance. In vivo calcium measurements show cytosolic calcium transients only at initiation of cooling after heat stress. Calcium chloride also induces thermotolerance when added after heating, as does ACC. These substances can induce early increases in ascorbate peroxidase activity after heating, and induce expression of antioxidant genes. Thus they may play a role in heat induced oxidative stress response pathways in recovery. Two different pathways induced during heating appear to result in the expression of genes for heat shock proteins. The predominant pathway induced at 30oC involves ABA, while that at 35-40oC involves an oxidative burst generated through the NADPH oxidase, atrbohB. Addition of SA can induce HSP (heat shock protein) expression, but there is little evidence that this occurs endogenously in plant cells, although nahG plants unable to signal via SA are thermosensitive. Results in this study indicate that there are several signalling pathways associated with heat shock. The pathway induced during recovery from heating involves calcium ions and ethylene, and results in increased antioxidant capacity. During heating two pathways induce expression of HSPs: one involving abscisic acid and possibly salicylic acid and one involving an oxidative burst. At least one further pathway is believed to exist, which involves protein kinases and phosphatases as heat shock causes up-regulation of expression of certain genes for these signalling components.

T cells are an essential part of the adaptive immune system and protect the body from intracellular infections. The specificity with which αßTCR-bearing T cells recognize cognate antigen presented on MHC molecules is paramount to maintaining the balance between mounting effector functions against pathogens and establishing peripheral tolerance to self. The mechanism by which T cells translate qualitative differences in TCR:pMHC binding to sensitive proximal signalling events which ultimately result in specific Tcell effector responses to infected cells but not to self is mostly unknown. To address how T cell signalling responds to qualitative differences in TCR triggering by pMHC, I established a system of stimulating T cells bearing the 1G4 TCR specifically in vitro with a panel of four NY-ESO-1_156-165 peptide variant MHC tetramers. Single amino acid substitutions to the NY-ESO-1_156-165 peptide conferred a maximum 35-fold difference in the monomeric affinity for the 1G4 TCR. The system allows the highly controlled investigation of very rapid TCR proximal signalling events simultaneously and quantitatively using flow cytometry. Stimulations with pMHC tetramers showed rapid sensitive sequential signalling responses which were able to confer ligand discrimination. Very early signalling events such as CD3ζ phosphorylation showed analogue responses to the different affinity pMHC tetramers. Later signalling events including phospho-ERK showed a distinct on/off switch-like response. The amplitude of the very early analogue signalling responses determined the extent of later digital ERK signals. This indicates that a certain analogue signalling threshold must be passed to result in T cell activation. The thymocyte protein Themis has been shown proximal TCR signalling to modulate thymocyte selection thresholds. Its deletion results in profound defects in positive thymocyte selection. Themis locates to the LAT signalosome of the TCR signalling cascade via Grb2, yet its molecular function is unknown. Employing the system I established, I demonstrate that Themis-k/d cells show increased levels of CD3z-chain phosphorylation, phospho-ERK signalling and signal-induced apoptosis which was independent of the ERK signal. This shows that Themis is a global attenuator of proximal TCR signalling. We are currently investigating possible associations of Themis to proteins phosphastases such as SHP-1 which could attenuate TCR proximal protein tyrosine signalling events.

Nucleotide synthesis is a conserved and highly regulated response to DNA damage, required for the efficient repair of DNA double strand breaks (DSB) by homologous recombination (HR). This is essential to prevent loss of heterozygosity (LOH) and maintain genome stability. The aim of this study was to identify new genes important for HR through roles in damage-induced nucleotide synthesis. A screen was performed to identify S. pombe gene deletion strains whose DSB sensitivity was suppressed by deleting the ribonucleotide reductase (RNR) inhibitor spd1⁺ to promote nucleotide synthesis. The screen identified a number of genes including ddb1⁺, cdt2⁺, rad3⁺ and csn1⁺ which have known roles in nucleotide synthesis. Distinct roles were identified for the DNA damage checkpoint in suppressing LOH. rad3⁺, rad26⁺, rad17⁺ and the rad9⁺, rad1⁺ and hus1⁺ genes encoding the 9-1-1 complex were required for DNA damage-induced nucleotide synthesis through Cdt2 induction to promote Spd1 degradation. The HR repair defect of rad3⁺ and rad26⁺ deletion strains was partially suppressed by spd1⁺ deletion. However, the HR repair defect of rad17⁺, rad9⁺, rad1⁺ and hus1⁺ deletion strains was not suppressed. An additional role was confirmed for Rad17 and the 9-1-1 complex in preventing LOH by promoting DSB resection. A role was identified for the Gcn5 histone acetyl transferase (HAT) protein module, consisting of Gcn5, Ngg1, Ada2 and Sgf29, in suppressing DSB sensitivity by promoting nucleotide synthesis. This was independent of Cdt2 or RNR protein levels. The Gcn5 HAT module was also found to regulate DSB repair pathway choice consistent with previous observations. Deletion of gcn5⁺, ngg1⁺ or ada2⁺ decreased HR and increased non-homologous end joining. Surprisingly, deletion of spd1⁺ in a gcn5∆, ngg1∆ or ada2∆ background also promoted HR. This predicts a role for nucleotide pools in regulating DSB repair pathway choice. Eleven other candidates showed repeatable suppression of DSB sensitivity following spd1⁺ deletion. However many of these candidates did not show reduced nucleotide levels. This suggests deleting spd1⁺ may also suppress DSB sensitivity by a different mechanism.