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The JAK/STAT signalling pathway plays a central role in numerous biological processes contributing to development and maintenance of homeostasis. Drosophila melanogaster offers a conserved JAK/STAT pathway with much lower redundancy. For this reason, the fruit fly was used as a model organism to investigate genetic interactions and functions of the JAK/STAT pathway in the context of the whole organism. However, very little is known regarding the molecular mechanisms governing the Drosophila JAK/STAT pathway. Here, we present a molecular analysis of the sole receptor of the JAK/STAT pathway in Drosophila, Dome. We show that Dome shares characteristics with different sub-families of mammalian cytokine receptors. Specifically, the identified JAK binding site in Dome is reminiscent of that found in IFN? receptor, while constitutive endocytosis leading to lysosomal degradation shares similarities with the Leptin receptor. An increase in tyrosine phosphorylation and a shift in the ubiquitination pattern of the receptor in response to ligand binding are also described. Furthermore, the structure-function analysis of socs36E, the only SOCS-like protein in the Drosophila genome that can potently suppress the JAK/STAT pathway, revealed two independent mechanisms of action. Firstly, SOCS36E affects stability of the receptor, most likely by forming ubiquitin ligase via the SOCS box domian, a mechanism well described for all mammalian SOCS proteins. Secondly, regulation of Dome phosphorylation by the N-terminal domain of SOCS36E contributes to suppression of the JAK/STAT pathway in a SOCS box independent manner. Finally, two alleles of the Drosophila JAK that give rise to a phenotype reminiscent of human leukaemia, hopTuml and hopT42, are shown to increase transcriptional activity of the pathway reporter without increasing phosphorylation of STAT. Both mutations cause constitutive activation of the kinase independently of the receptor. Moreover, autophosphorylation kinetics of both mutants are unaltered, compared to the wild-type Hop, suggesting non-canonical signalling to be the underlying cause of oncogenicity.

Cardiovascular disease (CVD) and cancer are two of the leading causes of mortality worldwide. The JNK pathway has been shown to play key roles at various stages of both of these diseases and therefore is an important protein to try and understand. In animal models of atherosclerosis and breast cancer, inhibition of JNK has been demonstrated to reduce pathogenesis and therefore targeting this protein may be key for developing treatments. JNK exists as three individual proteins, JNK1, JNK2 and JNK3 and studies are now showing that not only can these proteins work independently but also opposingly in some instances and therefore understanding the function of the individual isoforms is becoming critical to fully understanding this pathway. Although more research is focusing on JNK isoform function, characterisation of each JNK protein has not yet been carried out in human primary vascular cells or a human breast cancer cell line, an investigation which must be carried out to understand the role of this pathway in the pathogenesis of these diseases. In the current study lentiviral shRNA was used to target and knockdown JNK1 and JNK2 in both human umbilical vein endothelial cells (HUVECs) and MCF-7 breast cancer cells and the effects of knockdown on cell growth and cell death processes were analysed. In HUVECs knockdown of JNK2 caused an increase in pc-Jun levels and an increase in the percentage of multinucleated cells was observed, suggesting JNK2 may play a role in HUVEC cell growth. Unfortunately, the lentiviral infection itself caused detrimental effects which made it difficult to continue experiments and explore these findings further. In MCF-7 cells JNK knockdown did not produce any changes in cell growth or induced cell death when compared to non-target controls, suggesting that JNK does not play a key role in this breast cancer cell line.

Endogenous circadian clocks are a consequence of the periodic nature of the environment we live in. They allow organisms to anticipate daily environmental changes and organize a wide array of biological functions, such as daily activity, sleep and feeding. The molecular clock circuits driving rhythmic locomotor behaviour in Drosophila melanogaster consist of two interlocked negative-feedback transcription loops, with a conserved central role for the heterodimeric transcription factor CLOCK/CYCLE (CLK/CYC) and its inhibitor PERIOD (PER). Research presented in this thesis aims to describe underlying developmental requirements for clock function in adult D. melanogaster. It has been demonstrated previously that major oscillator components CLK, CYC and PER are present in larvae and some circadian modulation to behavioural is demonstrated in larvae themselves. Moreover, a light pulse administered early in development is enough to establish a phase of the locomotor rhythm of adult flies, pointing out to the connection between a function of the circadian clock during development and adulthood. This research reveals that adult circadian behaviour does not require either a functioning clock or the expression of per during prior development (Chapter 3). However, inhibition of CLK/CYC activity during metamorphosis (post-pupal formation), either by depletion of CYC or over-expression of its inhibitor PER, irreversibly affects clock-controlled locomotor activity in adult flies (Chapter 4 and 5). Even when PER over-expression is restricted to 18 ventral lateral clock neurons (LNvs) expressing the neuropeptide PIGMENT DISPERSING FACTOR (PDF), subsequent adult circadian behaviour is disrupted. A subset of small ventral lateral neurons (s-LNvs) was implicated as particularly sensitive to CLK/CYC inhibition through constitutive PER over-expression, suggesting that these cells require CLK/CYC function during development (Chapter 5). Circadian oscillations in the peripheral tissue of adult flies were less dependent on developmental CLK/CYC activity than locomotor behaviour (Chapter 5). Thus, the newly discovered developmental function for CLK/CYC appears to be specific to the neural clock circuits. Analysis of the daily rhythms of TIMELESS protein localisation within clock neurons revealed that molecular oscillator is severely disrupted in small ventral lateral neurons (s-LNvs). Therefore my research suggests CLK/CYC is necessary during metamorphosis to establish a proper function of the molecular oscillator in s-LNvs (Chapter 5). I hypothesized that genes downstream from CLK/CYC are involved in this process, with Pdp1? and Mef2 as the strongest candidates, however this has not been confirmed (Chapter 6). Moreover, it appears that chromatin modifications are not involved in mediating the phenotype observed as result of developmental CLK/CYC inhibition (Chapter 6). In summary, my work presents data confirming that CLK/CYC, but not PER activity, is required in PDF-expressing ventrolateral neurons during metamorphosis for establishing adult locomotor rhythmicity.

The Small Ubiquitin-like Modifier 1 (SUMO1) protein is a stress-inducible posttranslational modification present in all eukaryotic organisms. Conjugation of this modifier to a target protein results in the alteration of target protein function. The subsequent de-conjugation of SUMO from target proteins is conducted by a class of enzymes termed SUMO proteases. Previous research regarding the SUMO E3 ligases SIZ1 and HPY2 has inferred a connection between protein SUMOylation and auxin signalling (Huang, et al., 2009). Here, that connection has been strengthened through phenotypic analysis of Arabidopsis thaliana double knock-out mutant line for the SUMO proteases OVERLY TOLERANT TO SALT 1 and -2 (OTS1 and -2), ots1 ots2, and through the confirmation of the SUMOylation of several auxin cascade proteins. Loss of OTS1 and -2 was shown to result in an increase in auxin response in Arabidopsis thaliana seedlings exposed to exogenous auxin stimulus, indicating that an increase in global SUMOylation levels alter auxin homeostasis. Further investigation regarding components of the auxin signalling cascade revealed that the auxin receptor, TIR1, and two of the auxin-regulated transcription factors, ARF7 and ARF19, undergo SUMOylation under transient assay conditions. Mutations in TIR1 inducing lysine to arginine substitution of the SUMO-binding residues at each predicted SUMO site eliminated SUMO1 binding under transient assay conditions, further confirming that WT TIR1 is SUMOylated and that the predicted locations were correct. These non-SUMOylatable TIR1 mutant clones were then transformed into the auxin signalling Arabidopsis mutant line tir1/afb2/afb3 to further elucidate the role SUMOylation plays in auxin signalling in planta.

Quorum sensing molecules (QSMs) are involved in the regulation of complicated processes helping bacterial population benefit from their cell-density. This phenomenon has been recently studied in some fungal populations. Prokaryotes and Eukaryotes’ co-evolution raises the prospect of the existence of inter-kingdom signalling pathways. The involvement of hormone-like molecules such as QSMs in microbial cells communication promise potential role of QS process in inter-kingdom cross-talk. Bacterial antagonistic activity against fungi is considered as an important bio-control opportunity to control fungal invasion of plants. Several bacterial species such as Bacillus spp. have shown the ability to inhibit fungal growth. During the screening of antagonistic bacteria against Aspergillus flavus (A. flavus), Bacillus subtilis (B. subtilis) was identified as having high antifungal activity. The bacterium, Bacillus licheniformis (B. licheniformis) is related to B. subtilis genetically and is used at industrial-scale for production of the antimicrobial compound bacitracin. Although the comQXPA cluster involved in QS development has been identified in the genome sequence of B. subtilis and different B. licheniformis strains, the QS system in B. licheniformis was not previously investigated in detail, and its QSM (ComX pheromone) was not identified. In this context, and given the importance of this antagonistic bacterium as an industrial workhorse, this study was aimed to use B. licheniformis NCIMB-8874 as a model antagonistic bacterium to investigate its effect, and the effect of its ComX pheromone on potential inhibition of fungal growth. The results obtained from bioinformatics studies on B. licheniformis NCIMB 8874 genome sequence presented in this project confirmed the presence of essential quorum sensing-related genes, such as the comQXPA gene cluster. The cell-cell communication of B. licheniformis NCIMB-8874 was investigated through further elucidation of QS process in this bacterium. The detection of the QSM, ComX pheromone, was achieved through molecular biology and biochemical studies including over-production, purification and partial identification. Subsequently, the potential influence of ComX pheromone and Bacillus cells on the growth of A. flavus was examined and concluded that the QSM could cause a significant reduction in the growth of A. flavus strains (NRRL 3357 and ESP 15). This work reports for the first time the amino acid sequence of the purified ComX pheromone and its novel antifungal property. Pheromone as a QSM is a potential signal for communication of cells between kingdoms and could be applied for bio-control purposes. Identification of new antifungal peptides against A. flavus could lead to the development of biotechnological strategies which facilitate control of aflatoxin contamination.

Quorum sensing (QS) is a cell density dependant phenomena utilized as a communication process among microorganisms to regulate their physiological responses. QS depends on the production and release of low molecular weight, diffusible chemical signalling molecules, known as quorum sensing molecules (QSM) in the extracellular milieu. When the concentrations of the QSMs reach a critical threshold corresponding to a particular cell density, they bind to a receptor enabling them to function as transcriptional regulators. QS is widely studied in Gram-positive and Gram-negative bacteria in addition to unicellular fungi. Various recent studies report the presence of QS in filamentous fungi. Lipophilic molecules, including lactone-based molecules and the oxygenated poly-unsaturated fatty acids, oxylipins, are the major signalling molecules reported in filamentous fungi. This study correlates the cell-densities of filamentous fungi to the regulation of their different physiological responses. Two fungal species have been investigated. The first is Penicillium sclerotiorum. It was found that addition of ethyl acetate extracts from high cell densities of P. sclerotiorum culture increases sporulation delays the onset of hyphal branching and enhances the production of the secondary metabolite, sclerotiorin in shaken flasks as well as stirred tank bioreactors (STR). GC-MS analysis of the high cell-density extracts revealed several molecules including the oxylipin ricinoleic acid that might be involved in the regulation of the P. sclerotiorum physiological responses via QS. The second species studied was Aspergillus terreus. Here, the role of oxylipins as signalling molecules was investigated. It was found that the supplementation of linoleic acid, as an oxylipin precursor, enhances the production of the secondary metabolite lovastatin in shaken flasks and STRs. Studies using linoleic acid were extended, to investigate its effect on the cytosolic proteome profile of A. terreus. Results showed that several proteins were altered, mainly stress-related proteins and those involved in carbohydrate metabolism. Furthermore, the effect of different oxylipins was investigated on intracellular cAMP levels. It was observed that addition of oxylipins induces a burst in cAMP levels; in particular 9-HpODE, the linoleic acid-derived oxylipin in A. terreus, induces cAMP levels in a dose dependant manner. In silico analysis of A. terreus genome revealed the presence of genes encoding the different components of G-protein/cAMP-mediated signalling. It is speculated that the addition of 9-HpODE activates the signalling mechanism in A. terreus by binding to G-protein coupled receptors. Upon activation, the secondary messenger cAMP is produced. cAMP then induces the expression of different genes, triggering different cellular responses such as sporulation, and secondary metabolism in A. terreus.