Both plants and animals rely on nucleotide-­binding domain leucine-­rich repeat-­containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept in NLR biology is that “sensor” NLR proteins are often paired with “helper” NLR proteins to mediate immune signalling. However, the degree to which NLRs form signalling networks beyond sensor and helper pairs is poorly understood. In this thesis, I discovered that a large NLR immune signalling network with a complex architecture mediates immunity to oomycetes, bacteria, viruses, nematodes, and insects. Helper NLRs in the NRC (NLR-­required for cell death) family are functionally redundant but display distinct specificities towards diverse sensor NLRs. Several sensor NLRs, including Rx, Bs2 and Sw5b, signal via interchangeable NRC2, NRC3 or NRC4, whereas some other sensor NLRs have a more limited downstream spectrum. For example, Prf signals via interchangeable NRC2 or NRC3 but not NRC4, and Rpi-­blb2 signals via only NRC4. These helper/sensor NLRs form a unique phylogenetic superclade, with the NRC clade sister to the sensor NLR clades. The network has emerged over 100 million years ago from an NLR pair that diversified into up to one half of the NLRs of asterids. I propose that this NLR network increases evolvability and robustness of immune signalling to counteract rapidly evolving plant pathogens.

The period gene is an essential component of the circadian oscillator in D. melanogaster. A coding polymorphism in the Thr-Gly region of the gene, may contribute to the temperature compensation of the clock. The protein sequences flanking the repeat are believed to interact with the repeat and are structurally similar to it. Several in vitro deletion and insertion mutations of the Thr-Gly region were designed to test which regions flanking the repeat are required for temperature compensation. The locomotor activity analysis of flies transformed with these mutagenised constructs revealed that the removal of sequence 3' to the repeat unexpectedly causes shortening of the period. Insertion of sequence which disrupts the predicted beta turns in the middle of the repeat, or deletions of the repeat produced flies with long periods and unusual patterns of temperature compensation. These may be a result of disrupted interactions between the repeat and its flanking regions. The period changes in the deletion genotypes are likely to be a result of altered PER stability. The long period Thr-Gly mutants were not additive to the effects of the classic perLI mutant (which has delayed nuclear entry), signifying that the mutations affected a different part of the PER molecular cycle. Additionally, the LD activity patterns of the short and long period Thr-Gly mutants are different from those observed for perS and perLI, as the mutants show increasing shifts of activity into the night at high temperatures. The DD activity patterns concur with the observed LD patterns and reveal that at high temperatures the subjective night is shortened for most of the Thr-Gly genotypes. This suggests that the Thr-Gly deletions may disrupt an interaction with another factor that plays a part in establishing locomotor activity patterns.

The ability to entrain to environmental cycles and therefore anticipate and prepare for the changes they predictably bring is the preserve of the endogenous biological clock, most widely studied at the circadian level. Despite a rich history of research into the behavioural and physiological rhythms shown by many crustacean species, the underlying molecular system driving such traits is not well understood. The aim of this research was to develop our understanding of crustacean clocks through the study of two species, one of major ecological importance and the other a powerful model organism. The Antarctic krill Euphausia superba is a keystone species in the Southern Ocean ecosystem, and evidence suggests that the clock may influence both daily and seasonal rhythms. Using a variety of approaches, including the creation of a de novo assembled head transcriptome, a full suite of clock-related genes have now been cloned and characterised. Unlike many species Euphausia superba possesses orthologs of every canonical core clock gene, and cell culture assays indicate that the central feedback loop has the capacity for complete transcriptional inhibition via two separate pathways, raising the possibility that the krill clock may be an ancestral type or employ multiple oscillators to control rhythms of differing periods. In contrast to the relatively intractable krill, the amphipod Parhyale hawaiensis has simple maintenance requirements and an extensive genetic toolkit with the potential to enable sophisticated dissection of the molecular clock. With the aim of laying the groundwork for future research the clock genes of this species have also been identified, along with the development of a locomotor activity assay. Parhyale hawaiensis shows evidence of bimodal patterns of activity under the control of a molecular clock that combines mammalian-like characteristics with some unique features worthy of further investigation.

Differences in Drosophila courtship song components are considered to play an important role in species sexual isolation, because of the observed lack of variation within individuals of a species, and the relatively large differences between closely related species. When a male courts a female, a song is produced, usually consisting of a hum song and trains of pulses. Song recordings from the 8 members of the D. melanogaster subgroup, as well as from several types of interspecific hybrid crosses reveal that the Interpulse Intervals (IPIs) oscillate rhythmically about their mean, in a species-specific fashion, as courtship progresses. Various other song components, such as Intrapulse Frequency (IPF), Sine Song Frequency (SSF), Cycles per Pulses (CPP), Mean Burst Duration (MBD), and Mean Interburst Interval (MIBI) which are also thought to contribute to the maintenance of species-specific differences, thus sustaining species barriers were also studied in different species. Hybridisation between species gave evidence for possible X-linked factors in song rhythms, but generally, autosomal factors appear to be involved in controlling the other song characters. Another behavioural trait that may contribute to the species isolation of the members of the melanogaster subgroup are the circadian locomotor activity patterns. Locomotor activity profiles in constant darkness conditions revealed species-specific differences between the species in the period of their circadian oscillator, while locomotor activity profiles in light/dark cycles demonstrated phenotypic differences between the various species of the melanogaster subgroup. Hybridisations were used to assess the relative contribution of maternal and paternal factors. The overall species pattern of activity appeared to be determined by the sex-chromosomes, whereas other characteristics were primarily autosomally controlled. The implication of these findings on song and circadian behavioural cycles with respect to the current molecular analysis of circadian clock genes is discussed.

Extracellular signal-regulated kinases (ERKs) and the c-Jun N-terminal kinases/stress- activated protein kinases (JNKs/SAPKs) are activated by an array of extracellular signals to mediate a variety of cellular responses e.g. mitogenesis, differentiation, hypertrophy and apoptosis. The study investigated the regulation of ERK and JNK by agonist-mediated stimulation of the human m2-AChR or m3-AChR stably expressed in CHO cells. Stimulation of both receptors dramatically activated ERK, although stimulation by the m3-AChR was more sustained. The m3-AChR is efficiently coupled to JNK activation, whereas the m2-AChR is not. Activation of JNK in CHO-m3 cells was delayed and more sustained relative to that of ERK in either CHO-m2 or CHO-m3 cells. The dose-dependence for methacholine (MCh)-stimulated JNK activation by m3-AChR and ERK activation by both receptor subtypes were similar. Although pertussis toxin (PTX) had no effect on Ins(1,4,5)P3 accumulation in CHO-m3 cells, there was significant inhibition of agonist-induced ERK and JNK activation in CHO-m3 cells, suggesting that the m3-AChR was able to couple to Gi/o in addition to Gq. ERK activation was entirely PTX-sensitive in CHO-m2 cells. ERK activation in both cell types was shown to be independent of Ca2+. However, JNK activation by m3 receptors was shown to have both a Ca2+-depdnent and a Ca2+-independent component. PKC inhibition studies demonstrated a novel PKC- and an atypical PKC-component, but not a classical PKC-component in ERK activation in CHO-m2 cells, whereas, there appears to be a cPKC and an aPKC-component in CHO-m3 cells. In contrast to this, PKC appears to have an inhibitory role in m3-AChR-mediated JNK activation. The results presented demonstrate that the m2-AChR activate ERK and JNK via divergent mechanisms.

This study investigated G-protein coupled receptor (GPCR) and receptor tyrosine kinase (RTK)-mediated signal transduction via phospholipase C (PLC). A transient elevation of intracellular [Ca2+] ([Ca2+]i) was identified in response to PDGF-BB. Despite the recruitment and phosphorylation of PLC-, the mechanism was apparently novel and Ins(1,4,5)P3-independent. Muscarinic M3 receptor-dependent Ins(1.4.5)P3 and Ca2+ signalling was biphasic, consisting of a peak and plateau. Both agonist-occupied receptors relied on capacitative Ca2+ influx triggered by intracellular Ca2+ store release for maximum elevation of [Ca2+]i and initiated intracellular Ca2+ release from the same thapsigargin and Ins(1,4,5)P3-sensitive intracellular store. This study demonstrated that activation of muscarinic M3 receptors abolished PDGF-mediated elevation of [Ca2+]i by depleting the intracellular store, whilst PDGF receptors inhibited subsequent muscarinic receptor-mediated elevation of [Ca2+]i, possibly by inhibition of PLC- isoform. Furthermore, a PDGF-mediated elevation of [Ca2+]i was also identified in differentiated SH-SY5Y cells and hippocampal neurons. Muscarinic M3 receptors, PDGF receptors and epidermal growth factor (EGF) receptors activated mitogen-activated protein (MAP) kinase in SH-SY5Y cells. However, only PDGF-mediated MAP kinase activation had a Ca2+-dependent component suggesting that PDGF-mediated Ca2+ signalling may play other roles and an understanding of these functions emphasises the need to understand the mechanism of [Ca2+]i regulation by growth factor receptors in neurons.

No description available.

No description available.

No description available.

The presence of ethylene in a storage environment can undermine both quality and postharvest life of many fruits, often generating significant waste and associated economic losses. A demand for discovering alternative technologies capable of scavenging ethylene has led to the development of a new material, e+® Ethylene Remover, which has significant ethylene adsorption capacity. The material has been shown to remove ethylene to below physiologically active levels during fruit storage at 0-20 o C and consequently extend postharvest life for a variety of fresh produce types. Different formats incorporating e+® Ethylene Remover have been developed. Successful application of e+® Ethylene Remover in laboratory settings has created opportunities to test new formats of the product. To this end, work was conducted herein to attest whether e+® Ethylene Remover, which has been shown to maintain avocado (Persea americana cv. Hass) firmness in recent laboratory trials, could result in a meaningful extension of storage life in a commercial setting. It was shown that e+® Ethylene Remover coated sheets were a highly efficacious format for suppressing ethylene and extending storage life of imported avocado and pluot plums in a series of commercial trials. Moreover, the potency of the e+® Ethylene Remover treatment in retarding ethylene induced ripening was significantly enhanced when avocados were treated during the early stages of ripening. After a storage period of up to 31 days (5-6 o C), avocado fruits which had received an early treatment at source and then treated again in the laboratory were significantly more firm and greener compared to fruits treated following 5 weeks of transit alone . Strawberry is regarded as non-climacteric fruit; nevertheless, exogenous ethylene can negatively influence postharvest life. The low ethylene produced by non-climacteric fruits has been generally ignored and research reporting on the involvement of ethylene in these fruits is typically devoid in the literature. To this end, application of a highly sensitive laser based photoacoustic ethylene detector has revealed a possible role of ethylene in determining the postharvest life of strawberries. Moreover, fruit quality parameters including disease incidence, sugars, organic acids, phenolic compounds and plant phytohormones/metabolites were found to be profoundly affected by ethylene and likewise the removal of ethylene. Storage of strawberries in the presence of e+® Ethylene Remover was associated with lower disease incidence, significantly less red fruits and higher ascorbic acid content. In contrast, ethylene and 1-methylcyclopropene (1-MCP) treatments resulted in the higher postharvest disease. Ethylene-treated fruits were associated with lower level of sucrose and higher simple sugars (fructose and glucose) suggesting a role of ethylene in promoting the rate of senescence and concomitant reduced postharvest quality of strawberries. Changes in ABA, ABA metabolites and auxins within different tissues of ripe strawberry during storage were investigated. ABA was more abundant in the flesh than in the achenes, while auxins were undetectable in the flesh tissue. Auxins indole-3-acetic acid (IAA) and the conjugated form indole-3-acetylaspartic acid (IAAsp) were detected in high concentrations in the achenes and were affected by ethylene and storage length.