No description available.

Endosomal trafficking and autophagy are two fundamental processes of eukaryotic cell biology, from unicellular organisms such as yeast to multicellular metazoans such as C.elegans and Humans. Both processes are involved in a diverse number of physiological processes and implicated in a number of pathologies. A recent study has exhibited a mutation on the SM protein Vps45 as a cause of severe congenital neutropenia in humans. The same mutation in yeast causes defects in endosome to vacuole trafficking in S.cerevisiae as well as a temperature sensitive lethality at the non-permissive temperature. A null allele of vps-45 in C.elegans results in developmental arrest during the highly secretory phase of moulting in a similar temperature conditional manner to yeast and defects in yolk protein trafficking. The work presented in this thesis aims to provide basic understanding in an animal model of the impact of loss of Vps45 function that might be informative of the reason for the death of the highly secretory neutrophil cells under the absence of a functional Vps45 protein. The vps-45 and unc-51 mutants as well as a novel unc-51 vps-45 double mutant where possible, were characterised for lifespan, duration of post- embryonic development as well as moulting duration. Reduced embryonic viability, reduced lifespan as well as delays in the moulting process were identified. Data suggested that both autophagy and protein trafficking play a role in C.elegans development through unc-51 and vps-45 respectively. In addition to this, the seam cells of both the vps-45 and unc-51 defective C.elegans were observed during the moult using an autophagy marker. An increase in autophagic activity during the moult was observed, which was more pronounced in the case of the vps-45 mutant. As such the obtained data suggest autophagy and endosomal trafficking play an important role in the moulting process. Following up to previous work conducted in our lab in yeast defective for Vps45 trafficking which exhibited increased sensitivity to oxidative stress, the redox state of the vps-45 and unc-51 animals as well as their sensitivity to oxidative stress was assessed using a set of ER and cytosolic GFP markers and killing assays. Both the vps-45 and unc-51 mutants showed a higher sensitivity to oxidative stress, with the unc-51 exhibiting the more pronounced phenotype overall. These results came in agreement with the shorter lifespan phenotypes exhibited by both mutants in the previous experiments, possibly as a result of accumulation of ROS, as well as the severe defects of the double mutant. Finally, a suppressor identified for the moulting death of the vps-45 mutant was characterized for a set of phenotypes, in order to exclude suppression of any of the other phenotypes identified for the vps-45 mutant. Furthermore, the suppressor was identified as being autosomal and recessive and as thus an SNP full genome sequencing technique was employed, which gave rise to two suppression loci in two different chromosomes, along with two different subpopulations corresponding to these loci which exhibited different growing patterns.

Neuronal dysfunction and cell death occurs during neurodegeneration. Animal models that express human disease genes and show neurodegenerative-like pathologies are widely used to study particular molecular systems in early neurodegenerative changes. Axonal transport (AT) is perturbed in several prevalent neurodegenerative diseases. The development of a Huntington’s Disease (HD) model in Drosophila melanogaster larvae is described, in which disease gene expression is directed to motor neurons (Chapter 2). This results in stalling and accumulation of AT vesicles in live animals and a locomotion defect after additional environmental stress. The cause of AT disruption and neuronal dysfunction in most cases of neurodegeneration is unknown, but it is associated with protein misfolding and aggregation that overrides cellular defences such as the heat shock protein (HSP) molecular chaperone system. In addition to HD, this applies to human tauopathies such as Alzheimer’s Disease (AD), which involve axonal misfolding and aggregation of tau. Increased throughput assays to test larval locomotion are developed (Chapter 3) in a Drosophila larval model of tauopathy, in which locomotion defects are detectable under normal environmental conditions. Candidate chemical modulation of this locomotion phenotype is described that targets HSP induction (Chapter 4). The chemicals used result in no detectable change in hsp70 level, lower total tau levels, and worsening of the locomotion defect phenotype. Tissue-specific elevation of hsp70 after hypoxic stress (Chapter 5) protects from acute behavioural disability and reduced survival in aged adult Drosophila expressing human tau in the nervous system. These studies indicate some therapeutic potential for HSP elevation in tau mediated pathology. Nevertheless, further work is required if chemical chaperone induction, and the roles of HSPs in axonal transport and homeostasis during chronic neurodegenerative and acute environmental stress, are to be further explored in these models

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Genetic Programming is a form of Evolutionary Computation in which computer programs are evolved by methods based on simulating the natural evolution of biological species. A new generation of a species acquires the characteristics of previous generations through the inheritance of genes by sexual reproduction and through random changes in alleles by random mutation. The new generation may enhance its ability to survive by the acquisition of cultural knowledge through learning processes. This thesis combines the transfer of knowledge by genetic means with the transfer of knowledge by cultural means. In particular, it introduces a new evolutionary operator, memory operator. In conventional genetic programming systems, a new generation is formed from a mating pool whose members are selected from the fittest members of previous generation. The new generation is produced by the exchange of genes between members of the mating pool and the random replacement of genes in the offspring. The new generation may or may not be able to survive better than its predecessor in a given environment. The memory operator augments the evolutionary process by inserting into new chromosomes genetic material known to often result in fitness improvements. This material is acquired through a learning process in which the system is required to evolve generations that survive in a less demanding environment. The cultural knowledge acquired in this learning process is applied as an intelligent form of mutation to aid survival in a more demanding environment.

In a rat model of intrauterine growth restriction (IUGR) induced by maternal global undernutrition, adult offspring are obese with associated metabolic disturbances. These metabolic abnormalities are all augmented by feeding a high calorie postnatal diet and reversed by neonatal leptin treatment. Evidence is now accumulating which indicates that altered epigenetic regulation and gene expression may underpin the relationship between the early life environment and metabolic disturbances in adult life. Therefore, to determine the mechanism responsible for the alterations in energy balance in the IUGR rat, this study investigated the effect of maternal diet, neonatal leptin treatment and a postnatal high fat diet on the expression and DNA methylation of genes involved in energy balance in the liver and adipose tissue of adult offspring. These genes included the peroxisome proliferator activated receptors (PPARs) and their target genes; acyl-coA oxidase (AOX), carnitine palmitoyl transferase-1 (CPT-1) and lipoprotein lipase (LPL). Real time PCR indicated that the expression of several key genes involved in energy balance, including PPARα, PPARγ and their target genes, was not altered by maternal diet or postnatal diet in the liver or adipose tissue of these offspring. However, in adipose tissue, neonatal leptin treatment resulted in an increase in the expression of most genes tested, including PPARα, PPARγ and their target genes. The increased PPARγ and LPL would facilitate the uptake of fatty acids into the adipocyte, whilst the upregulation of PPARα and its target genes AOX and CPT-1, not normally expressed in adipocytes, would direct fatty acids taken up towards the β-oxidation pathway instead of storage. This would imply that the fat cell had transformed from a fat storing cell to a fat metabolising cell. Gene expression data therefore indicated that the phenotypic changes induced by neonatal leptin treatment, i.e. the reduced weight gain, could be due to increased expression of PPARα, PPARγ and their target genes in adipose tissue: Furthermore, the effects of this are persistent, due to the specific period of leptin administration during neonatal development. There was, however, no evidence of altered DNA methylation in the promoter regions measured which could account for these persistent effects. To investigate mechanisms underlying the regulation of the PPARα promoter by leptin, the rat PPARα promoter was mapped, cloned and characterised. As part of this process, six alternatively spliced variants were identified; one from adipose tissue (P1), two from the liver (P2, P3), one from the heart (P4) and two from the kidney (P5, P6). These transcripts were found to differ in their 5’untranslated region due to tissue specific promoter usage and alternative transcription start sites. The liver and adipose specific promoters were cloned and characterised using a reporter gene strategy. They were shown to differ in their basal activity, response to known activators of transcription and to neonatal leptin treatment. The regulation of the PPARα promoter by leptin was investigated and shown to function via a non-canonical mechanism requiring both signal transducer and activators of transcription (Stat3) and specificity protein-1 (Sp1), which act at a unique region of the liver specific P2 promoter. The adipose specific P1 promoter was shown to be unresponsive to leptin treatment. Furthermore, real time PCR with primers specific to the P1 and P2 PPARα transcripts indicated that the increased PPARα expression seen in leptin treated offspring was due to an increase in the P2 specific transcript, not the P1 transcript. This indicated that the neonatal leptin treatment facilitated a selective switch in promoter usage to increase the expression of PPARα and its target genes in a tissue in which they are not normally expressed, thus inducing an altered metabolism within the adipocytes of these offspring.

The effects of pupal cold shock on the life cycle and wing morphology of the Speckled Wood butterfly are examined and their genetic assimilation is investigated. Metamorphosis is modelled in terms of changes in stability, and the mediation of cold shock effects by hormones is considered. Current theories of pattern formation are evaluated for the species, and pattern is analysed using manual, photographic and digital methods. The development of wing morphology is modelled, and cold shock effects understood by comparison with normal development. Developmental canalisation is estimated as variability and fluctuating asymmetry. An index is developed that predicts the extent of assimilation. Likely modes of inheritance are suggested, and the possibility of natural cold shock and assimilation in the species is considered. Recent trends in biology indicate that neo-Darwinian concepts cannot adequately account for certain developmental and hereditary phenomena and that a new paradigm is emerging. The two schools are compared with particular reference to Weismann and Waddington, and the phenomenology is re-examined in the light of the new findings.

No description available.

Myzus persicae, more commonly known as green peach aphid, is an important pest in agriculture. Each year it causes great losses to agricultural and horticultural plants. It feeds on sap in the phloem, the main transportation route for plant nutrients. It also transmits more than 100 plant viruses, and as there are still no agrichemicals specifically against viruses, farmers often spray large amounts of pesticides to protect their crops. A group of widely-distributed plant proteins, termed lectins, have been recently studied because of their insecticidal properties against aphids. Snowdrop lectin, Galanthus nivalis agglutinin (GNA) was the first isolated monocot mannose-binding lectin, conferring properties of resistance to sap-sucking or homopteran pests. The aim of the project described in this thesis was to identify and express a novel lectin in Arabidopsis thaliana and to assess the performance of the green peach aphid, Myzus persicae, on both transgenic and wild type plants. A gene encoding a novel mannose-binding lectin was cloned from Allium triquetrum (wild garlic). The full-length cDNA of Allium triquetrum agglutinin (ATA) was 719 bp and contained a 522 bp open reading frame encoding a 173 amino acid polypepetide. Homology analysis showed that ATA has high similarity with other mannose-binding lectins and includes three putative mannose-binding subdomains, which suggests that ATA may also confer resistance against aphids. The pGreen 0029 vector and the 35S CaMV cassette were ligated to produce the expression construct. An expression vector was engineered using the pGreen vector together with CaMV 35S promoter and the novel ATA gene was inserted. Transgenic Arabidopsis thaliana plants were subsequently generated using the Agrobacterium-mediated floral dip method. Six homozygous ATA-transformed lines and one empty vector-transformed control line were obtained using kanamycin selection. Several key growth parameters of the transgenic plants were assessed to demonstrate that expressing ATA causes few III phenotypic changes to the host plant. The transgenic plants were subsequently used for aphid bioassays, including choice and non-choice behaviour tests, Mean Relative Growth Rate test, fecundity test, and survival test. The aphid bioassays revealed that (1) ATA does not change the behaviour of aphids to choose a host; (2) Adults are more sensitive to ATA than nymphs; (3) ATA has significant detrimental effects on aphid fecundity. The results described in the thesis show that the production of Allium triquetrum agglutinin in Arabidopsis confers partial resistance to the aphids, Myzus persicae. It is likely that this lectin in related Brassicaceae plants could be used, perhaps in conjunction with other pest-control measures in agriculture. Key words: Allium triquetrum agglutinin, aphid bioassay, Arabidopsis thaliana, choice test, fecundity test, lectin, non-choice test, mean relative growth rate, Myzus persicae, pleiotropic effects, resistance against aphids, survival test.

No description available.