Biological wastes as plant growth substrate amendments

Globally, agriculture is being intensified with mechanization and increased use of synthetic fertilizers and pesticides. There has been a scaling up of production to satisfy the demands of supermarket distribution. Problems associated with intensification of production, trade globalisation and a larger market demand for greater volumes of fresh produce, include consumers' concern about pesticide residues and leaching of nutrients and pesticides into the environment, as well as increases in the transmission of human food-poisoning pathogens on raw vegetables and in fruit juices. The first part of this research was concerned with the evaluation of a biological control strategy for soil-borne pathogens, these are difficult to eliminate and the chemicals of which the most effective fumigants e.g. methyl bromide, are being withdrawn form use. Chitin-containing crustaceans shellfish waste was investigated as a selective growth substrate amendment in the field, in glasshouse and in storage trials against Sclerotinia disease of Helianthus tuberosus, Phytophthora fragariae disease of Fragaria vesca and Fusarium disease of Dianthus. Results showed that addition to shellfish waste stimulated substrate microbial populations and lytic activity and induced plant defense proteins, namely chitinases and cellulases. Protective effects were seen in all crop models but the results indicate that further trials are required to confirm long-term efficacy. The second part of the research investigated the persistence of enteric bacteria in raw salad vegetables using model food poisoning isolates. In clinical investigations plants are sampled for bacterial contamination but no attempt is made to differentiate between epiphytes and endophytes. Results here indicate that the mode isolates persist endophytically thereby escaping conventional chlorine washes and they may also induce host defenses, which results in their suppression and in negative results in conventional plate count screening. Finally a discussion of criteria that should be considered for a HACCP plan for safe raw salad vegetable production is presented.

Investigations into selective metabolic aspects of bifidobacteria: carbohydrate metabolism, fatty acid biosynthesis and plasmid biology

The gastrointestinal tract (GIT) is a diverse ecosystem, and is colonised by a diverse array of bacteria, of which bifidobacteria are a significant component. Bifidobacteria are Gram-positive, saccharolytic, non-motile, non-sporulating, anaerobic, Y-shaped bacteria, which possess a high GC genome content. Certain bifidobacteria possess the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA) by a biochemical pathway that is hypothesised to be achieved via a linoleic isomerase. In Chapter two of this thesis it was found that the MCRA-specifying gene is not involved in CLA production in B. breve NCFB 2258, and that this gene specifies an oleate hydratase involved in the conversion of oleic acid into 10-hydroxystearic acid. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the colon. Key to the development of such novel prebiotics is to understand which carbohydrates support growth of bifidobacteria and how such carbohydrates are metabolised. In Chapter 3 of this thesis we describe the identification and characterisation of two neighbouring gene clusters involved in the metabolism of raffinose-containing carbohydrates (plus related carbohydrate melibiose) and melezitose by Bifidobacterium breve UCC2003. The fourth chapter of this thesis describes the analysis of transcriptional regulation of the raf and mel clusters. In the final experimental chapter two putative rep genes, designated repA7017 and repB7017, are identified on the megaplasmid pBb7017 of B. breve JCM 7017, the first bifidobacterial megaplasmid to be reported. One of these, repA7017, was subjected to an in-depth characterisation. The work described in this thesis has resulted in an improved understanding of bifidobacterial fatty acid and carbohydrate metabolism, Furthermore, attempts were made to develop novel genetic tools.

A small molecule activator of p300/CBP histone acetyltransferase promotes survival and neurite growth in a cellular model of Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterised by motor and non-motor symptoms, resulting from the degeneration of nigrostriatal dopaminergic neurons and peripheral autonomic neurons. Given the limited success of neurotrophic factors in clinical trials, there is a need to identify new small molecule drugs and drug targets to develop novel therapeutic strategies to protect all neurons that degenerate in PD. Epigenetic dysregulation has been implicated in neurodegenerative disorders, while targeting histone acetylation is a promising therapeutic avenue for PD. We and others have demonstrated that histone deacetylase inhibitors have neurotrophic effects in experimental models of PD. Activators of histone acetyltransferases (HAT) provide an alternative approach for the selective activation of gene expression, however little is known about the potential of HAT activators as drug therapies for PD. To explore this potential, the present study investigated the neurotrophic effects of CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), which is a potent small molecule activator of the histone acetyltransferase p300/CBP, in the SH-SY5Y neuronal cell line. We report that CTPB promoted the survival and neurite growth of the SH-SY5Y cells, and also protected these cells from cell death induced by the neurotoxin 6-hydroxydopamine. This study is the first to investigate the phenotypic effects of the HAT activator CTPB, and to demonstrate that p300/CBP HAT activation has neurotrophic effects in a cellular model of PD.