Brassica vegetables, such as broccoli, are characterised by the presence of sulphur-containing compounds, termed glucosinolates, which are associated with potential health benefits for humans. Glucosinolates are metabolised in the gut by members of the gut microbiota, producing biologically active breakdown products, such as isothiocyanates. The effects of consuming Brassica on the composition of the gut microbiota, and the bacterial mechanisms employed for glucosinolate metabolism, are unclear, and forms the basis of the research presented in this thesis. Culturing human faecal microbiotas in an in vitro batch fermentation model identified the bacterial-mediated reduction of glucoraphanin and glucoiberin to glucoerucin and glucoiberverin, respectively. An Escherichia coli strain was found to exhibit reductase activity on glucoraphanin and the broccoli-derived compound S-methylcysteine sulphoxide, through the reduction of the sulphoxide moiety. Within this fermentation model, the relative proportions of members of the genus Lactobacillus were found to significantly increase when the microbiota was repeatedly exposed to a broccoli leachate, and 16S rDNA sequencing identified these as L. fermentum. Metabolite analysis detected relatively high concentrations of lactate and short-chain fatty acids when faecal microbiotas were cultured in the presence of broccoli leachates, compared to a glucose control media. A human dietary study investigating the effects of Brassica on the microbiota composition revealed a significant association between dietary Brassica and changes to the relative proportions of a number of bacteria, many of which belong to the Clostridiales. Further studies are required to reveal the nature of this association, and whether the presence of glucosinolates may have been a factor. The work presented in this thesis highlights the strong connection between diet, the gut microbiota, and the potential health benefits to the host that may be derived from the bacterial metabolism of dietary compounds.

All living organisms contain genes. Turning these genes on and off at the appropriate times controls much of an organism’s development and its responses to environmental conditions. In recent years, chromatin has emerged as an important player in orchestrating gene regulation. This thesis focuses on the role of chromatin in the maintenance of gene expression states and their inheritance through cell division. FLOWERING LOCUS C (FLC) in the plant Arabidopsis thaliana is repressed by the prolonged cold of winter, and repression is maintained in subsequent warm conditions. The molecular complexes involved in modulating FLC chromatin are vital for FLC regulation and are conserved among plants and animals, making FLC a paradigmatic system for understanding of the role of chromatin in gene regulation. After cold, FLC chromatin adopts a distinct configuration. In this study, experiments are used to show that this local chromatin ‘state’ instructs its own inheritance through cell division in growing plants. Thus, memory of winter cold is stored in the chromatin of the FLC gene. Mathematical models developed in this work focus on understanding how chromatin states are maintained and also re-established after DNA replication. Minimal models are used to investigate if a particular set of interactions between chromatin and chromatin-modifiers can give rise to the qualitative behaviours, and quantitative results that are observed experimentally. Models developed here make predictions for the FLC system, and more generally show how cis and trans determinants of gene expression can be integrated by chromatin. The role of transcription in determining chromatin states is also examined experimentally by studying the chromatin-associated protein LHP1. LHP1 is required for FLC repression and binds to modified histones associated with repressed FLC chromatin. In this work, it is shown that LHP1 also binds RNA and that this is important for its in vivo function.

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Understanding the mechanisms regulating root development under drought conditions is an important question for plant biology and world agriculture. This thesis examines the effect of osmotic stress on the plant hormones abscisic acid (ABA), cytokinin and ethylene responses and how they mediate auxin transport, distribution and root growth via PIN proteins Root growth is reduced under osmotic stress, and ABA responses increase. Root growth can be rescued by inhibiting ABA biosynthesis, indicating its critical role in the regulation of growth under stress. There was also a reduction in cytokinin signalling under stress. The inhibition of root growth under osmotic stress does not require ethylene signalling, however auxin can rescue growth. Osmotic stress also modulates auxin transporter levels, particularly PIN1, which regulates auxin transport to the root tip. As PIN1 levels are reduced under stress in an ABA-dependent manner, overriding the ethylene effect on PIN1 levels, and auxin responses decrease under stress, I present the hypothesis that ABA is limiting auxin transport to the root under stress to reduce growth. However, the interplay between ABA, ethylene, cytokinin and auxin is tissue-specific, with the result that PIN1 and PIN2 differentially respond to osmotic stress. Combining experimental analysis with extensive literature searches allowed the systematic construction of interaction networks, incorporating the known interactions between the hormones and stress. This network analysis reveals that ABA regulates root growth under osmotic stress conditions via interactions with cytokinin, ethylene and auxin demonstrating complicated non-linear relationships and providing a framework for further kinetic modelling. Kinetic modelling (using differential equations to simulate these interactions) of ethylene and ABA effects on PIN1 levels reveals that the hormones most likely act on the same pathway to regulate PIN1 levels. The work presented here provides novel insights into how root growth is regulated by hormones under drought and osmotic stress conditions.

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