Clubroot disease, caused by the soil borne microorganism, Plasmodiophora brassicae is a significant disease of cruciferous crops as it results in stunted plant growth and reduced yield. The aim of this thesis was to investigate the effect of environmental factors on the viability of P. brassicae resting spores. Methods of detecting and quantifying the organism were optimised, and several different methodologies were investigated as potential bases for a viability assay. A molecular based seedling assay which quantified the rate of germination and subsequent root infection was determined to be an effective assay system. Investigation of several factors within a laboratory setting demonstrated that calcium stimulated the germination of resting spores, but subsequently reduced the level of infection in Brassica root tissue. Magnesium displayed the same effects but was not as significant as calcium. Ammonium displayed the same effects as calcium and magnesium however the level of ammonium applied was also significant. The commonly reported occurrence of lower incidence of clubroot disease at high pH was supported by finding less spore germination at high pH. Temperature was significant to P. brassicae spore germination and subsequent root infection, with the optimum temperature for germination and infection combined being 18.5-19oC. Low temperature (5oC) reduced spore germination but did not have a significant effect on the zoospores infective capability following germination. Prediction of environmental conditions within field settings was achieved using a simulation model specifically designed to incorporate parameters anticipated to influence P. brassicae viability. Monitoring the DNA equivalent levels of P. brassicae spores in soils over the course of crop growth allowed determination of predictive significant factors, and the results from these investigations supported the findings of the seedling assay. Calcium, magnesium, nitrogen and pH were among the factors significant to the level of P. brassicae spores within the soil.

The pheromone mating pathway in baker’s yeast Saccharomyces cerevisiae enables this organism to initiate a developmental response upon detection of a mating pheromone. Stimulation of the Ste2 receptor by an external trigger molecule in one of the haploid mating cells of yeast MATa, results in the transcriptional induction of a subset of yeast genes. The aim of this study is to convert this system into a readily measurable response, to function as a biosensor. It is crucial for the improved system to be exclusive from the wild-type response. A successful improved system should be able to detect other peptides, such as peptide markers in diseases. An invivo luciferase activity assay utilizing a PFUS1:LUC construct, was developed to report the activation of the pathway. Deletions of both FAR1 and SST2 genes were proven to provide an increased signal to noise ratio. As an attempt to decouple Ste2 and its ligand, three α-factor analogues; N3G, E7 and C3G, were found to stimulate the mating pathway in decreasing order. A modified Ste12 (Ste12mod) transcription factor coupled with a modified pheromone response element (PREmod2) partnership was also designed. A library of Ste12mod was constructed by random mutagenesis of the N-terminal DNA-binding domain. The mutant Ste12 plasmids were transformed into a far1 sst2 ste12 strain complemented with pFUS1(mod2)K. Screening for mutants with an increased resistance to G418 resulted in 17 unique mutations. Further screening by mating and luciferase assays narrowed down to one mutation (F45I) that responds to modified PRE. However, this strain successfully mated with the wild-type, which warrant further modifications and screening to achieve a full orthogonal system. The results in this study not only provided more insight to the physical interaction between Ste12 and PRE, but also the possibility of uncoupling a pathway in an already developed system.

Meiosis is a specialised cell division that results in the formation of four genetically unique haploid daughter cells from one diploid parent cell. This is achieved by one round of DNA replication followed by two rounds of nuclear division. This is in contrast to mitosis, which produces genetically identical diploid daughter cells. Errors during meiosis can result in aneupolidy. In humans, aneuploidy can cause miscarriage and disease such as Patau, Edwards and Down syndromes (trisomies 13, 18 and 21, respectively). Therefore, understanding how meiosis is regulated is of great importance in understanding the causes of aneuploidy and disease in humans. In this thesis, I have used the model organism budding yeast to study how meiosis is regulated in yeast cells, with a view to understanding how meiosis is regulated in human cells. PP2ACdc55 is a highly conserved phosphatase and its role in meiosis was not addressed in any organism before my work. I constructed a meiotic null allele of CDC55 (cdc55-mn) by replacing its promoter with the mitosis-specific PCLB2. By carefully characterising the phenotype of cdc55-mn strains, I showed PP2ACdc55 is crucial for timing the activation of the FEAR network. I have demonstrated that premature activation of FEAR during meiosis (caused by a lack of PP2ACdc55 activity) blocks spindle assembly and nuclear divisions. In cdc55 meiotic null (cdc55-mn) cells, the Cdk-counteracting phosphatase Cdc14 is prematurely released from the nucleolus concomitant with hyperphosphorylation of Net1. I have found that a mutant form of Net1 that lacks 6 of the Cdk phosphorylation sites rescues the meiotic null defect of cdc55-mn cells. Therefore, I have shown that phosphoregulation of Net1 by PP2ACdc55 is essential in order to prevent precocious exit from meiosis I. In my work described in Chapter 4, I isolated mutant alleles of cdc55 that suppressed the spo12Δ dyad phenotype confirming the opposing roles of Net1-phosphorylation by Cdc55 and Spo12 in the FEAR pathway. I also isolated alleles of CDC55 that suppressed the spo11Δ spo12Δ spore lethality. These alleles affected reductional segregation during meiosis I in achiasmate cells but had no effect in wild type cells. Investigating these alleles further might shed insights into mechanisms that work with chiasmata in ensuring efficient monopolar attachment during meiosis I.

Botrytis cinerea is an economically important necrotrophic pathogen which causes disease in hundreds of species of plants during pre- and post-harvest conditions. This thesis investigates the transcriptional responses of B. cinerea and the host Arabidopsis thaliana during the infection through the analysis of microarray and RNA-seq data sets. This work develops techniques for clustering time series expression profiles and identifying direct gene targets using microarray data; and techniques for identifying differentially expressed and differentially spliced genes using RNA-seq data. A clustering algorithm which uses Gaussian process regression to capture the time series structure of microarray data was developed and analysed. Features which are not considered by standard clustering algorithms were added, specifically the ability to include replicate data by using replicate information to inform a prior distribution for noise, and the ability to consider outlier values by using a mixture model likelihood. This algorithm is shown to produce more coherent and biologically meaningful clusters than standard algorithms when applied to publicly available time series data. This algorithm was also used to cluster A. thaliana transcription factors with similar expression profiles during B. cinerea infection. The transcription factors CAMTA3 and MYB108 are known to play a role in the A. thaliana defence response to B. cinerea. Mutant A. thaliana plants were generated which constitutively express the CAMTA3 gene, and these are shown to be more susceptible to B. cinerea infection than wild-type plants. Microarray data sets from mutant CAMTA3 and MYB108 A. thaliana plants were generated and used together with a time series data set of A. thaliana infected with B. cinerea to identify the most likely direct targets for these two transcription factors. Possible regulatory motifs to which these transcription factors bind were also identified. RNA-seq data sets of A. thaliana infected and mock-infected with B. cinerea at three key infection stages were generated. 2,081 novel splice junctions were identi fied for A. thaliana from the data. Differentially expressed genes for A. thaliana and B. cinerea were identified between the key infection stages using existing methods, however these methods are limited to pairwise testing. An improved method using generalised linear models was developed to enable the incorporation of both time and infection stage factors, which identified 12,940 A. thaliana genes differentially expressed due to B. cinerea infection. Different isoforms of A. thaliana genes were identified at a transcript level, at an event level and at a splice junction level. Generalised linear models were then used with the multinomial distribution, which considered both time and infection stage factors, to identify 928 A. thaliana genes which are likely to be differentially spliced due to B. cinerea infection.

Nodules and lateral roots are both key organs for the uptake of nutrients by plants. During nodulation, leguminous plants form root nodules, housing symbiotic Rhizobial bacteria able to fix atmospheric nitrogen, allowing the plant to utilise it. Lateral roots are formed by all plants and allow the root system to be extended laterally, increasing the region of soil from which nutrients may be taken up. Formation of lateral roots and nodules share developmental features such as single cell-type origins of the primordia, and hormonal and nutrient regulatory mechanisms, so it is hypothesised that the evolution of nodulation co-opted elements of pre-existing genetic mechanisms of lateral root formation. To test this hypothesis, Arabidopsis thaliana (non-legume) genes similar to known Medicago truncatula (legume) nodulation genes were screened for phenotypic effects. Mutants of Arabidopsis NODGS and a GRAS-domain SCR-like transcription factor were found to confer lateral root phenotypes, suggesting evidence for the co-option hypothesis. The mutants were examined further using cell-type specific transcriptomics through Fluorescence-Activated Cell Sorting (FACS) to identify genomic components underlying the possible co-option. For the purposes of future research, the translation of FACS transcriptomics to Medicago was evaluated, validating microarray probe design for the most recent genome annotation but also highlighting challenges faced in analysing more complex plant roots. The GRAS-domain SCR-like transcription factor mutant was found to modulate lateral root development through pathways involving the phytohormone gibberellic acid (GA). Treatment with GA rescued some components of the GRASdomain SCR-like transcription factor phenotype, indicating a potential role for the gene in activating GA biosynthesis. A second mutant, of NODGS, was also found to affect lateral root development with some dependence on nitrate level. Existing knowledge suggested a role in root morphogenesis and flagellin-triggered signalling, and this work implies a level of cell-type specificity in gene function.

The herbal extract of Artemisia has been regarded to be anti-hyperglycaemic since olden times and is commonly used by diabetics in Libya. The present work was designed to evaluate, test and determine which fraction or component of the herb had the hypoglycaemic effects in normal and streptozotocin-induced diabetic rats. The plant extract was administered to the animals in their drinking water and body weight, food and fluid intake and urine volume were all monitored daily. Food and fluid intake and body weight gain in normal rats were not altered by treatment with the plant extract but there was a rise in the urine glucose in the first six rats but rats 7, 8 and 9 were not affected by treatment with plant. Urine volume was increased in all rats suggesting Artemisia judaica is a mild diuretic. The streptozotocin-induced diabetic rat model, used in this study, was associated with the characteristic diabetic symptoms of hyperphagia, hyperglycaemia, polydipsia, weight loss and urinary glucose excretion. When a crude aqueous extract of Artemisia was given in their drinking water, it had little effect on these symptoms after 10 days of treatment. Urine glucose was reduced in the last two days and ketones in the urine were abolished by this treatment. Diabetes mellitus is known to affect many and varied parameters in rat liver. Insulin, biguanides and sulphonylureas are known antidiabetic diabetic treatments. Artemisia judaica extract was tested for its effect on hepatic steroid metabolism and glycogen phosphorylase a activity in comparison with the above drugs. Clearly Artemisia does act as an insulin-mimetic in these assays by reversing all the effects produced by the administration of streptozotocin. In particular the changes in the enzyme activities of cytochrome P-450 (2E1, 2B and 2C) on androst-4-ene-3, 17-dione metabolism are all reversed by the administration of Artemisia extract to diabetic rats.

In recent decades, the ecological impact of recreation in woodlands and forests has been a subject of considerable world-wide interest. However, there are few studies examining the effects of recreation on woodland vegetation, soils and fauna in Britain. This thesis identifies recreational trampling as a major contributor in facilitating ecological change in urban fringe semi-natural ancient temperate woodlands of Warwickshire, England. Relationships with trampling intensity are generally curvi- linear, suggesting that the rates of damage are most rapid at initial stages of trampling. Biotic communities are shaped so that their structure and diversity is related to the type, intensity and frequency of impact. The impact of trampling on vegetation is the most precise indicator of recreational use. Multi-variate analyses indicates that trampling is the primary organisational gradient operating on ground vegetation, with trail centres dominated by secondary plant associations at equilibrium with the trampling pressure. Trail margins are dominated by vegetation that is tolerant of low levels of trampling and high rates of competition. Experimental trampling experiments show that the ecological carrying capacity of woodlands for recreation are lower than previously thought; from below 150 people per year in Rubusfruticosus agg. and Pteridium aquilinum dominated stands to below 75 people per year in coniferous stands with Hyacinthoides non-scripta ground flora. The ability of vegetation to tolerate trampling is related to plant anatomy, morphological adaptations, plant strategies, growth rate, position of the perennating bud, environmental conditions such as canopy density and is more a function of the ability to recover from trampling rather than to resist. By virtue of their delicate morphology, stands dominated by shade tolerant species are the most vulnerable to trampling. Increases in soil compaction and decreases in pore space and oxygen content are recognised as important in shaping woodland vegetation and fauna, and the reduction in soil inhabiting invertebrate and micro-organism populations have consequences for woodland processes. A bioindicator index to assess soil damage is provided using Acari body length. Models summarising the ecological changes associated with trampling and the ecological carrying capacity of woodlands are provided, along with a woodland management checklist and an index of vulnerability for resource managers to assess the potential of woodland stands to withstand recreational use.

Circadian clocks involve feedback loops that generate rhythmic expression of key genes. Molecular genetic studies in the higher plant Arabidopsis theliene have revealed a complex clock network. We begin by modelling the first part of the Arabidopsis clock network to be identified, a transcriptional feedback loop comprising TIMING OF CAB EXPRESSION 1 (TOCl), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). As for many biological systems, there are no experimental values for the parameters in our model, and the data available for parameter fitting is noisy and varied. To tackle this we construct a cost function, which quantifies the agreement between our model and various key experimental features. We then undertake a global search of parameter space, to test whether the proposed circuit can fit the experimental data. Our optimized solution for the Arabidopsis clock model is unable to account for significant experimental data. Thanks to our search of parameter space, we are able to interpret this as a failure of the network architecture. We develop an extended clock model that is based upon a wider range of data and accurately predicts additional experimental results. The model comprises two interlocking feedback loops comparable to those identified experimentally in other circadian systems. We propose that each loop receives input signals from light, and that each loop includes a hypothetical component that had not been explicitly identified. Analysis of the model predicts the properties of these components, including an acute light induction at dawn that is rapidly repressed by LHY and CCAL We find this unexpected regulation in RNA levels of the evening-expressed gene GIGANTEA (GI), supporting our proposed network and making GI a strong candidate for this component. We go on to develop reduced models of the Arabidopsis clock to aid conceptual understanding, and add a further proposed feedback loop to develop a 3-loop model of the circadian clock. This 3-loop model is able to reproduce further key experimental data.

Studies on the molecular organisation and properties of the first three enzymes of pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPTase), aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase), in various organisms have been reviewed. The molecular organisation of these three enzymes has been investigated in wheat using gel filtration chromatography. CPSase activity could not be detected in gel filtered extracts and in crude extracts from wheat seedlings was shown to be highly labile. ATCase and DHOase activity was detected and the molecular weights of these enzymes were estimated to be 1.03 x 105 ( 1.4 x 10^4) and 8.6 x 10^4 (6 x 103), respectively. At no time during these investigations were high molecular weight species (consistent with the presence of a multifunctional complex containing these enzymes) detected. During the course of these investigations, a protease was detected which was shown to co-migrate with ATCase and DHOase activities. This protease was shown to be insensitive to the serine protease inhibitor PMSF, but was partially inactivated by iodoacetamide, consistent with the protease being a member of the cysteinyl protease family. Inclusion of iodoacetamide during chromatography also failed to reveal high molecular weight species of these enzymes. Antisera were raised against purified wheat ATCase and were characterised by their ability to inactivate the enzyme. These antisera were then used to probe western blots of crude extracts from wheat seedlings and screen a wheat cDNA expression library to ATCase sequences. Western blotting failed to show any immunoreactive species in extracts prepared under conditions which suppressed protease activity (SDS, -mercaptoethanol), although a low molecular weight (approximately 3.7 x 10^4) ATCase could be detected in samples obtained after gel filtration chromatography. Antisera also showed very little cross-reactivity with the ATCase from E.coli a result consistent with studies on the enzyme from B. subtilis.

Four endemic medicinal plants from the Yucatan peninsula belonging to genera with little pharmacological and phytochemical reported information and used for medicinal purposes by local communities were selected. The species selected included Jacquinia flammea Millsp. ex Mez, Sideroxylon foetidissimum Jacq. subsp. gaumeri, Serjania yucatanensis Standl., and Serjania adiantoides Radlk. The root, stem/bank and leaves of each plant species were extracted using ethanol and the resulting crude extracts were tested for their cytotoxic effect using the modified MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay followed by a bioassay-guided fractionation of the most active extracts in order to identify the active metabolites. The initial cytotoxic evaluation against HeLa cells at two fixed concentrations (100 and 33.3 µg/mL) identified the root extracts f J. flammea, S. foetidissimum subsp. gaumeri and S. yucatanensis, and the stem/bank extract of S. adiantoides as the most active extracts. The crude extract of roots of J. flammea was subjected to solvent partition using solvents of ascending polarity (pet. ether, CHCI3, EtOAc, BuOH and water). The resulting fractions were tested for their cytotoxic activity. The water fraction of the solvent partition showed the strongest activity against HeLa cells (IC50 = 28.61 ± 2.27 µg/mL). When tested against RAW 264.7 cells, the water fraction also showed significant activity (IC50 = 10.60 ± 1.83 µg/mL). The water fraction was subjected to chromatographic fractionation using open silica gel columns resulting in the isolation of a saponin as the most active metabolite against RAW 264.7 cells (IC50 = 4.76 ± 0.32 µg/mL). The isolated compound was identified using 1D (1H and 13C and DEPT-135) and 2D (COSY, HMBC, HSQC and NOESY and ROESY) NMR and mass spectrometry analysis as sakurasosaponin. The molluscicidal and antifungal activities of sakurasosaponin have been reported but no studies on its cytotoxic activity have been previously reported. The crude extract of roots of S. foetidissimum subsp. gaumeri was subjected to solvent partition using solvents of ascending polarity (pet. ether, CHC13, EtOAc, and BuOH). The resulting fractions were tested for their cytotoxic activity. The BuOH extract of S. foetidissimum subsp. gaumeri showed the strongest activity against RAW 264.7 cells (IC50 = 35.12 ± 4.32 µg/mL) and it was subjected to further chromatographic fractionation using open silica gel columns yielding mixtures of saponin-containing fractions. The crude extract of roots of S. yucatanensis was subjected to solvent partition using solvents of ascending polarity (pet. ether, CHCI3, EtOAc, and BuOH). The resulting fractions were tested for their cytotoxic activity. The crude extract of S. adiantoiodes did not show cytotoxic activity when tested against RAW 264.7 cells.