The Target of Rapamycin Complex 1 (TORC1) is a key and conserved regulator of cell growth and proliferation. The xenobiotic compound rapamycin is a potent inhibitor of TORC1 in yeast. The EGO complex, a non-essential activator of TORC1 is required for recovery of cells following rapamycin treatment. Why? Here, we find that rapamycin is in fact only a partial inhibitor of yeast TORC1; wild-type cells are able to maintain slow proliferation in the presence of high concentrations of the drug (i.e. concentrations multiple times the minimum inhibitory concentration). We find that this residual, rapamycin-insensitive, proliferation is dependent on the EGO complex and on TORC1 activity. We show that the ability of cells to maintain slow proliferation in the presence of rapamycin dictates their ability to recover. We find that rapamycin is not actively detoxified in yeast; instead, rapamycin is cleared by dilution-by-proliferation. The cell-associated intracellular pool of rapamycin is stable, decreasing only very slowly following washout of the drug and only diminishing at the rate of cell proliferation. The rapamycin-insensitive growth rate also persists long after rapamycin washout, indeed, until cells recover from the drug. The rapamycin-insensitive growth rate is not only able to quantitatively account for the observed kinetics of recovery from the drug in wild-type cultures, but also explains the severity of the ego- recovery defect. We contributed to a large-scale genetic screen seeking mutants that, like ego- mutants, fail to recover from rapamycin treatment. We find that loss of any one of 10 proteins identified results in a rapamycin recovery defect and a slow rapamycin-insensitive growth rate. Our data propose important or novel roles of the core HOPS/CORVET complex, threonine biosynthesis, Vps15p, Vsp34p, Ccr4p and Dhh1p activities in modulating the activity or efficiency of TORC1. Overall our results reveal that rapamycin is only a partial inhibitor of yeast TORC1, that persistence of the drug within the cell limits recovery and that rapamycin is not actively detoxified in yeast. Instead, recovery occurs due to dilution-by-proliferation and distribution of the drug among an increasing number of progeny cells. We also identify a set of potentially novel regulators of TORC1 activity.

Absence seizures (ASs) are non-convulsive epileptic events which are common in pediatric and juvenile epilepsies. They consist of EEG generalized spike-and-wave-discharges (SWDs) accompanied by an impairment of consciousness and are expressed within the thalamocortical network. This thesis initially focused on investigating the modulation of ASs by two serotonin receptors (5-HTRs), 5-HT2A and 5-HT2C, in a polygenic (i.e. Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and a pharmacological (i.e. γ-hydroxybutyrate, GHB) model of ASs. It was found that, in GAERS, pharmacological activation of 5-HT2A/CRs blocked ASs, whereas 5-HT2AR antagonists increased seizure length. However, experiments on the GHB-model revealed that GHB induced not only ASs but also a period of sedation/hypnosis, a behavioural state that had been neglected in the literature. Thus, the rest of this thesis was devoted to further characterizing the GHB-model. The main result was that GHB-elicited ASs can be distinguished at the level of both EEG and behaviour. In vivo characterization of thalamic firing during GHB-elicited ASs and hypnosis via silicon probes in freely moving animals revealed that both states were accompanied by a decrease in firing rate. In particular, contrary to what was predicted by in vitro and in vivo experiments under neurolept anaesthesia, T-type Ca2+ channel-dependent burst firing in thalamic neurons was found in <10% of spike-and-wave complexes of SWDs. The prevalent activity of nucleus reticularis thalami neurons during ASs was either silence or tonic firing. Indeed, thalamic application of the potent T-type channel antagonist, TTAP-2, by reverse microdialysis did not affect GHB-elicited ASs. Finally, the development of an algorithm to classify GHB-elicited ASs demonstrated that the spectral properties of SWDs can be used to discriminate hypnosis and SWDs. Moreover, spectral coherence can be used in different experimental models of ASs to characterize SWDs according to their waveform regularity.

The interface between urban combinations and associated industry with fragile ecosystems delivering significant ecosystem services denotes one of the critical frontiers for ecological genomic investigation. The major issue when evaluating diffuse pollution generated at this interface revolves around the possible interactions between mixtures of contaminates that individually remain below trigger level but together may result in significant environmental impact. To determine whether mixture effects need to be considered, it is essential to define geochemical parameters by performing a survey for major classes of contaminates and to evaluate their penetrance into the food chain. The coastal marine environment of the Saudi Red Sea is subject to direct and indirect influences of major populations and industrial facilities found along the coast such as those discovered in proximity to Jeddah City in Saudi Arabia. Sampling of both sediment and sea water was performed at contrasting sites representative of near-shore with off-shore locations. Possible food-chain transference of any contaminates was evaluated by sampling fish (L.nebulosus) and plankton at the off-shore sites. Biomarkers are mostly useful in the evaluation of progressive diseases that apparent their symptoms long after exposure to the initiating factor. In such cases, traditional early warning symptoms of developing disease may be lacking. Thus, detection of earlier events can provide a valuable timely warning of risk. It is important to identify and address the growing environmental problems being faced by the community and address it before it takes the shape of an epidemic. To assess toxicity of single and paired metals to the nematode C. elegans, toxicity tests were designed to first determine the impact of single metal exposure Copper, Zinc and Aluminium and then nematodes were exposed to paired combinations. Exposures with paired metals showed a variety of interactions which ranged from antagonistic to synergistic effects.

The enteric bacterium \(Escherichia\) \(coli\) is exposed to nitric oxide (NO) in its oxygen-limited environment. Various transcription factors regulate gene expression to provide protection against nitrosative stress, but their respective roles remain controversial. Key questions answered in this thesis were whether S-nitrosylated OxyR directly regulates the expression of NO-regulated genes; whether NsrR is required for the synthesis of an important protective system; and whether FNR is a physiologically relevant sensor of environmental NO. Transcription from the NO-activated \(hcp\) promoter was almost totally dependent upon a functional FNR protein, which is inactivated by oxidative stress, but unaffected by deletion of the \(oxyR\) gene. This indicated that the effects of an \(oxyR\) mutation on resistance to nitrosative stress are indirectly due to inactivation of FNR rather than to direct activation of \(hcp\) transcription by S-nitrosylated OxyR. The NO-sensitive repressor, NsrR, is not essential for protection against nitrosative stress. Nitric oxide produced during nitrite reduction had no effect on the ability of FNR to activate or repress gene expression, which is primarily due to NO sensing by NsrR, not by FNR. Some NO accumulates in the \(E. coli\) cytoplasm even in mutants that lack known sources of NO. The source of most of this residual NO is the NsrR-regulated protein, YtfE. Contrary to the proposal by others that YtfE repairs iron-sulfur centres by replacing iron atoms released during nitrosative stress, it is proposed that YtfE releases NO from nitrosylated iron-sulfur proteins, and that Hcp reduces this NO to the less toxic N2O.

PolyA signals located at the 3’ end of eukaryotic genes drive the cleavage and polyadenylation reaction to the nascent pre-mRNA. Although these sequences are expected only at the 3’ end of genes, we found that strong polyA signals are also present within the 5’ untranslated regions (UTRs) of many Drosophila melanogaster mRNAs. Although the polyA signals in 5’ UTRs show little activity of triggering 3’ end processing in the endogenous transcripts, they are very active when placed at the 3’ end of reporter genes. We further investigated these unexpected observations and discovered that both these novel polyA signals and standard polyA signals become functionally silent when they are positioned close to transcription start sites in either Drosophila or human cells. This suggests that the transcriptional stage when the polyA signal emerges from the polymerase II (Pol II) transcription complex could determine whether a putative polyA signal is recognized as functional. The data suggest that this mechanism, which probably prevents cryptic polyA signals from causing premature transcription termination, depends on low Ser2 phosphorylation of the C-terminal domain of Pol II and inefficient recruitment of processing factors.

The first chapter in this thesis describes research on the asymmetric [1,2]-sigmatropic rearrangement of benzylic ammonium ylids. Our group previousely developed method showing that DMSO as solvent, and BTPP as base, in the presence of 5Å molecular sieves, dramatically improves the yield of the reaction. Hence, we applied the developed the method using8-(–)-phenylmenthol and (2S)-camphorsultam as chiral auxiliaries. In the second chapter, a new application of Pd-catalysed allylation is reported. This enabled the synthesis of (30)of branched sp3-functionalised sulfonamides, a compound class for which few reported methods exist. By reacting benzyl sulfonamides with allylic acetates in the presence of Pd0 catalysts and a base, at room temperature, direct allylation was efficiently performed, yielding products that are analogues of structural motifs seen in biologically active small molecules. The reaction was performed under mild conditions and could be applied to nanomolar sigma-receptor binders, thus enabling a late-stage functionalisation and efficient expansion of drug-like chemical space. The third chapter described a synthesis of benzosultam without recourse to transition-metal catalysis, or stoichiometric amounts of organometallic building blocks. Iodomethane sulfonylamide adds to benzyne (generated using fluoride sources), and then the formed intermediate undergos an intramolecular cyclisation to afford sultam. Using this method that procceds under simple reaction conditions, (11) benzosultams were synthesised in modest yield.

The family Hantaviridae, of the Bunyavirales order, contains many important human pathogens of which Puumala virus (PUUV) is the most widely distributed member in Europe. It causes nephropathia epidemica, a milder form of haemorrhagic fever with renal syndrome and mortality rates of up to 1% have been reported. They are enveloped viruses, with a tripartite single-stranded negative sense RNA genome, that replicate solely in the cell cytoplasm. Several factors have been proposed to play a role in hantavirus pathogenicity, including regulation of innate immune responses, cell signalling and enhancement of endothelial cell permeability. The work presented in this thesis describes biological and molecular characterisation of the mechanisms behind a hantavirus infection. Transcriptome analysis was a valuable tool that allowed the investigation of the broader picture of the effect of PUUV on the host cell. 549 and as many as 7,283 genes were differentially expressed at 24 and 48 hours post infection, respectively, in PUUV-infected cells, revealing extensive transcriptional change. By 48 hours normal cellular function appeared severely disrupted. Most genes involved in mitochondrial functioning were down-regulated, suggesting a reduced cellular energy level. Dysregulation of an important signalling hub such as mitochondria might have a more global impact on cellular functions, consistent with findings in this study. Intrinsic apoptosis pathway, which is mediated by mitochondria, appeared inhibited. Whereas, death receptor signalling was activated. Pathways associated with actin formation, organisation and signalling also appeared inhibited. Members of Rho family of GTPases, which are key regulators of actin dynamics, were down-regulated overall. Furthermore, integrin signalling, which mediates Rho GTPase activity, was also inhibited. Immunofluorescence studies revealed marked morphological changes in mitochondria and substantial remodelling of the actin cytoskeleton. Further analysis revealed a direct interaction between PUUV N protein and anillin, a scaffolding protein that mediates formation and organisation of actin filaments, suggesting a potential novel mechanism behind actin cytoskeleton reorganisation. Biological interferon (IFN) assays enabled the identification of two IFN antagonists encoded by PUUV, the cytoplasmic tail of the Gn glycoprotein and the non-structural protein, NSs. The Gn tail inhibited type I IFN induction at the level of TRAF3-TBK, in agreement with previous studies on other hantaviruses. Whereas, NSs was found to block IFN induction downstream of IRF3, suggesting it was able to disrupt transcription or translation. Utilising immunofluorescence and chromatin immunoprecipitation methods, it was found that PUUV NSs possessed a potential mechanism to inhibit transcription by blocking serine 2 phosphorylation at the C-terminal domain of RNA polymerase II in a similar manner to the previously described Bunyamwera virus NSs. The data presented in this thesis illustrates the broad range of mechanisms employed by PUUV to alter cell function to aid virus replication and subvert innate immune responses.

Transposable elements (TEs) are almost ubiquitous components of eukaryotic genomes that have long been considered solely deleterious or ’junk DNA’. They are split into two main forms, retro-transposons and DNA transposons, depending on the method of replication employed. Hosts have developed strategies for combating TEs including RNAi, methylation and copy number con-trol. TEs have also evolved ways of persisting in the genome in order to survive, such as target site specificity. Two additional ways which may be utilised by TEs, positive selection and horizontal transfer, were investigated here primarily using the budding yeasts in the Saccharomyces sensu lato complex. These species typically contain up to five families of retrotransposons, designatedTy1-5, and multiple subfamilies, all of varying transpositional activity. Discoveries of insertions evolving under positive selection and providing benefits to their hosts have been sporadic and serendipitous findings in a number of organisms. Full genome screenings for such insertions are rarely published, despite the impact TE insertions have upon their hosts. A population genomics approach was performed to address this issue in the genomes of Saccha-romyces cerevisiae and sister species S. paradoxus. Signatures of positive selection acting upon Ty insertions were identified using Tajima’s statistical D test. Neighbouring genes were also analysed to ascertain the true target of selection where hitchhiking linked the two. A subset of LTR-gene pairings were explored using qPCR in order to identify any effects on host gene expression the occupied loci may cause. Two genes displayed significantly increased levels of expression, which may be due to the presence of positive selection candidate LTRs, which in turn may contribute to improving host fitness. This thesis further documents the systematic screening for Ty-like elements of all available genomes of budding yeast and related species. Extensive phylogenetic analyses estimated evolutionary relationships and possible horizontal transfer events of elements between the species. Evidence for in excess of 75 horizontal transfer events was uncovered here, around half of which were successful in propagating in new genomes. The occurrence of horizontal transfer of TEs in the genomes of budding yeast is therefore far more common than previously documented. During screening of genomes, a further potential method of avoiding host defences was uncovered. The divergence of the highly active Ty4 family, which coincided with population isolation of multiple Saccharomyces species into subfamilies, was surprising given previous reports of this family being of particularly low activity. Such events are rarely recorded in eukaryotic genomes, and may also illustrate the compulsive spread of a new subfamily via horizontal transfer. The investigations reported here represent the first genomic screening of Ty insertions in Saccharomyces for signatures of positive selection, and an updated, comprehensive search for evidence of HT between species of budding yeast. Both may act as methods for TE families to persist in the genomes of their hosts, and represent far more than simply ’junk DNA’.

The relationship between ecosystem functioning and the biodiversity of microorganisms has been a central focus of recent ecological studies, and fungi are considered to play a key role in this relationship. For example, the Mycorrhizae’s association with two-thirds of terrestrial plants makes them one of the most common forms of symbiosis on Earth. Although Arbuscular Mycorrhizal Fungi (AMF) have been shown to be important in nutrient cycling, soil stability and enhancing plant growth by increasing root mycelia, much of the information we have on them is restricted to a small sample of woodland and grassland habitats. Consequently, the mechanisms regulating the diversity and community structure of fungi remain poorly studied across many habitats. This is particularly true for salt marshes, which are key conservation priority habitats in many countries, including the UK. Using the latest sequencing technologies, this work examined the community ecology of fungi across six different salt marsh habitats over two locations in Essex and Lancashire, UK and related this to local environmental variables and sediment nutrient statuses. In addition, by linking with other datasets from the same sites, this study also examined the role of biotic factors that are likely to influence the relationship of rhizosphere fungal communities with each other. Although a range of local abiotic factors significantly influenced the composition of fungal communities within each salt marsh, at larger scales, the compositions of these fungal communities were significantly affected by the pattern of the biotic factors that also differed over seasons. Indeed, the ability of these variables to predict fungal richness and abundances differed greatly between sites, suggesting that drivers of fungal II community structure may be site-specific to some extent. Indeed, fungal richness in relation to abiotic or biotic factors explained considerably different amounts of variation in each site, and generalised poorly to other sites. Therefore, it is possible now to argue against deriving general environment-diversity relationships without empirical validation in multiple sites. Finally, functionally similar fungi ponded similarly to the environmental gradients, suggesting a possible disconnect between functional redundancy and resilience in microbial communities.

The ogt gene encodes an O\(^6\)-alkylguanine DNA alkyltransferase, which is reported to repair DNA against methylation damage caused by reactive nitrogen species, which are generated during an anaerobic respiration in a presence of nitrate. This study examined transcription activation by NarL at the ogt promoter using biochemical techniques with various semi-synthetic ogt promoters. The ogt promoter has two crucial DNA sites for NarL dimers at positions -78.5 and -45.5 relative to the transcript start site. An interaction between NarL and αCTD was investigated using site-specific mutagenesis. Locations and orientations of NarL and RNA polymerase in the transcript initiation complex were also confirmed in this study. It was found that NarL, at position 45.5, is located on the different DNA helical face from αCTD, which binds to the minor groove immediately upstream the -35 element. This unrecognized promoter architecture allows residue 273 of αCTD to interact with residue 178 of NarL.