Modelling and reconstruction of genetic regulatory networks has developed in a wide field of study in the past few decades, with the application of ever sophisticated techniques. This thesis looks at how models for genetic networks have been developed from simple Boolean representations to more complicated models that take into account the inherent stochasticity of the biological system they are modelling. Statistical techniques are used to help predict the interaction between genes from microarray data in order to recover genetic regulatory networks and provide likely candidates for interactions that can be experimentally verified. The use of Granger causality is applied to statistically assess the effect of one gene upon another and modifications to this are presented, with bootstrapping used to understand the variability present within the parameters. Given the large amounts of data to be analysed from microarray experiments, clustering techniques are used to help reduce the computational burden and novel algorithms are developed to make use of such clustered data. Variability within clusters is also considered, by developing a novel approach with the use of principal component analysis. These algorithms that are developed are implemented with an observed dataset from Xenopus Laevis that has many genes but few timepoints in order to assess their effectiveness under such limited data. Predictions of likely interactions between genes are provided from the algorithms developed and their limitations discussed. Using extra information is considered, where a further dataset of gene knockout data is used to verify the predictions made for one particular gene.

Meiotic recombination in eukaryotes requires two orthologues of the E. coli RecA proteins, Rad51 and Dmc1. Both genes play an important role in the binding of single strand DNA, homology search, strand invasion and strand exchange resulting in Holliday junctions which are resolved into crossovers or non-crossovers events. Even though both genes are well characterized in a variety of organisms including plants, very little information is available from hexaploid wheat. In most diploid plant species, deletion of either the RAD51 or DMC1 orthologues leads to sterility but wheat being a polyploid, offers a unique opportunity to examine the effects of the deletion of specific homoeologue, while maintaining a degree of fertility. The transcript expression profiling of RAD51 and DMC1 genes in Arabidopsis, rice and wheat using available microarray databases indicated higher levels of expression in mitotically and meiotically active tissues compared to other tissues. However, the possible function of the DMC1 gene in mitotic-active tissues needs to be investigated further. Previously cDNA sequences of TaRAD51 and TaDMCl of hexaploid wheat were cloned and reported. In this study, it has been demonstrated that the reported TaRAD51A1 and TaRAD51A2 cDNA sequences are (D) and (A) homoeologues of TaRAD51 respectively and TaDMCl cDNA sequence is (D) homoeologue of the TaDMC1. This study also found that the amino acid sequences and evolutionary relationships of RAD51 and DMC1 cDNA homoeologues are highly conserved across eukaryotes. Functional characterization of TaRAD51 and TaDMCl gene homoeologues was undertaken in planta using Forward Genetics, Reverse Genetics and Complementation methods. Forward and Reverse Genetic screening of a subset of a Highbury mutant population could not identify any mutants that have deletions in TaRAD51 and TaDMC1 genes. However, Reverse Genetics screening of Paragon mutant population identified mutant lines that tested as having deletions for all the three homoeologues of TaRAD51 and TaDMCl. However, most likely due to high mutational load and a deleterious phenotype, only a few mutant lines survived. Phenotypic and cytogenetic analysis indicated the probable functional redundancy of TaRAD51 (B) homoeologue in meiosis, although the unknown size of the deletion and limited phenotype makes it impossible to completely certain of this. The single mutants for TaDMC1 (B) and (D) indicated a reduction in pollen viability and ear fertility compared to wild-type. The cytological examination of these mutants indicated low levels of abnormal diakinesis, resulting in the formation of dyads. However, the single mutants were still able to produce normal tetrads. This suggests that there is a possible dosage effect of these homoeologues in hexaploid wheat. Unless deletion lines for the (A) and (D) homoeologues of TaRAD51 and (B) homoeologue of TaDMC1 can be recovered and characterized the above assumptions will remain inconclusive. The results of complementation assays using over-expressing CaMV35S::TaRAD51(D)±GFP constructs demonstrated a very low (-14% and -2%, respectively, with +GFP and -OFP constructs) functional complementation in terms of seed set compared to 0% in homozygous Atrad51 mutants. One explanation of these results is that the wheat genes are not complete functional orthologues for the inactivated Arabidopsis genes. The functional complementation experiments could not be performed for TaDMC1 gene because of time limitation, although the transformants were produced in AtDMC1/atdmc1 background. Finally, overexpression of the TaRAD51 gene suggests 2-fold increase in genetic distances in Arabidopsis using CaMV35S::TaRAD51(D) construct. This was done by crossing the appropriate transformant with fluorescent tetrad lines. However the results need to be confirmed by a large scale analysis.

The control of pollen viability and release is of major commercial importance in the development of crops for hybrid seed production and selective breeding. It has been shown that key transcription factors in Arabidopsis particularly MALE STERILITY1 (MS1), are functionally conserved in rice (Li et al., 2011), therefore extending this comparative analysis and controlling fertility in temperate cereals, such as barley, is the long term goal of this project. Although anther and pollen development of barley seems morphologically similar to Arabidopsis, the genes involved and how they are regulated are currently unknown. Arabidopsis MS1 is a tapetum specific transcription factor which is expressed exclusively from the tetrad stage to early microspores release. Identification and accurate staging of barley anther development is essential for expression analysis and functional characterisation of genes involved in pollen development. Therefore, a complete morphological study of barley development was conducted. External characteristics have been described in parallel to anther development in order to predict anther stages by the observation of external stages phenotypic traits. Characterization of the barley orthologue of MS1 (HvMS1) has been conducted. Recently a new grass genome has been released, Brachypodium distachyion. This new resource has been used to aid primers design alongside the rice OsPTC1 sequence, the orthologue of MS1 (Li et al., 2011). Genome sequencing has indicated that the Brachypodium genus is more closely related to wheat and barley than it is to rice, Due to the close relationship between Brachypodium and barley, this new grass has been used as intermediary to identify the OsPTC1 orthologue in barley as well as downstream MS1 targets. A highly similar sequence to OsPTC1 was found in Brachypodium, Bradi4g31760. This new gene, as a result of its similarities to OsPTC1, was considered as its putative orthologue gene in Brachypodium. Therefore, the most conserved areas between OsPTC1-Bradi4g31760 were used for primers design to successfully amplify equivalent gene in barley (HvMS1). The characterization of this barley gene showed a similar expression pattern to the MS1 putative orthologue in Arabidopsis of tapetum specific expression. In addition, RNAi silencing of this gene has revealed that it is essential for the normal development of pollen, with a lack of viable pollen produced in the putative HvMS1 silenced transgenic lines.

The human BTG/TOB protein family comprises six members (BTG1, BTG2/PC3/Tis21, BTG3/Ana, BTG4/PC3B, TOB1/Tob, and TOB2) that display anti-proliferative activity in a number of cell types. They are characterised by a conserved N-terminal BTG domain that mediates interactions with the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylases. It was unclear whether the anti-proliferative activity of the BTG/TOB proteins was mediated through interactions with Caf1a (CNOT7) and Caf1b (CNOT8). To address this we further characterised the amino acid residues located along the BTG2 and TOB1 interaction surface with Caf1a (CNOT7)/Caf1b (CNOT8) to identify residues required for the interaction. We then analysed the role of BTG2 and TOB1 in the regulation of cell proliferation, translation and mRNA abundance using a mutant that is no longer able to interact with Caf1a (CNOT7)/Caf1b (CNOT8). We conclude that the anti-proliferative activity of BTG/TOB proteins is mediated through interactions with the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase enzymes. We also demonstrate that recruitment of BTG2 and TOB1 to mRNA leads to reduced protein levels and mRNA degradation. Furthermore, we show that the regulation of mRNA abundance and protein levels is dependent on Caf1a (CNOT7)/Caf1b (CNOT8), but does not appear to require other Ccr4-Not components, including the Ccr4a (CNOT6)/Ccr4b (CNOT6L) deadenylases, or the non-catalytic subunits CNOT1 or CNOT3.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) are a family of proteins that are closely related to the matrix metalloproteinases (MMPs). It has been suggested that the proteins have a critical role in the breakdown of articular cartilage during osteoarthritis (OA). More recently it has been suggested that their actions could potentially regulate atherosclerotic plaque stability. Atherosclerosis is a chronic, inflammatory disorder characterised by lipid and cholesterol accumulation and the development of fibrotic plaques within the walls of large and medium arteries. The stability of the plaques is very important because clinical symptoms are only presented after rupture of the unstable plaques, leading to thrombosis and ischemia. During the current study, immunohistochemical analysis confirmed that ADAMTS-­1, -­4 and -­5 were being expressed within human carotid atherosclerotic lesions; macrophages were identified as major contributors to their expressions. Following on from this THP-­1 macrophages were stimulated with transforming growth factor-­β (TGF-­β), interferon-­γ (IFN-­γ), TNF-­like protein 1A (TL1A), interleukin (IL)-­17A and IL-­33. The regulation of ADAMTS-­1, -­4 and -­5 expressions were analysed using quantitative polymerase chain reactions (QPCR) and western blots. It was shown that TGF-­β increased the expressions of ADAMTS-­1 and -­5 and decreased the expression of ADAMTS-­4. IL-­33 decreased the expressions of ADAMTS-­1, -­4 and -­5 and IFN-­γ also decreased the expression of ADAMTS-­1. TL1A and IL-­17A stimulation of macrophages had no regulatory actions over ADAMTS-­1, -­4 or -­5 expressions. Looking at evidence from previous studies, TL1A and IL-­17A were identified as agents that could potentially act in synergy to amplify pro­inflammatory cytokine responses. To investigate this further, THP-­1 macrophages were stimulated with TL1A and IL-­17A, TL1A and IFN-­γ and also IL-­17A combined with IFN-­γ. TL1A and IL-­17A were shown to act in synergy to increase the expressions of ADAMTS-­1, -­4 and -­5 in macrophages. The regulation of ADAMTS-­1, -­4 and -­5 expressions in macrophages by IL-­33 was studied further. The mechanism of signal transduction was studied using RNA interference (RNAi) targeting extracellular signal-­‐regulated kinases (ERK)-­1, ERK-­2, p38, c-­Jun N-­terminal kinases(JNK)-­1/2, c-­Jun, phosphoinositide 3-­kinase(PI3K)-­γ, PI3K-­δ, p50, p65 and Janus kinase(JAK)-­1/2. It was determined that the attenuation of ADAMTS-­1, -­4 and -­5 expressions occurred through transcriptional regulation that was dependent on the ST2 receptor. ERK-­1, ERK-­2, JNK-­1/2, c-­Jun, PI3K-­γ and PI3K-­δ were also involved in the signal transduction of the response. The cellular roles of ADAMTS activity within atherosclerotic disease progression remain poorly understood. During the current study adenoviral vectors were created that delivered shRNA-­targeting ADAMTS-­1, -­4 and -­5. The adenoviral vectors were utilised in studies designed to investigate the roles of ADAMTS-­1, -­4 and -­5 during macrophage migration and foam cell formation. The studies showed that knockdown of ADAMTS-­1, -­4 and -­5 had no effect on macrophage migration or foam cell formation. More research is required into the cellular roles that ADAMTS proteases play during atherosclerotic disease progression. The field of research is now growing and could potentially provide some exciting opportunities for novel therapeutics of the future.

Recent studies have indicated the presence of a high degree of cryptic genetic diversity in some clitellate sentinel species. One of these species, the earthworm Lumbricus rubellus, has been recently found to comprise two divergent clades in the UK, and are possibly cryptic species. L. rubellus is commonly used in ecotoxicological assays, where undetected differences in contaminant responses between cryptic lineages may lead to confusing or misleading results. Furthermore, given the key role that earthworm species play in the soil ecosystem, a better understanding of cryptic diversity is necessary to investigate whether divergent lineages play different roles within their ecosystems. In this study, the phylogenomics of the acid-tolerant, cosmopolitan, epigeic species Lumbricus rubellus was investigated, with regard to demography during the glacial stages of the Pleistocene and the recent post-glacial colonization of North Europe using mitochondrial DNA markers, next-generation sequencing and environmental niche modelling tools. The niche suitability of L.rubellus during the last 120.000 years was inferred, allowing hypotheses on survival and recolonisation to be constructed. Phylogenetic, population structure and coalescent-based analyses resulted in the discovery of 11 deep divergent lineages (with levels of divergence up to 18% for mitochondrial markers), which most likely survived in refugia during Pleistocene glaciations. Signatures of expansions point to a possible recolonisation of central Europe during the last Glaciation, survival of one of the clades in a northern cryptic glacial refugium and a consequent recolonisation of northern Europe during the last 10,000 years. Genetic evidence and divergence time ultimately suggest that L. rubellus is a cryptic species complex, which clades diverged as far as ~5MY ago. The entire mitochondrial genome of the species complex is described here for the first time, and a survey of the deep phylogenetic signal over the mitochondrial genomes of eight selected individuals was carried out, supporting and deepening the phylogeny constructed using only two mitochondrial genes. Finally, whole genome analysis of genetic divergence supported the hypothesis of cryptic divergence for the two most divergent lineages selected

I have analysed the regulation and function of the Him gene to gain new insights into Drosophila heart development and its controlling factors. My results show that Him is important during the early specification of pericardial cells and cardioblasts. Loss of Him leads to a reduced number in both of these cell types by the end of embryogenesis. Over-expression of Him throughout the heart results in supernumerary pericardial cells. Him is expressed in all embryonic pericardial cells from embryonic stage 12 to approximately stage 15. I have identified an enhancer fragment that reproduces this expression pattern. Phylogenetic footprinting revealed three highly conserved regions within this sequence. I undertook an extensive mutational analysis of this enhancer to identify regulatory elements within it. I identified Tinman as a direct activator of Him expression. My data indicate that Him is activated in a widespread area of the dorsal mesoderm and the amnioserosa and is actively limited to the pericardial cells. A 5 bp mutation within the enhancer sequence allows for expression within the cardioblasts. Both heart cell types develop from the dorsal mesoderm and some share immediate progenitors. By stage 13, Him is pericardial cell specific and Mef2 is cardioblast specific. This is essential for normal heart development. If Him is not excluded from the cardioblasts, expression of the muscle-cell specific differentiation gene myosin is disrupted, similar to what has been described for Mef2 null mutants. If Mef2 is expressed in pericardial cells, the larval development of the pericardial cells is severely disturbed. A possible explanation for these data is that Him is part of a genetic program that prevents the premature differentiation of heart cells and its down-regulation permits the pericardial cells to undergo their correct development.

No description available.

Chemical control of mosquito vectors, via indoor residual spraying or insecticide treated bed nets, is an integral component of malaria control strategies. Limited availability of insecticides licensed for public health and the rapid development of resistance in mosquito populations to these insecticides, in particular to some pyrethroids, may compromise vector control efforts. With the exception of mutations in the insecticide target sites, relatively little is known about the genetics of pyrethroid resistance in malaria vectors. In some populations candidate effector genes, e.g. cyp6p3 or cyp6m2 in An. gambiae s.s. from Akron, Benin, have been identified as being over expressed in resistant strains but the underlying mechanisms responsible for the increased expression remain unknown. In this study, a combination of quantitative PCR, genetic mapping and microarray tools were used to investigate the mechanisms responsible for pyrethroid resistance in two African major malaria vectors, Anopheles gambiae and An. arabiensis. The current work was unable to confirm an association of these known candidates in either a laboratory colony established from Akron or in recent field caught material. Therefore a genetic mapping approach was adopted using field collected mated females to generate F2 isofemale lines. A major QTL on chromosome 3R was identified which coincides with a genomic region previously implicated in pyrethroid resistance in East African populations. This is the first genetic mapping of insecticide resistance using natural out-bred populations of Anopheles and the advantages and limitations of this approach are discussed. In a second experiment, genetic loci involved in permethrin resistance in An. arabiensis were mapped by establishing genetic crosses between a permethrin resistant strain from Chad and a susceptible strain from Mozambique. A single QTL on chromosome 2R was identified in the F2 progeny that accounts for ~24% of the phenotypic variance. This QTL coincides with a large cluster of detoxification genes. Pyrethroid resistance is not associated with target-site mutations in this population. Finally, microarrays were used to identify genes differentially expressed between a backcross population, generated by crossing the F1 population from the resistant Chad strain and the susceptible Mozambique strain of An. arabiensis back to the parental resistant strain, with the susceptible strain. A number of candidate genes were identified, including the P450 genes cyp4h24 and cyp9j5, but neither of these were located within the boundaries of the QTL on 2R. These findings support the presence of metabolic resistance in this population and fine mapping of the identified QTL as well as further investigation of the microarray hits is warranted.

Carboxylesterase 1 (CES1) is the main human liver esterase and is involved in the metabolism and disposition of numerous endogenous and pharmacological compounds. Some of the substrates of this enzyme are widely prescribed agents such as clopidogrel (Plavix®), methylphenidate (Ritalin®) and oseltamivir (Tamiflu®). However, there is much uncertainty regarding the genetic variability within CES1, and its regulation and involvement in drug-drug interactions (DDI). Polypharmacy is frequent in elderly, HIV and tuberculosis infected populations, and the risk of harmful DDIs is high, especially when these populations overlap. The role played by CES1 on the treatment of all these three pathologies and vice versa needs to be better characterized. In this thesis the role of CES1 genetic variability and its potential role in DDIs are explored both in isolation and in conjunction with other genetic, demographic, physio-pathological and iatrogenic factors. The impact of CES1 genetic variability was assessed on the anti-platelet effect of clopidogrel as well as on isoniazid pharmacokinetics in acute coronary syndrome (ACS) and HIV/Tuberculosis co-infected populations respectively. DDIs mediated by CES1 were explored in a HIV positive cohort treated with clopidogrel and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Also, in vitro experiments with primary hepatocytes were used to investigate CES1 intracellular expression in the presence of prototypical PXR inducers used in tuberculosis treatment. The results of this thesis show that the CES1 rs2244613 SNP does affect clopidogrel anti-aggregant activity and may contribute to treatment non-response. Another CES1 variant, rs3815583, was found to be associated with changes in isoniazid pharmacokinetics. The studies did not indicate that NNRTI coadministration with clopidogrel would impair the anti-platelet activity since no relevant changes in exposure of the antiplatelet agent were identified. In the same way, the results do not anticipate DDIs between CES1 substrates and rifamycins, since no induction of expression was identified after incubating primary human hepatocytes in vitro with rifampicin, rifabutin and rifapentine. In conclusion, the results shown in this thesis support the idea that CES1 genetic variability may play a bigger role than previously suspected in treatment response but may not be a mediator of clinically relevant DDIs.