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Colorectal cancer (CRC) is the second most lethal cancer worldwide and treatment options remain limited for these patients. The backbone of chemotherapy is 5-Fluorouracil (5FU), but only 10-15% of patients respond and whilst this rises to 40-50% when used in combination with other chemotherapies, drug resistance remains a major obstacle in the treatment of CRC. Therefore, a better understanding of the mechanisms driving 5FU resistance in CRC is vital. Metabolic reprogramming is now an accepted 'Hallmark of Cancer' and more recently has been shown to alter therapeutic susceptibility. Altered metabolic programs have been reported for CRC, but the importance of this in contributing to therapy resistance remains unclear. Our hypothesis was that specific metabolic programs could be mediating response to 5FU in CRC and a more in-depth understanding of the metabolic impact of 5FU may help us predict 5FU resistance clinically. Using a multi-omics approach we mapped response to 5FU-based treatments in human and murine CRC models and uncovered a significant role for mitochondrial metabolism in mediating 5FU response and that this promotes cell survival. We also found that this metabolic reprogramming drove acquired 5FU resistance and was predictive of intrinsic resistance in CRC patients. Finally, this resulted in a 5FU-imposed metabolic dependency that can be targeted therapeutically to improve responses across in vitro and in vivo models.

There is an inter-individual variation in drug response to anti-cancer agents because it is a complex trait controlled by multiple genes and environmental factors. This thesis explores the genes controlling variation in response to chemotherapy drugs 5-fluorouracil and oxaliplatin as well as cancer chemoprevention agents aspirin, salicylic acid, metformin, disulfiram with copper and curcumin by using quantitative trait loci (QTL) mapping in a model organism Saccharomyces cerevisiae that could potentially identify targets for future human studies. This approach was feasible due to the conservation of genes between these two organisms. A panel of 111 F₁₂ meiotic recombinant segregants from a four-parent S. cerevisiae advanced intercross lines cross previously generated and genotyped by whole genome sequencing was used. Segregant growth under different drug treatments was measured using PHENOS. Subsequently, linkage-based fine QTL mapping was performed to locate associated regions of the genome and identify causative genes. In this study, linkage analysis has mapped hundreds of genetic loci in the yeast genome responsible for the variation in response to the agents tested. Conserved homologs to human genes were identified. Some associated hits are supported by previously reported studies such as the effect of aspirin and metformin on TOR1 (mTOR in humans) and 5-fluorouracil on CDC21 (TYMS in humans) thus validating this screening approach. Identified genes and pathway enrichment revealed mechanisms by which these agents may exhibit their anti-cancer properties. Candidate genes were selected and functionally validated by reciprocal hemizygosity. Furthermore, the yeast gene deletion library was screened to discover additional pathways of aspirin's response. Detection of genetic variants influencing the differences in drug response could help identify individuals at risk or benefit of using anti-cancer agents. This study could aid the development of biomarkers for drug response, identify targets for genetic testing and validate the repurposing of drugs for cancer prevention.

This body of work served to investigate the mechanisms driving chemoresistance in a series of in vitro TNBC cell line models. Molecular characterisation and comparison of drug resistant models allowed for the identification of promising druggable targets, whose efficacy in restoring chemosensitivity was assessed via a number of methods, including RNAi, CRISPR, and targeted inhibition. In all, the preliminary identification of a means to sensitise an otherwise highly aggressive cancer to chemotherapeutics, represents an essential step in order to improve current standard of care practices of an otherwise intractable disease.

The urgent need to improve therapies for people with drug-resistant epilepsy (DRE) requires improved understanding of the aetiopathophysiology of epilepsy and the mechanisms responsible for pharmacoresistance. Autoantibody-mediated processes may explain DRE in a proportion of cases. Autoantibodies can be detected in people with isolated epilepsy (without encephalitis) but their prevalence rate, associated clinical features and overall clinical significance are unknown. Glutamic acid decarboxylase 65 (GAD65) autoantibodies are the commonest such example described in the literature and GAD65 autoantibody-positive epilepsy is thought to be particularly treatment-resistant. Laboratory techniques for detecting GAD65 autoantibodies are calibrated for the concentrations observed in patients with type 1 diabetes mellitus (T1DM) and it is unclear how well different assays perform at the higher levels of autoantibodies found in autoimmune neurological conditions. It is also unknown whether other immune factors, such as cytokines or lymphocyte subsets, are associated with DRE. Studies of these parameters offer novel potential therapeutic insights. The aims of this work were to: • estimate the prevalence of serum autoantibodies in epilepsy, and search for distinguishing clinical and paraclinical features of seropositive patients; • develop and optimise a novel cell-based assay (CBA) for the detection of GAD65 IgG autoantibodies in neurological conditions; • apply the novel CBA to cohort screening to identify DRE patients with GAD65 autoantibodies for clinical phenotyping; • characterise plasma cytokine profiles and immunocellular phenotypes in DRE. Clinical and experimental data from two large prospective studies of adults with epilepsy are presented. The first focussed on new-onset focal epilepsy (NOFE) and the second on DRE. The observed autoantibody prevalence rate in adult outpatients with epilepsy was 9% (42/479). Neuronal surface-directed autoantibody (NSA) status was best predicted by older age, ictal piloerection, epilepsy risk factors, mood problems, attention domain of the Addenbrooke's Cognitive Examination-Revised (ACE-R) and limbic system changes on MRI. A fixed and permeabilised CBA was developed to detect GAD65 IgG autoantibodies in serum, plasma, CSF and supernatants of cultured peripheral blood mononuclear cells (PBMCs). Serum from 170 healthy controls tested negative. High endpoint titre GAD65 autoantibodies were present in 7/206 (3.4%) patients with DRE but in none of 213 with NOFE. The CBA demonstrated inter-rater reliability and compared favourably to commercial GAD65 autoantibody radioimmunoprecipitation and enzyme-linked immunosorbent assays. To try and enhance the CBA, molecular cloning techniques for antigen tagging and cell surface expression for use in a live and non-permeabilised CBA were applied. However, antigen surface expression was unsuccessful. Our novel assay identified seven patients with DRE and high titre GAD65 autoantibodies. All were female and demonstrated continuous GAD65 autoantibody production throughout long-term follow-up. Five of seven had temporal lobe epilepsy, three had T1DM and two had had bilateral hippocampal sclerosis. Pain was reported as a seizure trigger in three patients, an unusual finding in epilepsy. Cytokine concentrations measured by a multiplex bead-based immunoassay combined with flow cytometry showed GAD65 autoantibody-positive DRE patient plasmas were enriched for IL1b, IL4, IL10, IL15 and TNFa, consistent with multifaceted immune system involvement. Mass cytometry of PBMCs suggested DRE GAD65 autoantibody-positive patients with high GAD65 autoantibody titres may represent a distinct subgroup. This work provides valuable insights into potential clinical and immunological aspects of epilepsy, with implications for clinical practice and future epilepsy research.

Intratumour heterogeneity is a phenomenon during cancer progression in which cancer cells diverge and form clonal populations with distinct phenotypic, genetic, or epigenetic states within the same tumour. This intrinsic heterogeneity provides a fuel for cancer evolution enabling tumour cell populations to adapt to selective pressures imposed by the tumour microenvironment or therapeutic interventions. Lineage-tracing approaches shed light into the clonal dynamics of complex populations, but generally lack the ability to directly associate clonal lineage with measurements that infer phenotype such as epigenetics and transcriptomics. In contrast, single-cell sequencing techniques can provide insight into the makeup of complex biological ecosystems, revealing the presence of rare cell populations that are typically masked in bulk analyses, but lack the ability to link these cell populations to clonal lineages. To address this challenge, we developed the WILDseq platform, a novel approach that allows clonal characterisation at the single-cell transcriptomic level while facilitating the prospective analysis of dynamic regulation of phenotypic heterogeneity under the selective pressure of therapeutic intervention. WILDseq relies on uniquely labelling individual cells with a heritable, expressed DNA barcode coupled with high-throughput single-cell RNA-sequencing. Importantly, this lentiviral-labelling approach can be deployed in any model system that is susceptible to viral transfection. Thus, this platform allows the comprehensive and systematic characterisation of clonal phenotypic di fferences within complex populations. Here we demonstrate how this technology can be used to determine clonal populations which are sensitive or resistant to a particular therapeutic intervention, identify transcriptomic signatures that correlate with these phenotypes and analyse how these cells adapt their transcriptomes to escape therapy. We have applied WILDseq to the study of di fferential clonal responses to chemotherapy in the heterogeneous 4T1 model of breast cancer and validated transcriptomic signatures of therapeutic resistance and sensitivity in primary patient data. We additionally used WILDseq to study the clonal response to the epigenetic regulator JQ1 which revealed intrinsic signatures that primed clones to JQ1 sensitivity. We observed JQ1-dependent depletion of CD8+ cytotoxic T-cells and suggest that this drives changes in clonal distribution. Finally, we are working on developing a high throughput FISH assay to leverage the WILDseq technology for mapping clonal and transcriptional identities spatially. Collectively, this thesis contributes to the characterisation and understanding of breast cancer heterogeneity and the impact of clonal architecture on tumour progression and response to therapy.

Plasmodium falciparum remains one of the greatest global public health challenges. Current malaria control efforts face the persistent challenge of drug resistance and, to date, the malaria parasite has been able to develop resistance to almost every drug with which it has been challenged. One strategy to tackle the problem of drug resistance is to systematically identify new antimalarials to which the parasite has not yet developed resistance. Many novel compounds with antimalarial activity are in development. However, in most cases, the targets and the mechanisms of action of these compounds are unknown. Understanding which compounds are targeting new pathways for which no resistance exists is critical to prioritising novel drug candidates. Understanding drug resistance in more depth has the added advantage that targeting pathways for which resistance comes at a high fitness cost could prolong drug efficacy in the field. There may also be cases where drug resistance mutations confer hypersensitivity to new antimalarials compounds. Identifying cases where this occurs would allow us to develop better combination therapies. Currently, it is only practical to test the effectiveness of new antimalarials against a handful drug- resistant mutants at a time. To develop a new platform to address this problem, I set out to use BarSeq technology and CRISPR-Cas9 editing to create a pool of barcoded drug resistant mutants that covered a diverse representation of the currently known malaria resistome. These mutants would include three main groups: 1. Lines with clinical significance: mutations that confer resistance to antimalarial compounds currently being used in the field. 2. Lines with significance to compounds in the Medicines for Malaria Venture (MMV) pipeline: mutations that confer resistance to compounds currently in the drug development pipeline. 3. Putative future drug targets: resistance mutations generated to "toolbox" compounds not currently in the drug development pipeline. By utilising CRISPR-Cas9 genome editing technology, barcode cassettes were inserted into the genome of drug-resistant lines. These lines were then combined into one master pool that would allow all lines to be interrogated in parallel. Amplicon sequencing was then used to compare the relative growth of each mutant over time in the presence and absence of query compounds. This would allow multiplex competition assays to identify the targets or mechanisms of resistance to novel antimalarials. The ultimate aim is to develop a comprehensive cross-resistance screening platform for rapid compound profiling. The initial part of my work focused on improving the efficiency of the CRISPR-based barcode insertion reaction, targeting the Pfrh3 locus, that had previously been developed in the lab. When I attempted this approach on a larger scale, it was found that editing was too inefficient with only 5% of transfections being successfully barcoded. Several different editing strategies to increase barcoding efficiency were explored. In the end, a positive and negative selection approach was used to enrich for edited parasites and to insert barcodes into a new nonessential locus, Pfpare. This increased the percentage of successfully barcoded transfections to over 50% with all parasites being edited in these successful transfections. This increase in efficiency has allowed me to insert 53 barcodes into 47 different drug-resistant lines, covering the lines of clinical significance, lines with significance to compounds in the MMV pipeline, and the majority of the putative future antimalarial targets Drug resistant mutants were pooled together and grown in competition in the absence of the drug to measure fitness costs of resistance. The pool was tested with well-characterised antimalarials GNF179 and NITD609 to optimise screening parameters including selection concentration and time of harvest. The pool was then validated using a panel of blinded compounds with different mechanisms of action supplied by the MMV. Testing of the pool revealed that it could be used to select out specific mutant lines that had been shown to confer resistance to these compounds. Antimalarial compounds with novel mechanisms of action were successfully identified using the BarSeq approach because of their profile of total killing of the pool, indicating no relevant resistant lines were present. However, to provide insight into their mechanism of action, in vitro evolution experiments or other compound profiling experiments would still need to take place. The rate at which resistant mutations can be generated is limited by the mutation rate of the parasites in the selection. The use of so called hyper-mutator lines that have lost the ability to proofread during transcription has been established as a way to increase mutation rate and therefore the rate at which resistance is able to develop in yeast and bacteria. Recently our lab generated a Plasmodium falciparum line with this phenotype. Drug selection assays have shown that it is capable of developing resistance at a lower inoculum of parasites than its wildtype parent line. In this thesis, I investigated whether introducing a second source of mutation, in this case UV radiation, could accelerate this process even further. I established a protocol for treating parasites with UV radiation, then challenged UV and non-UV treated hyper-mutator and wildtype parasites with the antimalarial compound NITD609. The hyper- mutator line once again proved more able to develop resistance at lower concentrations than the wildtype parental line. The UV treated parasites returned more quickly after selection, however with only a modest improvement in time after selection. Nonetheless, this approach may be beneficial for compounds for which resistance is more challenging to generate.

In this thesis, molecular cloning techniques were used to obtain the sequences of natural antimicrobial peptides and their synthesis was achieved using solid-phase synthesis (SPPS). In addition, the peptides were purified and identified by high-performance liquid chromatography (HPLC) and MALDI-TOF MS, and their biological activity was determined. In the third chapter, a natural active peptide, QUB-1369, was identified and isolated from Amolops hainanensis, and it was found to be more effective against the Gram-positive bacterium, S. aureus, and less effective against the Gram-negative bacterium, E.coli and yeast, in a series of functional assays. This peptide was structurally-modified and it was found that the antimicrobial spectrum of the analogue, QUB-1464, was expanded to a certain extent, and the antimicrobial function was significantly improved. In addition, there was an inevitable decrease in haemolytic activity. Therefore, the rational design modification of this AMP helped to improve its therapeutic activity. In Chapter 4, an antimicrobial peptide was identified in Agalychnis callidryas with a known sequence, and because it contained a specific sequence, it was necessary to investigate the relationship between its structure and function. Firstly, changing the size of the side chain groups of its amino acid residues had a large effect on the activity. Secondly, the balance between the hydrophobicity and the number of charges also had a powerful influence on the cytotoxicity as well as on the activity. In the study in Chapter 5, the idea of a modified hybrid peptide (chimaera) was presented. The N terminus of different families of AMPs was selected for fusion with the fixed C terminus, and the changes in functional activity and toxicity were observed to identify the hybrid peptides with a relatively high therapeutic potential. The results showed that this modification idea played a very positive role in future studies of AMPs.

Human demand for novel antimicrobials has increased due to the rapid development of antibiotic-resistant bacteria. Because of their remarkable antimicrobial capacity, antimicrobial peptides are at the forefront of new antibacterial drugs. In our research, we are working with the isolation and identification of small molecule peptides with potent bioactivity from frog skin secretions and performing functional experiments on them. In chapter 3, DRP-AC4, a novel dermaseptin family peptide was isolated and identified. DRP-AC4 possesses broad-spectrum antimicrobial activity and inhibits the proliferation of a wide range of cancer cells. In chapter 4, another novel peptide named Temporin-LTd was identified. Temporin-LTd is a linear cationic short alpha-helical peptide with strong antimicrobial activity against Gram-positive bacteria but weak activity against Gram-negative bacteria. In chapter 5, three analogues of DRP-AC4 were designed to employ the influence of cationicity, hydrophobicity and C-terminal amidation on bioactivity. Extended hydrophobic face exhibited higher haemolytic activity and anti-proliferative activity. cationicity-enhanced analogue exhibited higer antimicrobial activity without increasing haemolytic activity.finally, C-terminal amidation removed analogue lost most of its activity. In chapter 6, eight derivatives of Temporin-LTd template were designed and compared their helical content regarding their biological activity. The helical content of Temporin-LTd analogues is associated with their haemolytic and anti-cancer cell proliferative activity, but not associated with their antibacterial activity. Furthermore, we found that the cationic residue type could affect the activity and helical content of Temporin-LTd. Surprisingly, our addition of leucine over glycine at position 9 resulted in the loss of the antimicrobial activity of Temporin-LTd. Finally, we found that disruption of the C-terminal helical structure provides initial thoughts for the development of novel anti-fungal drugs.

Introduction: Glioblastoma multiforme is the most common aggressive primary malignant brain tumour in adults. The current gold standard treatment comprises of surgery followed by radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. Prognosis is poor, with a median survival of 14.6 months following diagnosis. Treatment often fails due to intrinsic or acquired TMZ resistance of a small population of cells termed glioma cancer stem cells (GCSCs). A promising target for glioblastoma therapy is the Notch signalling pathway, which can be suppressed using gamma-secretase inhibitors such as dibenzazepine (DBZ). We investigated the effects of the Notch ligands, Delta-like 4 (DLL4) and Jagged-1 (JAG1) on tumour resistance to TMZ and GCSCs. Methods: U87 and U251 glioblastoma cells were transduced with empty vector-, DLL4, and JAG1-encoded retroviruses. Cells were cultured under 2D, 3D, and CSC culture conditions and response to single (TMZ/DBZ) and combination (TMZ and DBZ) treatment was assessed. A patient derived GCSC line, CSC-5, was also used to evaluate the effect of single and combination treatment by neurosphere recovery assay. Results: DLL4 and JAG1 overexpression promotes resistance by increasing the TMZ IC50. Neurosphere formation, recovery, and secondary neurosphere formation is increased following DLL4 and JAG1 overexpression and is reversed upon combination TMZ and DBZ treatment. TMZ has little effect on the self-renewal of GCSCs, however single DBZ and combination DBZ and TMZ treatment significantly reduces GCSC self-renewal. Consequently, Notch inhibition reduces GCSC marker expression and may promote GCSC differentiation. Conclusions: These data show the importance of the Notch pathway, in particular the ligands DLL4 and JAG1 in resistance to TMZ chemotherapy and GCSC self-renewal. The addition of Notch inhibitors to current treatment is a promising approach to overcome TMZ resistance and decrease brain tumour recurrence and encourages further translational and clinical studies.

Fibroblast Growth Factors (FGFs) play a vital role in development and tissue regeneration. There are at least 23 distinct members of the FGF family that bind to at least four tyrosine kinase receptors known as FGF Receptors (FGFRs). FGF/FGFR signalling plays an important role in variety of processes and is tightly regulated. However, some cancer cells hijack the control of FGFR pathway, causing deregulation of FGFR signalling. FGF and their receptors, such as FGFR1 and 2, have been involved in susceptibility of breast cancer and progression as well as in a number of other cancers. Cervical cancer (CC) is one of the most commonly diagnosed cancers in women worldwide. One of the main causative agents of CC is the human papillomavirus (HPV). However, not all females infected with HPV develop CC demonstrating that other factors might be important. Although some studies have emphasised the possible involvement of FGFR signalling in CC, the aetiology and the molecular mechanisms related to CC and FGFR signalling remain poorly understood. The main aim of this study was to investigate FGFR signalling in cervical cancer cell lines (HeLa, SiHa and CaSki cell lines; CCCLs). A detailed understanding of the mechanism correlated to CC will make possible to use specific FGFR inhibitors that are currently in clinical trial, for CC treatment either on its own or in conjunction with the existing treatment, in order to increase success rates. In addition, FGFR signalling could be of early prognostic value. In order to examine gene and protein expression of FGFRs and associated ligands in CCCLs, PCR and immunocytochemistry were performed. Downstream extracellular signal-regulated protein kinase (ERK) signalling in CCCLs was investigated using Western blotting. Functional FGFR studies carried out through cell proliferation, migration and apoptosis assays. The above experiments were performed using stimulated and unstimulated CC cells in the presence and absence of FGFR inhibitors PD173074 and SU5402. RNAi mediated FGFR1 and FGFR2 knockdown was also performed to further assess function. The human CCCLs expressed FGFR1, FGFR2, FGFR4, FGF2, FGF4 and FGF7 mRNA and protein, with 'b' and 'c' isoforms of FGFR1 and FGFR2 confirmed using gene expression analysis. The FGFR1 and FGFR2 protein in CCCLs was localised within the nucleus with a distinct speckled arrangement. Importantly, ELISA revealed that all CCCLs secreted FGF2 ligand only, but not FGF4 and FGF7 suggesting that FGFR was activated in a paracrine manner. Activation of the ERK pathway occurred in response to FGF2, 4 and 7 exposures in all three CCCLs and the activation was eradicated in the presence of PD173074 or SU5402 inhibitor, suggesting that the effect is specific to FGFR signalling. Functional studies revealed that FGFR signalling enhanced cell proliferation and migration. Furthermore, knockdown of FGFR1 and FGFR2 by RNA interference or blockade using PD173074 inhibited cell proliferation, cell migration and also promoted the apoptosis CC cells. Drug resistant CCCLs (HeLa, CaSki and SiHa) were established following long-term exposure to PD17307. Collectively, this work supports an association between CC progression and FGFR signalling, laying the foundations for developing FGFR inhibitors for CC therapy. The drug resistant CCCLs will be valuable for further investigations into the mechanisms involved in acquired drug resistance.

The multidrug transporter Pdr5 is one of the major contributors to multidrug resistance in Saccharomyces cerevisiae and it is highly homologous to the multidrug transporter Cdr1 in the pathogen Candida glabrata. Over-expression of PDR5 is one of the mechanisms of acquired multidrug resistance. Fully understanding the mechanisms of PDR5 gene expression regulation is important for understanding and tackling multidrug resistant yeast pathogens. In this thesis I also show a homolog of Rtt106 in the pathogenic C. glabrata, which is required for long term azole antifungal resistance. In this study I show the histone chaperone Rtt106 is strongly recruited to the PDR5 promoter in a transcription factor Pdr3 dependent manner to support basal PDR5 expression. Rtt106 and Pdr3 act in the same pathway to influence expression of Pdr3 regulated genes, including PDR5. Both Rtt106 and Pdr3 are involved in the recruitment of the chromatin remodeler complex SWI/SNF to the PDR5 promoter. All three are required to support basal PDR5 expression and to confer long term resistance against the azole antifungal drug ketoconazole. Rtt106 and Pdr3 are not involved in ketoconazole-induced rapid PDR5 expression, while the transcription factor Pdr1 and SWI/SNF are required for that induction. Genome-wide analysis of Rtt106 binding shows it displays three distinct modes of promoter association. The first mode of Rtt106 association is displayed at promoters containing DNA binding sequences called pleiotropic drug resistance elements (PDREs), which are recognised by the transcription factors Pdr1 and Pdr3. The second mode of Rtt106 association is displayed at promoters of histone genes and the third mode at a distinct subset of gene promoters. On a genome-wide level Rtt106 displays both stimulatory and suppressive influence on transcription. To sum up, my research shows a novel role for the S. cerevisiae histone chaperone Rtt106 in contributing to multidrug resistance by regulating the gene expression of the multidrug transporter PDR5 in a Pdr3 dependent manner. The Rtt106 homolog in C. glabrata is also required for azole antifungal resistance, which makes it a potential therapeutic target in the fight against fungal pathogens.

Chronic lymphocytic leukaemia (CLL) is an indolent B cell malignancy infiltrating the lymphatic system and invariably the bone marrow (BM). Although treatment options for patients with advanced disease have significantly increased in the past years and improved life-expectancies, the disease remains incurable and after emergence of therapy resistant disease patients succumb to infections due to secondary BM failure. Survival of CLL cells depends on protein kinase C- (PKC) expressed in activated BM mesenchymal stromal cells (BM-MSCs) which display activation of inflammatory pathways, as well as BM endothelial cells (BMEC) (Lutzny et al., 2013). This activation provides a nurturing environment, which not only contributes to disease progression, but likely also disrupts normal haematopoiesis, leading to insufficient blood production. I hypothesised that disease-induced alterations in the tumour microenvironment of CLL cells contribute to BM failure by skewing hematopoietic stem and progenitor cell (HSPC) fate choice commitments and tissue residency. In my study, the analysis of the peripheral blood (PB) of 67 patients with predominantly early stage CLL revealed that short term (ST) HSCs and Common Myelo-Erythroid Progenitors (CMP/MEPs) are found in higher frequencies and are associated with a skewed differentiation into myeloid cells in vitro and progenitor cells are less likely to produce erythrocytes. These findings provide evidence for the mechanisms underlying BM failure in patients with CLL. Our current knowledge of how malignant B cells interact with stromal cells within the BM microenvironment is limited. I hypothesised that BMEC play a fundamental role in nurturing malignant B cells during disease progression. In a murine model for CLL, I identified that malignant B cells home preferentially to the BM in close proximity to EC, and during disease progression, sinusoidal, but not arterial vessels, upregulate the stem cell marker, SCA1. RNAseq analyses of the remodelled sinusoids revealed activation of inflammatory and cytokine regulation pathways. In lymphoid malignancies, minimal residual disease (MRD) in the BM has prognostic value to predict disease relapse after patients received chemotherapy, suggesting the BM microenvironment is not only important for disease progression, but also plays an essential role for chemoresistance. Our lab recently demonstrated that BM-MSCs contribute to drug resistance via PKC mediated activation of lysosomes, which is required for tumour-stroma cell interactions (Park et al., 2020). However, it still is unclear which type of stromal cells are involved in this environment mediated drug resistance (EMDR) mechanism. I hypothesised that BMEC are the main drivers of EMDR in CLL and dependent on the activation of PKC. My results show indeed that BMECs activate lysosome production as a drug resistance mechanism and these effects are mitigated by pharmacological inhibition of PKC. Malignant B cells are found in close proximity to EC after treatment, suggesting the endothelial niche provides a sheltering from the action of cytotoxic drugs. In vitro experiments demonstrated that pharmacological inhibition of PKC sensitises CLL to the action of cytotoxic therapies. Overall the two projects carried out during my PhD have provided novel insights; first revealing that the disease-induced inflammatory environment could affect normal blood production in patients, and second that BMECs become remodelled. This reprogramming of BMECs is likely to play a fundamental role in the process of EMDR. Results from my work are important to improve current therapies for patients with CLL as well as the prevention of BM failure.

ABCG2 is a polyspecific membrane transporter of the ATP-binding cassette (ABC) superfamily, which is responsible for the efflux of a wide array of structurally unrelated molecules from mammalian cells. Physiologically, the protein is thought to serve a protective role; ABCG2 is expressed in certain stem cell populations, as well as at "barrier sites" throughout the body. Thus, ABCG2 expression is important from a pharmacokinetic perspective: expression in the gut reduces the bioavailability of orally-administered substrates, whilst its expression at the blood-brain barrier and placenta prevents drug accumulation in the brain and the foetus, respectively. ABCG2 expression can also be problematic in certain cancers, where it causes chemotherapy resistance. Recent structural information about ABCG2 from cryogenic electron microscopy has revealed conformational changes associated with transport. However, there has been little investigation of the driving forces behind the process. ATP hydrolysis alone is traditionally thought to drive ABC transporters, but recent research shows that a transmembrane chemical proton gradient (ΔpH) contributes in certain bacterial ABC multidrug transporters. Therefore, this work aimed to elucidate the effect of ΔpH on ABCG2-mediated transport. First, extensive characterisation of a frequently used fluorescent ABCG2 substrate, Hoechst 33342, revealed that it was unsuitable for this purpose. Despite its efficient transport by ABCG2, the dye's local pH can drastically affect its fluorescence when associated with lipid and DNA. Therefore, ethidium was favoured as a substrate that does not suffer from these problems. Assays in intact (human) Expi293F cells demonstrated that ABCG2 effluxes ethidium and identified carboxyfluorescein succinimidyl ester (CFSE) as another novel substrate. Experiments with proteoliposomes containing purified inside-out oriented ABCG2 revealed that an interior alkaline ΔpH was necessary and sufficient for ABCG2-mediated ethidium transport. These results are relevant physiologically in the gut where, in acidic regions, enterocytes maintain a substantial ΔpH (interior alkaline) and ABCG2 expression is the highest. The importance of an abnormal ΔpH (interior alkaline) is also well-established in cancer. Furthermore, single-cell expression data suggests that embryonic stem cells might also contain such a proton gradient. Findings from this work are therefore relevant from a drug development perspective, as they point to novel mechanisms by which ABCG2 activity can be modulated in health and disease.

It is estimated that over 150 million individuals suffer from serious fungal infections, resulting in 1.5 million deaths a year. Aspergillus fumigatus is a filamentous saprophytic fungus. However, in hosts with an altered immune system it can cause a variety of diseases. The most serious is invasive aspergillosis, which is estimated to cause over 200,000 life-threatening infections annually with high mortality rates. Fungal diseases are increasing due to the expansion of the immunocompromised cohorts of patients. The current antifungal arsenal is limited and paired with severe sides effects. Additionally, antifungal resistance is on the rise. To develop new antifungals, a mechanistic understanding of A. fumigatus pathogenicity is required. This thesis will address current methodologies available to phenotype large collections of mutants for fitness, infection-related stresses and invasion and will identify transcription factors required for these processes by utilising the genome-wide transcription factor knockout library. Gene expression is tightly regulated at the transcriptional level. This project outlines the importance of transcription factors in azole resistance as a pilot phenotype screening and explores the regulatory mechanism of the multi-drug resistant negative cofactor transcription factors, NctA and NctB. These key regulators control many processes, including ergosterol biosynthesis, a direct target of the azoles. Additionally, the transcription factor knockout library is screened under infection-related stresses, for detachment and cytotoxicity of epithelial cells. This first in field screening identifies previous uncharacterised transcription factors and explores new phenotypes for previous characterised transcription factors. Furthermore, regulators required for environmental adaptation are a distinct set from ones required for epithelial invasion. This provides the first evidence for the accidental pathogen hypothesis. Lastly, the genome editing technique CRISPR-Cas9 is explored to allow marker-free transformation in A. fumigatus. This technique can be used for gene replacement and epitope tagging without the need of labour- and time-intensive construct generation. Furthermore, marker-free transformation will reduce the chance of off-target effects.

Fasciolosis, a debilitating disease caused by flatworm parasites belonging to the genus Fasciola, is a prominent threat to global food security due to its widespread infection of agriculturally significant ruminants. Recent studies have identified a population of proliferative cells within Fasciola hepatica (Fasciola proliferative cellsFPCs) that resemble neoblast-stem cells, both morphologically and behaviourally. These cells are the only proliferating cells in Fasciola hepatica and can be localised using thymidine analogue 5-Ethynyl-2 ́-deoxyuridine (EdU). This thesis further characterises these neoblast-like stem cells, investigating their dynamics, supportive roles in growth and development and identifying putative regulatory effectors. This study reports increased juvenile growth, which correlated with increased proliferation of these stem-like cells, in juveniles maintained in chicken serum supplemented media (CS+). The comparison of FPCs between in vitro maintained and ex vivo maintained juveniles further supports the link between FPCs and juvenile development, with the larger, more developed ex vivo juveniles exhibiting significantly more proliferating cells. FPCs were found to be radiation sensitive enabling us to selectively deplete them and examine downstream effects. Using an RNA interference (RNAi) platform, the function of a key effector (fgfrA) was investigated and silenced juveniles exhibited a significant reduction in the number of EdU+ cells. The findings of this study highlight the stem-like characteristics of FPCs, their facilitative roles within fluke growth and indicate their importance to fundamental fluke biology. The disruption and dysregulation of this cell population may hold the key to developing novel control therapies.

Fasciolosis, caused by liver fluke parasites of genus Fasciola, is responsible for reducing global agricultural yield in livestock animals and is a neglected tropical disease of humans. A limited portfolio of flukicide drugs are available, however treatment of the highly pathogenic acute stage is unique to the benzimidazole, triclabendazole (TCBZ). Reports of TCBZ-resistant Fasciola hepatica populations in cattle, sheep and humans, could undermine fluke control and necessitates the exploration of new treatments. The availability of genomic/transcriptomic datasets complemented by an in vitro culture platform and the amenability of F. hepatica to RNA interference (RNAi)-induced gene silencing permits the interrogation of putative novel drug targets. This thesis herein explores avenues for overcoming TCBZ-resistant phenotypes in juvenile F. hepatica, through impeding detoxification and regenerative processes. ATP-binding cassette (ABC) transporters are implicated in multidrug resistant cancers through the active export of drugs from cells. Twenty-four putative ABC transporter sequences were identified in the F. hepatica genome. Pharmacological inhibition and RNAi-induced silencing of selected ABC transporters potentiated the efficacy of TCBZ and TCBZ metabolites, which was shown to overcome resistance in vitro. The previous development of the in vitro culture platform highlighted neoblast-like cells to be the drivers for fluke growth. Here, it was shown that these neoblast-like cells exhibit stem cell-like features, including the capacity for self-renewal and asymmetric division. Through irradiation and silencing of the cell-cycle regulator histone-2b, it was shown that these cells could be selectively depleted, which undermined juvenile growth. Furthermore, it was shown that TCBZ and TCBZ metabolites hinder juvenile growth and development through the inhibition of neoblast-like cells – furthering our understanding of TCBZ’s mode-of-action and calling into question the relevance of stem-cell mediated regeneration in the resistance phenotype. Corroborating this hypothesis, TCBZ-resistant juveniles were found to possess increased proliferative potentials in comparison to susceptible fluke. Subsequently, it was shown that the TCBZ-susceptibility status of juveniles could be altered through changes in neoblastlike cell dynamics – highlighting neoblast-like cells as a likely source of drug targets. Finally, the F. hepatica kinome was explored for genes implicated in growth and development. This highlighted an orthologue of polo-like kinase 1, which was vital for maintaining the neoblast-like cell population. Overall, this thesis contributes to our understanding of TCBZ-resistance mechanisms and emphasises interring with detoxification and stem-cell mediated development as favourable candidates for drug intervention.

Antimalarial drug resistance is an unrelenting obstacle to malaria control programs. In Southeast Asia (SEA), parasites have developed some degree of resistance to nearly every malaria drug currently available, with the most recent emergence to artemisinin combination therapies (ACTs). ACTs are the recommended front-line treatments for Plasmodium falciparum malaria worldwide and decreased susceptibility of parasites to both artemisinin and one of the widely used partner drugs, piperaquine, have been reported in multiple locations in SEA. It is therefore necessary to have reliable methods for detecting and evaluating resistant phenotypes. The purpose of this study was to combine clinical data from Cambodia with findings from genomic studies to evaluate putative markers of piperaquine resistance. The study first developed high-throughput assays to reliably detect one of these markers, a copy number variation (CNV) in the plasmepsin 2 and plasmepsin 3 (PM2-PM3) genes, in parasites likely to be PPQ-resistant. In addition to assay development, this study used gene overexpression techniques and CRISPR-Cas9 gene editing to examine the functional role of molecular markers of piperaquine resistance, including the PM2-3 CNV, and two gene candidates with nonsynonymous single nucleotide polymorphisms (SNPs): a putative exonuclease protein (exo-E415G) and a putative mitochondrial carrier protein (mcp-N252D). This research fills a knowledge gap in the lack of functional data for molecular markers of piperaquine resistance by examining the phenotypic relevance of the genotypes observed in contemporary isolates. To complement these functional studies, this doctoral work has also used a P. falciparum hypermutator parasite line to select for a piperaquine-resistant phenotype in vitro. Whole genome sequencing analysis (WGS) of the piperaquine-resistant lines obtained through these experiments has identified nonsynonymous SNPs in gene candidates that have been reported to play a role in antimalarial drug resistance, including SNPs in the chloroquine resistance transporter gene (pfcrt) and the multidrug resistant protein 1 (pfmdr1) transporter. SNPs in pfcrt have been reported to confer piperaquine resistance in the field and in vitro and our recent drug-pressure experiments provide additional evidence to support these findings. The WGS analysis also discovered novel SNPs in gene candidates not previously reported to modulate the piperaquine-resistant phenotype that will require further evaluation. Such work has enabled the possibility of examining whether genetic changes observed in patient isolates can also be investigated and observed in vitro. By combining functional molecular approaches with genomic analyses, this study provides new insights into the mechanisms of piperaquine resistance.

Despite remarkable advances in our understanding of the drivers of human malignancies, new targeted therapies often fail to show sufficient efficacy in clinical trials. Indeed, the cost of bringing a new agent to market has risen substantially in the last several decades, fuelled partly by lack of efficacy in late phase clinical trials. Even in cases where a new agent is deemed 'successful', the development of resistance is often seen as inevitable and clinical responses can be fleeting. Typically, resistance to targeted therapies is thought to arise from pre-existing populations within the tumour, rather than from de novo evolution, yet few studies have experimentally tested this understanding. Indeed, recent reports in the literature have described epigenetically regulated drug tolerant populations within cancers, defined by cell-cycle regulation and/or quiescent repopulation dynamics, drug induced chromatin remodelling or differential transcription factor binding, that can be transient or permanent in nature. This thesis will outline experiments using high complexity molecular barcodes to trace the fate of individual cellular clones in the development of drug resistance. With this technique, cellular clones can be uncoupled from their genomic backgrounds, giving a new depth to our understanding of clonal selection in cancer. In particular, high complexity barcodes are used to identify a pre-existing tamoxifen resistant population in the MCF7 cell line. This resistance phenotype is then linked to the induction of embryonic transcription factor OCT4. Finally, we use our molecular barcoding technique to interrogate the repopulation dynamics of a breast cancer PDX model, supporting their use as complex model systems suitable for studying the origins and consequences of tumour heterogeneity.

Neoadjuvant chemotherapy has become standard practice in patients with high-risk early breast cancer as it improves rates of breast conservation surgery and enables prediction of recurrence and survival by using response to treatment as a surrogate. Previous studies have focused on generating molecular datasets to develop prediction models of response, though little is known on how tumours and their microenvironments are modulated by neoadjuvant chemotherapy. The thesis aims at molecularly characterising tumour changes during neoadjuvant chemotherapy in a cohort of 168 patients. Serial tumour samples at diagnosis, and, when available, midway through chemotherapy and on completion of treatment were profiled by shallow whole genome sequencing, deep exome sequencing and transcriptome sequencing, resulting in the generation of an unprecedented genomics dataset with tumours in situ while patients received chemotherapy. Molecular predictors of response to chemotherapy were inferred from the diagnostic biopsy. Several novel observations were made, including previously undescribed associations between copy number alterations, mutational genotypes, neoantigen load, HLA genotypes and intra-tumoural heterogeneity with chemosensitivity. Possible mechanisms of chemoresistance included LOH at the MHC Class I locus, decreased expression of MHC Class I and II genes and drug influx molecules, as well as increased expression of drug efflux pumps. A complex relationship between proliferation, tumour microenvironment composition (TME) and response to treatment was explored by deconvoluting bulk RNAseq data and performing digital pathology orthogonal validation. Clonal and microenvironment dynamic changes induced by/associated with chemotherapy were then modelled. Two types of genomic responses were identified, one in which the clonal composition was stable throughout treatment and another where clonal emergence and/or extinction was evident. Validation by multi-region deep sequencing confirmed the dynamics of the clonal landscape. Clonal emergence was shown to be associated with higher proliferation and decreased immune infiltrate, with an increase in genomic instability and homologous recombination deficiency during treatment. The immune TME composition and activity mirrored response to treatment, with cytolytic activity and innate and adaptive immune infiltrates linearly correlating with the degree of residual disease remaining after chemotherapy. Finally, the circulating tumour DNA (ctDNA) genomic landscape was explored by using shallow whole genome sequencing and targeted sequencing of plasma DNA. Tumour mutations detected on exome sequencing were also detected in ctDNA in plasma, supporting the use of liquid biopsies as a biomarker for monitoring response to therapy and detection of minimal residual disease.

The repeated emergence of drug resistance in Plasmodium falciparum underscores the importance of understanding the genetic architecture of current resistance pathways, as well as any associated fitness costs. Why resistance emerges in particular regions of the world has been linked to particular genetic backgrounds that better tolerate resistance-associated polymorphisms; this is likely to play a key role in driving the epidemiology of drug resistance, however is infrequently studied at a large scale in a laboratory setting. The first results chapter establishes a barcoding approach for P. falciparum with the aim of tracking parasite growth in vitro. The strategy used was adapted for P. falciparum by using a pseudogene (PfRh3) as a safe harbour to insert unique molecular barcodes. These libraries of barcoded P. falciparum vectors were also used as a readout of transfection efficiency. The second chapter establishes a proof of principle for phenotyping by barcode sequencing, using a panel of barcoded parasites generated in different genetic backgrounds that comprise sufficient genetic diversity to pilot the method. These were grown in the presence and absence of antimalarial compounds, and growth phenotypes were measured in parallel using BarSeq. The third results chapter studies the contribution of mutations in Pfkelch13, a molecular marker of artemisinin resistance, to parasite fitness. Combining CRISPR/Cas9-based genome editing and high throughput sequencing, the impact of Pfkelch13 alleles on fitness in the context of particular strain backgrounds is revealed. In particular, the impact of genetic background in the emergence and spread of drug-resistant lineages (referred to as KEL1) in Southeast Asia carrying a Y580 Pfkelch13 allele. Overall, given the current pace of genome sequencing of pathogenic organisms such as P. falciparum, it will be important to increase the scale of experimental genetics, in order to tackle in real-time natural variation that might be under constant selection from drugs, thus anticipating the emergence of drug resistance in changing parasite populations. Through this work, tools were developed to facilitate parallel phenotyping by measuring in vitro growth using high-throughput sequencing. The work also develops novel approaches to address the importance of genetic background and a potential role for positive epistasis in a lineage responsible for the recent outbreak of drug-resistant malaria in Southeast Asia.

High grade serous ovarian cancer (HGSOC) comprises 75-80% of all ovarian cancer cases and is characterised by p53 mutation and genetic heterogeneity. There are two major molecular categories: homologous recombination repair (HR) pathway proficient and deficient. Most cases are treated with a combination of surgery and chemotherapy. Patients with HR deficient cancers respond well initially, but the majority experience relapse after first line chemotherapy due to drug resistance. As p53 is mutated in virtually all HGSOC cases, these cancers rely heavily on the G2/M checkpoint for DNA damage repair and survival. The G2/M checkpoint is controlled by the phosphorylation status of Cdk1. Wee1 kinase responds to DNA damage by phosphorylating Cdk1 and causing G2 arrest, giving time for DNA repair before entry into mitosis. We and others have hypothesised that combining a DNA damaging agent with Wee1 kinase inhibition would be particularly effective in HGSOC. AZD1775 is a small molecule inhibitor of Wee1 kinase that forces cells into mitosis without the repair of damaged DNA. The overall aim of the thesis was to characterise mechanisms of resistance to AZD1775 in cultured HGSOC cells and identify ways to overcome resistance that could be ready to deploy as the inhibitor moved into the clinic. It was hypothesised that resistance to AZD1775 could occur in a number of different ways through changes in key DNA damage response and cell cycle control pathways. To address the additional hypothesis that mechanisms of resistance to AZD1775 could be different in HR-deficient and proficient HGSOC, cell lines from each category were used to generate resistant clones for mechanistic studies. Given the increased use of the PARP inhibitor olaparib as a therapy for HGSOC, olaparib resistant clones were also isolated from the same HR-deficient and proficient HGSOC cell lines with the supplementary aim of investigating the effectiveness of AZD1775 therapy in olaparib resistant HGSOC. I first investigated the effect of AZD1775 as a single agent and in combination with the DNA damaging agent cisplatin and olaparib in a panel of twelve HGSOC cell lines in cell growth and viability assays. AZD1775 enhanced the sensitivity to cisplatin and olaparib in three cell lines from the panel, two of which were HR pathway deficient. To study mechanisms of resistance to Wee1 inhibition, two cell lines, ES-2 and OVCAR8, were chosen representing HR proficient and deficient HGSOC to isolate clones resistant to AZD1775, AZD1775 in combination with cisplatin and olaparib alone. Compared to the parental lines, AZD1775 resistant clones retained normal cell cycle profiles in the presence of AZD1775. No evidence was found for mutation in the coding exons of the Wee1 kinase gene in resistant clones. Western blot analysis revealed reduced Cdk1 expression in many of the resistant clones, while others showed increased Wee1 kinase expression. RNA sequencing based comparison of AZD1775 resistant clones with the parental ES-2 cell line showed an upregulation of TGFβ signalling that fed into the cell cycle control pathway via multiple cell cycle inhibitors. An alternative potential mechanism of resistance in some ES-2 and OVCAR8 clones involved upregulation of the TNF and NF-κB signalling pathway leading to evasion of apoptosis. An attempt to overcome AZD1775 resistance by blocking TGFβ or TNFNF- κB signalling pathways was successful for one resistant ES-2 clone using a TGFβR1 inhibitor. Western blotting of olaparib resistant clones isolated from ES-2 and OVCAR8 showed known changes responsible for olaparib resistance, including reduced expression of 53BP1 and down regulation of Poly(ADP-ribose)glycohydrolase. All but one of the olaparib resistant clones tested remained sensitive to AZD1775. In conclusion, mechanisms of resistance to two important inhibitors for the improved treatment of HGSOC have been identified and ways to overcome resistance in the clinic have been suggested.

Breast cancer remains the most lethal type of cancer for women. A quarter of breast cancer cases display overexpression of the HER2 protein, which is associated with poor prognosis. A major drug for treating HER2 (human epidermal growth factor receptor 2)-positive breast cancer is Herceptin, which reduces HER2 dimerization, thereby inhibiting the major cell proliferation pathways. Despite its effectiveness, resistance to Herceptin is a very significant clinical problem: only a third of patients respond to Herceptin, and two thirds of these relapse within one year due to resistance to the drug. Specific members of the Dual-specificity phosphatases (DUSPs) family act downstream of the HER2 protein in breast cancer and contribute to development of Herceptin resistance. I aimed to model the role of dual specificity phosphatases (DUSPs) and the effect of their inhibition in signalling pathways in HER2-positive breast cancer and resistance to Herceptin. This thesis focuses on the implementation of a multi-disciplinary approach to identify a specific profile of DUSPs, whose inhibition could reduce Herceptin resistance in breast cancer cells or resensitize resistant cells. A further aim was to assess druggability among these DUSPs to guide future drug discovery efforts. First, using Boolean modelling and experimental measurements of DUSP expression during initial treatment of HER2-positive breast cancer cells with Herceptin, I was able to predict regulatory mechanisms of DUSPs, where these mechanisms were partially known. Next, I used a systems biology approach together with experimental data to investigate how DUSP overexpression could favour cell proliferation in resistant HER2-positive breast cancer and to predict how this mechanism could be reversed by targeted inhibition of selected DUSPs. After showing that our Boolean models correctly accounted for resistance when overexpressed DUSPs were kept activated, I then simulated inhibition of both individual and combinations of DUSPs, and determined conditions under which the resistance could be reversed. Further on, I investigated whether inhibiting certain DUSPs resensitized Herceptin-resistant breast cancer cells to the drug. I built a series of kinetic models incorporating the key players of HER2 signalling pathways and simulating a range of inhibition intensities. I observed that when silencing certain DUSPs, resistant HER2-positive breast cancer cells became more responsive to the drug, showing a decrease in resistance, in agreement with the model predictions. Finally, by applying molecular docking and virtual screening of compound libraries together with predictions of pockets and protein-protein interactions, I could assess the druggability among DUSPs in order to guide future drug discovery efforts. Overall, this thesis highlights the value of combining a systems biology approach with wet lab experimentation and computational screening of chemical compounds, with regards to the identification of specific DUSPs playing a role in resistance to Herceptin of HER2-positive breast cancer cells.

The fibroblast growth factor (FGF) signalling pathway contributes to the regulation of a variety of cellular functions, affecting differentiation, migration, proliferation, and survival. Unsurprisingly, cancer cells can hijack this pathway for growth or survival advantages, through alterations in ligands, receptors or regulatory molecules. Sequencing consortia have highlighted how mutation, amplification, translocation or loss of elements in the FGF signalling network can contribute to tumourigenesis, and the pathway is a major clinical target. Many FGF receptor (FGFR) driven cancers develop resistance against commonly used receptor tyrosine kinase (RTK) targeted therapeutics and dissection of the mechanisms that underlie this, both in the cancer cell, and also via stromal crosstalk in the tumour microenvironment, is of utmost importance to the development of therapeutic approaches to treat FGFR-driven cancers. In this work, I focus on the mechanisms of FGF deregulation and the impact of stromal cells. I aim to identify the implications of FGFR2 aberrations on gastric and endometrial cancer, and FGFR1 aberrations on lung cancer. Furthermore, I aim to dissect the mechanisms by which targeted cells may develop drug resistance both in two-dimensional (2D) and in a more physiomimetic three-dimensional (3D) co-culture model. It was established that cancer cells with FGFR aberrations were sensitive towards FGFR inhibitors such as PD173074 (PD), AZD4547 (AZD) and BGJ398 (BGJ). Cancer cells could be killed with increasing concentration of these drugs and exhibited sensitivity to FGFR inhibitors by decreased p-AKT and p-ERK signalling. However, with prolonged exposure to FGFR inhibition, certain cells began to acquire resistance in 2D. To study drug resistance in a more physiomimetic environment, cells were grown alone or co-cultured with fibroblasts and treated with FGFR inhibitors PD, AZD or BGJ and imaged using fluorescent live cell imaging. These cultures were then fixed, paraffin embedded and immuno- or Haematoxylin and Eosin (H&E) stained. To study the emergence of drug resistance in 3D with and without stromal support, cells were seeded into Alvetex® scaffolds and treated with increasing concentrations of BGJ until resistant populations were observed via confocal fluorescence microscopy. Cancer cells grown in co-culture with fibroblasts acquired resistance faster than monoculture cells. Significantly regulated genes between resistant and parental mono- and co-culture cells were identified using ribonucleic acid sequencing (RNA-Seq) and bioinformatics. The majority of significantly regulated pathways in FGFR2-amplifed gastric cancer cells, were metabolic pathways including retinol metabolism, starch and sucrose degradation, which was mainly driven by sucrose-isomaltase (SI) and aldolase B (ALDOB), resulting in glucose generation. Targets were then modified using different approaches such as knockdown, overexpression and drug treatments to observe how this affects resistance of FGFR-driven cancers. Blockade of ALDOB resulted in lower cell numbers within 2D cultures in drug-resistant gastric cancer cells, possibly by slowing down cell growth rather than inducing cell death. However, the effect of glucose or sucrose on cells is possibly through a SI-independent manner. When analysing gene expression in cells grown on 2D versus 3D substrates, expression levels varied considerably, and this underlines the importance of investigating biological processes in a physiomimetic in vivo-like setting. Drug resistance in cancer is an increasing issue for successful therapy and a number of interesting targets have been identified in FGFR2-amplified gastric cancer that could be responsible for drug resistance towards FGFR inhibitors. With stromal support, cancer cells acquired resistance faster, which highlights the influence of fibroblasts and the extracellular matrix (ECM) environment on resistance. It was shown that sugar metabolism plays a substantial part in resistance, together with the retinol pathway. Possibly, these genes and pathways are intertwined, ultimately aiding cancer cells to grow in presence of inhibition via cytoprotective mechanisms and also generation of metabolites to ensure sustained proliferation of cells. The importance of these genes and pathways need to be further evaluated. Therefore, drug combination therapy could be the pivotal way forward to hinder cancer cells from rewiring their pathways to overcome drug inhibition.

Following both significant advances and setbacks in the past decades of fighting malaria, the end goal of malaria elimination is now once again within sight. However, this endgame may prove the most challenging yet, as there are still significant gaps in our understanding of human malaria more generally and as we know from past experience that the malaria parasite is able to rapidly overcome any challenge thrown at it. Despite the huge international endeavour to understand the genomic basis of malaria biology, the genome sequences of two of the five human malaria parasite species, Plasmodium malariae and P. ovale, have remained essentially a mystery. Consequently, the implications of these sequences on aspects such as drug resistance have eluded us. However, even for human malaria parasite species that have been sequenced at large scale, such as P. falciparum, a better understanding of the impact of genetic variation on drug resistance is needed, especially in light of multidrug resistance in Southeast Asia. In this thesis, I have, in collaboration with others, explored these different aspects of human malaria parasites, showing to what extent sequencing data can inform our understanding of human malaria and aid us in our fight against this devastating disease. Initially I assembled reference genome sequences for both P. malariae and P. ovale, an analysis of which I present in Chapter 1. I show that the P. malariae genome is markedly different to other Plasmodium genomes and relate this to its unique biology. Using additional draft genome assemblies, I also confirm that P. ovale consists of two species that appear to have diverged millions of years ago. In Chapter 2, I use the newly assembled P. malariae reference genome in combination with clinical data to characterize a case of clinical recrudescence of a P. malariae infection, and suggest that drug resistance may have played a role. To better understand the ability of malaria parasites to acquire drug resistance, in Chapter 3 I harnessed a large dataset of P. falciparum whole genome sequences with associated phenotype data on mefloquine, an antimalarial drug. I show that the current outbreak of multidrug resistance in Southeast Asia is accompanied by a hyper-sensitization of the parasite population through the acquisition of a complex genetic architecture of mefloquine sensitivity. Finally, in Chapter 4, by incorporating phenotype data on additional drugs, including chloroquine, artemisinin and piperaquine, I identify a specific haplotype of the pfcrt gene that displays super-resistance to chloroquine and that acts as a genetic backbone to artemisinin resistance and to multidrug resistance in general. The approach taken in this thesis is one of extracting new information from layering on additional data. I begin by comparing genome sequences to each other in Chapter 1, I then layer on clinical metadata in Chapter 2, I add in phenotype data for one drug in Chapter 3, and finally, in Chapter 4, I harness phenotype data for multiple drugs. At each level, I identify new biology that both expands our understanding of human malaria in general and sheds light on the specifics of antimalarial drug resistance. The contributions made in this work will be of significant importance in the upcoming end game of malaria elimination.

Coccidiosis is an intestinal disease caused by the protozoan parasite Eimeria, of which E. tenella and E. maxima are a common cause of disease in the poultry industry, causing weight gain loss, decreased feed efficiency and mortality in poultry. Coccidiosis is usually controlled by the application of anti-coccidial drugs or by vaccination, but drug resistance in Eimeria has been reported and vaccines require the passage of live Eimeria oocysts through the birds and are therefore expensive and difficult to produce. Alternative solutions are to develop subunit vaccines and to breed chickens for resistance to Eimeria by identifying resistance biomarkers, both of which require characterisation of the chicken immune response to Eimeria. To characterise immune responses to Eimeria, this study aimed to investigate the response of antigen presenting cells (APC) to Eimeria and determine which chicken pathogen recognition receptors (PRRs) recognise Eimeria vaccine candidates using in vitro techniques. The role of T helper (Th) 17 cells during E. maxima and E. tenella infection was also investigated in vivo through infection of a commercial broiler line. This study also aimed to identify biomarkers of Eimeria resistance by characterising the immune response to E. maxima and E. tenella in chicken lines which exhibit differential resistance and susceptibility to both these Eimeria spp. The development of chicken bone marrow-derived macrophage (BMM) and dendritic cell (BMDC) cultures provides an opportunity to study the responses of host-derived APC to Eimeria antigens and potential vaccine candidates in vitro. Here, both BMM and BMDC responded to an E. tenella oocyst crude lysate by upregulating mRNA expression of proinflammatory mediators (IL1B, IL6 and NOS2), BMM appeared more regulatory in nature (upregulated IL10 mRNA expression) and BMDC appeared more Th1-promoting (upregulated IFNG mRNA expression). Immune mapped protein 1 (IMP1) and apical membrane antigen 1 (AMA1) are two Eimeria vaccine candidates that have been shown to elicit protective immunity to Eimeria. In response to vaccine candidates IMP1 and AMA1, BMM responded in an inflammatory fashion through increased expression of IL6 and NOS2 mRNA. These results indicate that chicken macrophages and dendritic cells can recognise Eimeria and Eimeria vaccine candidates and facilitate inflammation though production of proinflammatory cytokines, but also have roles in promoting Th1 responses and in immune regulation. In order to trigger innate immune responses, pathogen associated molecular patterns (PAMPs) must be recognised by host PRRs, present on the surface of APC. Currently it is not known which Eimeria PAMPs are detected by which chicken PRRs. Use of a reporter gene assay identified that recombinant IMP1 and AMA1 are recognised by Toll-like receptor (TLR)1LB/2A heterodimers however further investigation is needed to determine other Eimeria PAMPs that are recognised by other chicken PRRs. Th1 responses are known to be important for the resolution of Eimeria infection however Th17 responses during Eimeria infection are less well characterised. Thought to contribute to immunopathology during Eimeria infection, Th17 responses represent a potential target in improving the outcome of Eimeria infection in chickens. Surprisingly, RT-qPCR analysis revealed no changes in the mRNA expression of Th17-associated cytokines in the gut of E. maxima- or E. tenella-infected Ross 308 broilers with the exception of IL21, indicating that IL-21 is acting in another capacity than as a Th17 effector during Eimeria infection. IL-21 is a highly pluripotent cytokine and further study would be required to characterise the role of IL-21 during Eimeria infection. In order to breed chickens for resistance, biomarkers of Eimeria resistance must first be identified. Line 15I and C.B12 chickens display inverse resistance and susceptibility to E. maxima and E. tenella. To identify biomarkers of resistance to E. maxima and E. tenella, the immune response of these lines to both Eimeria spp. was phenotyped. A higher increase in serum IL-10 was observed in E. maxima-infected susceptible line 15I than line C.B12 supporting a previous report that IL-10 is involved in susceptibility to E. maxima. RT-qPCR analysis revealed earlier increases of IFNG, IL10 and IL21 mRNA in the gut of resistant line C.B12 birds following E. maxima infection, indicating that a prompt immune response is a factor in resistance to E. maxima. No biomarkers of resistance to primary E. tenella infection were identified and further interrogation of the immune responses of these lines is required, particularly in response to secondary E. tenella infection. The results of this study have furthered our understanding of the role of APCs during Eimeria infection and following vaccination with IMP1 and AMA1 and support IMP1 and AMA1 as suitable vaccine candidates. IL-21 was identified as an important cytokine during Eimeria infection and further study is required to assess if IL-21 is beneficial or damaging to clearance of the parasite and to evaluate its potential as a therapeutic target. This study also confirmed previous findings that IL- 10 is involved in susceptibility to Eimeria and identified that a rapid response is important for resistance to E. maxima, providing a basis for further study to identify biomarkers of Eimeria resistance.

One of the major constraints limiting the efficiency of sheep production is the control of gastrointestinal nematode parasites. Parasite control by strategic use of anthelmintics is threatened by the emergence of nematode populations that are resistant to the drugs available. It is therefore increasingly apparent that steps toward maintaining sustainable productivity in the growing face of anthelmintic resistance (AR) is required by farmers. To facilitate the uptake of sustainable approaches to parasite management, a comprehensive understanding of the various factors that may influence farmers’ decision-making processes is required. In order to establish which factors are influential, and determine their impact on farmers’ roundworm control behaviours, both qualitative and quantitative research methods were used including focus groups as well as attitudinal and behavioural questionnaires. A retrospective analysis was initially conducted of historical surveys designed to identify farm specific characteristics and parasite management practices including anthelmintic usage. The objectives of the analysis were to identify factors associated with uptake of best practice advice including farm characteristics and information sourcing. In addition, the implementation of sustainable roundworm control practices was investigated using two surveys conducted in 2000 and 2010. Logistic regression models were applied for univariable and multivariable analysis of dependent and independent variables. The next step was to conduct a series of focus groups in different geographic regions of Scotland. The main purposes were to explore sheep farmers’ attitudes towards different aspects of roundworm management, as well as to identify potential motivators and barriers to uptake of sustainable parasite control practices. The findings aided in the development of an attitudinal questionnaire used to canvass opinions representative on a national level. The concluding study involved a telephone survey of 400 Scottish sheep farmers, designed to elicit attitudes regarding roundworm control, AR and sustainable roundworm control practices. A quantitative statistical analysis technique (Structural Equation Modelling) was then used to test the relationships between socio-psychological factors and the uptake of sustainable roundworm control practices. The analysis of historical questionnaire data demonstrated evidence of a shift towards the use of practices to reduce the rate of AR development, most notably a decline in the practice of ‘dose and move’ as well as an overall reduction in treatment frequency. Statistical analysis identified significant associations between farm characteristics and specific treatment strategies. For example, larger farms were more likely to adopt a set treatment regimen (P=0.036), compared with smaller farms, which were more likely to treat based on clinical signs of infection (P=0.021). Sourcing of roundworm control information primarily from veterinarians was most associated with treatment timings with no parallels between time points. From the qualitative focus group studies conducted we identified four overarching themes impacting on sheep producers’ attitudes to roundworm control and best practice advice. These themes comprised: a lack of perceived need to change, the complexity of advice, the ease of implementation of recommended practices and the effectiveness of extension approaches. Additionally, the most important and implementable guidelines identified by sheep farmers were: ‘working out a roundworm control strategy with an advisor’ and ‘administering anthelmintics effectively’ with regard to correct drenching practice i.e. appropriate dosing, administration and drug storage procedures. These findings exhibited similarities with veterinarians’ rankings with also, ‘testing for AR’, ‘preserving susceptible parasites’ and ‘reducing dependence on anthelmintics’ receiving the lowest rankings for importance and implementability. The quantitative analysis from the attitudinal/behavioural questionnaire identified eleven factors with significant influences on the adoption of sustainable roundworm control practices. The key influences on overall adoption were farmers’ baseline understanding about roundworm control and self-reported confirmation of anthelmintic resistance in their flock. Additional positive influences included, positive attitudes to veterinary services, enterprise type and perceived risk of AR. Factors that were shown to have the greatest relative effects on individual parasite control practices included; the perceived resource requirements for implementing a quarantine strategy, farmers’ AR suspicions for instigating AR testing and the confirmation of AR for adopting faecal egg count monitoring. The findings have highlighted several factors which can influence sheep farmers’ decisions to reject or adopt recommended roundworm control practices. It is evident that the perceived complexity, lack of need and practicality of the current recommendations necessitates changes to how extension is designed and disseminated to farmers. The findings also suggest that improving farmers’ acceptance and uptake of diagnostic testing and improving underlying knowledge and awareness about nematode control is a significant target to influencing adoption of best practice behaviours. The importance of veterinarians as highly-trusted information resources validates the need for further engagement with veterinarians concerning sustainable parasite control approaches, to facilitate collaboration with farmers. The need for interaction between farmers and their advisors is key to resolving the issues raised to enable the necessary explanation, justification and execution of recommended practices to suit farmers’ needs and farming conditions.

Since the discovery of DNA packaging into chromatin, and McClintock's (1951) work on position-effect variegation providing evidence of non-mendelian inheritance, the principal of a genome maintaining 'on' and 'off' states has been widely adopted. However, the underlying mechanisms that regulate these dynamic chromatin states and their effect on disease are still poorly understood. DNA methylation and histone trimethylation at H3K9 and H4K20 are the core hallmarks of the heterochromatic constitutively 'off' state. Constitutive heterochromatin is predominantly comprised of repetitive satellite containing pericentromeric regions and telomeres and in mouse heterochromatin clusters into large chromocenters. These regions are cytologically more compact and generally transcriptionally silent across embryonic and differentiated mouse cell types. However, in addition to increased genomic instability, mouse tumour cells sustain increased satellite expression suggesting constitutive heterochromatin is disrupted. Therefore how constitutive heterochromatin is maintained has important implications for genome regulation and disease, and remains poorly understood. While satellite DNA sequences are not evolutionarily conserved, pericentromeric and telomeric heterochromatin occurs across species. Heterochromatin formation is therefore independent of the underlying DNA sequence, supporting the hypothesis that epigenetic components can regulate chromatin structure. DNA methylation is generally thought to be associated with transcriptional silencing and chromatin compaction. However, Gilbert et al (2007) showed that the complete loss of DNA methylation did not affect the compaction at heterochromatin or global genome compaction. The role of H3K9me3 in regulating heterochromatin has also been an area of keen interest. H3K9me3 patterns are established by suppressor of variegation 3-9 homologues and provide the binding site for heterochromatic protein 1 [HP1] which can in turn recruit Suv39h1. This Suv3-9h-HP1-H3K9 axis enables its propagation throughout heterochromatin. Peters et al (2001) demonstrated that in mice loss of suv39 homologues 1 and 2 caused a loss of H3K9me3 at constitutive heterochromatic domains. These Suv39h null mice demonstrated decreased genome stability, and an increased prevalence of oncogenesis. However cytological chromocenters are still present in the absence of H3K9me3. Therefore the function of H3K9me3 as a causative agent in heterochromatin formation is still debated. Broadly the aim was to investigate the phenotypic role of heterochromatic epigenetic components in cancer progression, and address whether H3K9me3 effects large scale chromatin structure. To identify heterochromatic gene silencing components, an inhibitor screen was performed in an artificial silenced reporter system. The reporter fluorophore was silenced by the presence of centromeric arrays from yeast/bacterial artificial chromosomes and human alpha satellite repeats enriched for H3K9me3. To address the function of the de-silencing components identified in cancer, the fitness of colon cancer cells [HCT116] was investigated before and after the development of resistance to the MEK inhibitor trametinib. The most intriguing result was that BET protein inhibition resulted in derepression of the reporter construct and trametinib resistant HCT116 cells were more sensitive to BET inhibitors, while subsequent investigation showed HP1 protein levels were altered. Analysis of publically available datasets of tumour drug resistance, showed elevated BET protein binding at HP1 promoters in resistant cell lines suggesting an indirect role in gene silencing. To investigate the consequence of H3K9me3 loss on chromatin structure, mouse embryonic stem cells that lacked both Suv39 homologues were used. Microccocal nuclease digestion and sucrose sedimentation demonstrated a global decompaction of large-scale chromatin fibres whilst re-expression of suv39h1 rescued H3K9me3 at chromocenters and global chromatin decompaction. Loss of Suv39h also increased chromatin associated RNA levels that were also rescued by Suv39h1 re-expression. This suggests that H3K9me3 has a role chromatin fibre compaction globally as well as at constitutive heterochromatin, potentially mediated by chromatin associated RNA. To conclude, multiple components were identified that are involved in transcriptional silencing. Evaluating their function in tumour progression demonstrated a possible role of BET proteins in the development of MEKi resistance that may be mediated through HP1 proteins. H3K9me3 and its binding partner HP1 affect global chromatin compaction. The global decompaction after Suv39h loss correlates with an increase in chromatin associated RNA, suggesting a possible mechanism for changes in chromatin compaction beyond H3K9me3.

Squamous cell carcinoma of the head and neck (HNSCC) is an extremely common and devastating disease with a bleak prognosis. Despite intensive research, survival rates have not improved over the past 30 years principally due to untreatable recurrent/metastasising disease. Feline oral squamous cell carcinoma (FOSCC) is an equally common disease in cats with an even less favourable prognosis than humans. Human and feline squamous cell carcinomas share similar etiopathogenesis, molecular markers, tumour biology and treatment thus making FOSCC an excellent model for HNSCC. Epithelial to mesenchymal transition (EMT), under the direction microRNAs (miRNAs/mirs) could be a key driver in oncogenic transformation and chemoresistance. The aim of this study was to induce resistance to characterise the EMT/resistance phenotype and to investigate whether common miRNA-mediated pathways are present in HNSCC and FOSCC that drive this phenomenon. We used epidermal growth factor (EGFR)-inhibitor gefitinib to induce resistance in HNSCC and FOSCC and investigated the associated EMT-related molecular changes. In vitro and in vivo invasive and migratory properties of both species were explored to determine whether resistance and/or EMT status conferred a functional advantage. We determined the miRNA expression pattern during acquisition of resistance to gefitinib in both species by next generation sequencing and screened candidate miRNAs as potential therapeutics. We found that gefitinib-resistance produced a previously unrecognised biphasic response that consisted of two distinct phenotypes, a highly invasive mesenchymal phenotype during early resistance, and a more epithelial phenotype associated with established resistance. The biphasic nature of this transition may prove critical in establishing effective therapeutic targets and the timing of treatment to overcome resistance or in preventing local invasion or metastatic spread of squamous cell carcinoma. We found that the major anti-apoptotic PI3K/AKT pathway was activated in transitioning and resistant cells of both species as demonstrated upregulation of AKT, pAKT and c-FLIP together with inactivation of PTEN by phosphorylation. This indicates that avoidance of apoptosis may be a major pathway in resistance that could be targeted therapeutically. We showed that three miRNAs were differentially expressed in both gefitinib-resistant human and feline cell lines: miR-107 was downregulated, and miR-551b and miR-574 were upregulated. These microRNAs provide potential therapeutic targets in the fight against drug resistance in head and neck cancer although much further research needs to be conducted to elucidate the complex network of interactions that may be affected by targeting these powerful regulatory molecules.

Castration-resistant prostate cancer (CRPC) remains an aggressive incurable disease in men mainly due to treatment resistance. Current treatments do not effectively eradicate cancer stem cells (CSCs), which play a pivotal role in tumour maintenance, progression and drug resistance. Aldehyde dehydrogenases (ALDHs) have been used in some tumour types as CSC markers. Their high expression and high functional activity found in CSCs is also associated with drug resistance. Emerging evidence suggests deregulation of certain ALDH isoforms have implications in cancer. The role of ALDHs in prostate cancer as potential biomarkers and therapeutic targets has not been fully explored yet. Accordingly, this study investigated the expression, regulation and function of selected ALDH isoforms in prostate cancer. This study showed that ALDH1A3, ALDH1B1, ALDH2 and ALDH7A1 are highly expressed in primary prostate cancer cells (n=9) compared to benign (n=9) prostate cells. The expression of ALDH1A3 was high in the stem cells (SCs) (n=3) as well as the more differentiated counterparts (n=16). Treatment of both benign and malignant primary prostate cancer cells with all-trans retinoic acid (atRA) also resulted in increased expression of ALDH1A3 and ALDH3A1, supporting a feedback loop between atRA and ALDHs. Furthermore, SerBob, Bob and LNCaP cells were sensitive to treatment with epigenetic drugs and led to significantly higher expression of ALDH1A2, ALDH3A1 and ALDH7A1 respectively. Importantly, siRNA suppression of ALDH1A3 and ALDH7A1 led to reduced SC properties of primary prostate cultures including reduced cell viability, migration and colony formation, and increased differentiation of transit amplifying (TA) cells to committed basal (CB) cells. Novel ALDH-affinic probes showed reduced cell viability of primary prostate epithelial cultures as a single agent and also when used in combination with docetaxel. The results indicate the potential of using ALDH-affinic compounds as single agents for therapeutic intervention or in combination with docetaxel to sensitise resistant cells to this anticancer drug. The data in this thesis provides novel findings, which supports ALDH1A2, -1A3 and -7A1 as potential biomarkers and/or therapeutic targets for drug intervention. Although, a study analysing a larger number of samples is necessary to fully understand ALDH isoform expression in CSC, TA and CB cells it is envisaged that an ALDH-targeted therapy have potential in future treatment strategies for prostate cancer.

Hospital-acquired infections are the most frequent adverse event in healthcare delivery worldwide. Seven in ten hospital-acquired infections exhibit resistance to at least one antibiotic, and three in five doctors have encountered an infection unresponsive to any treatment at all. This prolongs hospitalisation, increases suffering, and causes long-term disability and unnecessary death. At present the antibiotic pipeline is unable to meet the demand for novel antibiotics needed to treat these infections. Herein I discuss how the lack of new scaffolds, limitations of target-based screening, and poor target validation each contribute to the current bottleneck in the antibiotic pipeline. In turn, I argue that the development and application of chemical probes is a more fruitful way to make progress towards new antibiotics with novel mechanisms of action. This dissertation describes a combined chemical-biology study in the search for novel inhibitors of the human pathogens Pseudomonas aeruginosa and Staphylococcus aureus. First we extend organocatalysis to the field of diversity-oriented synthesis as a powerful means to generate molecular diversity and complexity in small molecule screening collections. We then study a family of potential biofilm inhibitors identified from a diverse screening collection, and for which we suggest a possible mode of action upon the quorum sensing receptors LasR and RhlR. Thereafter we provide evidence that a novel macrocycle, based upon the cylindrocyclophane family of natural products, inhibits methicillin-resistant S. aureus through action upon the respiratory chain. Finally, we also report insights into the structure and small molecule inhibition of a key P. aeruginosa drug target, malate synthase G. Together the findings in this dissertation encourage the development and application of divergent synthesis and unbiased screening methods in antibacterial discovery.

Despite therapeutic advances, colorectal cancer still has a 45% mortality rate, and one of the most crucial problems is the development of acquired resistance to treatment with anticancer drugs. Thus the aims of this project are to develop drug-resistant colon cancer cell lines in order to identify mechanisms of resistance for the most commonly drugs used in colorectal cancer: 5-fluorouracil, oxaliplatin, and irinotecan. Following evaluation of drug sensitivity to these agents in an initial panel of eight colorectal cancer cell lines, 3 lines (DLD-1, KM-12 and HT-29) were selected for the development of 5-FU (3 lines), oxaliplatin (2) and irinotecan (1) resistant sublines by continuous drug exposure, with resistance confirmed using the MTT assay. Consistently resistant sublines were subject to a „stable isotope labelling with amino acids in cell culture‟ (SILAC) approach and a MudPIT proteomics strategy, employing 2D LC and Orbitrap Fusion mass spectrometric analysis, to identify novel predictive biomarkers for resistance. An average of 3622 proteins was quantified for each resistant and parent cell line pair, with on average 60-70 proteins up-regulated and 60-70 down-regulated in the drug resistant sublines. The validity of this approach was further confirmed using immunodetection techniques. These studies have provided candidate proteins which can be assessed for their value as predictive biomarkers, or as therapeutic targets for the modulation of acquired drug resistance in colorectal cancer.

Cancer cells have been known for some time to have very different metabolismas compared to that of normal non proliferating cells. As metabolism is involvedin almost every aspect of cell function, there has been a recent resurgence ofinterest in inhibiting cancer metabolism as a therapeutic strategy. Inhibitors thatspecifically target altered metabolic components in cancer cells are being developedas antiproliferative agents. However, many such inhibitors have not progressedinto the clinic due to limited efficacy either in vitro or in vivo. In this study weexplore the hypothesis that this is often due to the robustness of the metabolicnetwork and the differences between individual cancer cell lines in their metaboliccharacteristics. We take a systems biology approach. We investigate the cellular bioenergetic profiles of a panel of five non-small celllung cancer cell lines before and after treatment with a novel inhibitor of theglutaminase-1 (GLS1) enzyme. Additionally, we explore the effects of this inhibitoron intracellular metabolism of these cell lines as well as on the uptake and secretionof glucose, lactate and amino acids. To be able to do the latter robustly, wehad to modify the experimental assay considerably from procedures that seemto be standard in the literature; using these earlier procedures the metabolicenvironment of the cells was highly variable, leading to misleading results onthe metabolic effects of the inhibitor. We reduced cell density, altered mediumvolume and changed the time-window of the assay. This led to the cells growingexponentially, appearing indifferent to the few remaining changes. In this newassay, the metabolic effects of the glutaminase inhibitor became robust. One of the most significant results of this study is the metabolic heterogeneitydisplayed across the cell line panel under basal conditions. Differences in themetabolic functioning of the cell lines were observed in terms of both theirbioenergetic and metabolic profile. The amount of respiration attributed tooxidative phosphorylation differed between cell lines and respiratory capacity wasattenuated in most cells. However, the rate of glycolysis was similar betweencell lines in this assay. These results suggest that the Warburg effect arisesthrough a greater diversity of mechanisms than traditionally assumed, involvingvarious combinations of changes in the expression of glycolytic and mitochondrialmetabolic enzymes. The effects of GLS1 inhibition on cellular bioenergetics and metabolism alsodiffered between cell lines, even between resistant cell lines, indicating that theremay also be a diversity of resistance mechanisms. The metabolomic response ofcell lines to treatment suggests potential resistance mechanisms through metabolicadaptation or through the prior differences in the metabolic function of resistantcell lines. Part of the metabolome response to GLS1 inhibition was quite specificfor sensitive cells, with high concentrations of IMP as the strongest marker. Our results suggest that the metabolome is a significant player in what determinesthe response of cells to metabolic inhibitors, that its responses differ between cancercells, that responses are not beyond systems understanding, and that thereforethe metabolome should be taken into account in the design of and therapy withanti-cancer drugs.

Heat shock protein 90 (HSP90), a conserved molecular chaperone, has become a potential molecular target for cancer therapeutics. HSP90 inhibition (HSP90i) causes inhibition of several oncogenic pathways simultaneously and leads to anti-cancer activities in multiple cancers including in triple-negative breast cancer (TNBC). TNBC is a subtype of breast cancer with poor prognosis and lack of approved targeted therapies. Although HSP90i has shown promising initial clinical data, resistance to HSP90i can still arise in TNBC patients and its resistance mechanisms are not yet understood. In this study, using an in vitro system, we report for the first time the isolation of TNBC cells with acquired resistance to HSP90i. Proteome and whole transcriptome profiling, and a bioactive small molecule screen were performed to identify the molecular basis of resistance to HSP90i and potential therapeutic approaches to overcome acquired resistance to HSP90i in TNBC cells. Two independent HSP90i-resistant clones were acquired through prolonged exposure of a TNBC cell line (Hs578T) to HSP90i. The clones showed significant resistance to HSP90i, notably to resorcinol-based HSP90i. The HSP90i-resistant clones also shared genomic sequence variants, suggesting a pre-existing population of resistant cells within the parental cells. We demonstrate that upregulated expression of UGT1A9, possibly due to an increased intrinsic oxidative stress, is associated with acquired resistance to resorcinol-based HSP90i in TNBC cells, and sensitivity to HSP90i can be restored with a competitive inhibitor of UGT1A9. The HSP90i-resistant clones also exhibited slower growth and upregulated IL6- mediated JAK2-STAT3 survival signalling pathway, which might contribute to the crossresistance to chemotherapeutics and other targeted therapies seen in the clones. Finally, we demonstrate that inhibition of JAK2-STAT3 signalling pathway is able to increase the cytotoxic effects of HSP90i to TNBC cells. We conclude that by using in vitro assays, we are able to identify potential mechanisms of acquired resistance to HSP90i in TNBC cells. We propose that expression of UGT1A9 or STAT3 might be a potential biomarker of sensitivity to HSP90i in TNBC cells. A combined inhibition of HSP90 and JAK2 might be a potential therapeutic approach for the development of effective targeted therapies in TNBC patients.

The typical response of cancer patients to treatment is only temporary, and is often followed by relapse. The failure of various therapeutic strategies is commonly attributed to the emergence of drug resistance. The response patterns for patients under such treatments indicate that complex dynamics regulate the response of the tumour to the therapy. The environment in which the tumour lives (the stroma) is known to be a modulator of multiple mechanisms that lead to drug resistance and seems to be a likely candidate for explaining some of this complexity. Understanding the role of stromal cells in the promotion of drug resistance is critical for the design of optimal treatment strategies, and for the development of novel therapies that selectively target both the tumour and the stroma. In this thesis we design two novel mathematical models that describe cancer growth within its environment and the evolution of drug resistance within spatially complex and temporally dynamic tumours. A compartment model captures clinically observed dynamics and allows direct comparison with experimental data, facilitating model parametrisation and the understanding of inter-tumour heterogeneity. An individual cell-based model highlights the key role of local interactions, determining heterogeneity at the tissue scale, that will eventually determine treatment outcome. A non-spatial approximation of this second model allows us to find analytic guidelines for the design of effective therapy. These tools allow the simulation of a range of treatment strategies (including combination of different drugs and variation of schedule) as well as the investigation of therapy response based on patient- or organ-specic parameters. The work developed in this dissertation is based on the paradigmatic biology of melanoma and non-small cell lung cancer. Its results are therefore applicable to a variety of cancer treatments that target similar processes, and whose therapeutic failure can be attributed to environment-mediated drug resistance.

Influenza A viruses are important pathogens of humans, other mammals and birds. Swine are considered to be the ‘mixing vessel’ for influenza viruses because of their susceptibility to infection with not only swine influenza viruses but also human and avian influenza viruses. After infection of pigs with different influenza viruses, reassortment events between genomic RNA segments and point mutations can take place which can result in novel influenza virus strains capable of causing human pandemics. To combat infections, vaccination is available in many countries for humans, but not typically used in pigs. However, anti-influenza drugs have been used to treat livestock, and mutations conferring drug resistance occur in circulating strains. The mechanisms responsible for the emergence and spread of drug resistant mutations against amantadine and oseltamivir have been studied previously but often gave conflicting results. Therefore, this PhD thesis focused on resolving the mechanisms responsible for this rapid drug resistance spread. In chapter one I examine the extent of reassortment events in swine influenza A viruses by analysing within subtype reassortment and extrapolating the results for the between subtype reassortment. Reassortment is one of the mechanisms that can be responsible for mutations, conferring resistance to drugs, to spread between strains, and thus spread in the host population. The findings of this chapter show that the genomic segments most prone to reassortment code for a polymerase (PB1) and both glycoproteins, within all three subtypes studied. Since particular mutations in the matrix protein (MP) segment cause resistance to amantadine, my study focused on MP compared to other segments and revealed moderate level of reassortment. MP reassorts well with polymerases, both within and between subtype, while nonstructural (NS) is least likely to reassort. Chapter two of this thesis aimed at resolving the origin and spread of the most common drug resistance conferring mutation in swine influenza viruses which causes amantadine resistance. I show first that this mutation occurred in swine influenza viruses and was therefore not transmitted from the recently ancestral avian influenza strains, and second that the prevalence of resistance in swine influenza viruses is due to functional linkage of mutations at other sites and not by direct drug pressure. In chapter three I examine the mechanisms responsible for the rapid rise and spread of oseltamivir resistance in human influenza H1N1 viruses which arose in the absence of drug use. The primary mutation lies in the neuraminidase glycoprotein but because of the close functional interaction I focus on changes in haemagglutinin that occurred in association with resistance. The results showed several mutations in haemagglutinin were associated with resistance suggesting selection acting on haemagglutinin in order to balance the activity of both glycoproteins. Overall these results show the importance of functional linkage between segments as a mechanism for the occurrence of drug resistance conferring mutations, and reassortment as a means of spreading these mutations into newly emerging strains.

Genetic polymorphism in Plasmodium falciparum is a considerable obstacle to malaria intervention. Parasites have repeatedly evolved to overcome every front-line antimalarial deployed throughout history, and artemisinin resistant populations are expanding in Southeast Asia. Promising vaccine candidates routinely fail when challenged by the genetic diversity of natural parasite populations, and a recent trial using a blood-stage antigen showed immunity was allele specific. Modern sequencing technologies have revolutionized our understanding of parasite genomics and population genetics by providing access to single nucleotide variation, but characterizing more complex polymorphism remains a key challenge. Solving this problem is important because the selective pressures from drugs and host immunity often create complex polymorphism in the most clinically relevant genes that is missed using standard genotyping methods. In three sections, this thesis is a narrative about 1) encountering complex variation, 2) overcoming it with novel tools, and then 3) innovatively applying those tools to old and new questions. I first show examples of complex variation in a vaccine candidate (EBA-175) and a drug resistance gene (pfcrt) while reporting SNP based analyses of Kenyan and Tanzanian field isolates. While introducing this complex variation I also describe biological insights discovered in these populations. In Kenya I show evidence that chloroquine resistance selects for parasites that are primaquine sensitive, use a GWAS approach to discover new drug resistance loci, and catalogue variation in known resistance genes. In Tanzania I describe the population structure and allele frequencies of parasites from two geographic regions. In the second section of the thesis I develop methods for accessing complex variation and demonstrate their utility by producing de novo assemblies of eba-175, pfcrt, ama1, and msp3.4 from thousands of sequenced samples. Finally, in the third section I apply these tools in depth to eba-175. I comprehensively characterize the SNP and structural variation in eba-175 using an alignment of 1419 de novo assemblies. I use this resource to illustrate the profiles of positive selection across the gene, and corroborate these signals of balancing selection by showing the geographic distribution of the F/C indels and a lesser known 6bp indel positioned between the DBL domains. I then use the alignments to design Sequenom genotyping assays that facilitate a genome wide association study, testing for human associations with the eba-175 indels in the infecting parasite. I close by reporting a potential association on human chromosome 14 with the 6bp indel in eba-175.

Efficient control of sea lice is a major challenge for the sustainable production of farmed Atlantic salmon (Salmo salar (Linnaeus, 1758)). These marine ectoparasites feed on mucus, skin and blood of their hosts, thereby reducing the salmon’s growth rate and overall health. In the northern hemisphere, the most prevalent species is Lepeophtheirus salmonis (Krøyer, 1837). In 2006, global costs of sea lice infections are estimated to have exceeded €300 million, with the majority spent on a limited number of chemical delousing agents. Emamectin benzoate (EMB; SLICE®), an avermectin, has been widely used since its introduction in 2000, due to its convenient administration as an in-feed medication and its high efficacy against all parasitic stages of L. salmonis. However, over-reliance on a single or limited range of medicines favours the emergence of drug resistance and, as a result, the efficacy of this compound in treating L. salmonis has decreased in recent years, as reported from e.g. Chile, Norway, Scotland and Canada. Declining efficacy underlines the need for an improved understanding of the molecular mechanisms underlying EMB drug resistance in L. salmonis. Elucidation of these mechanisms would allow for improved monitoring tools, earlier detection of developing resistance, extended usability of current delousing agents and development of new parasiticides. The work described in this thesis sets out to examine the molecular mechanisms underlying EMB resistance in L. salmonis. In earlier studies, research in nematodes and arthropods has linked drug efflux transporters belonging to the family of ATP-binding cassette (ABC) transporters to ivermectin (IVM) resistance, a parasiticide with high chemical similarity to EMB. ABC transporters such as permeability glycoprotein (P-gp), transport a wide range of substrates, including drugs, and have been suggested to provide a potential molecular mechanism through which EMB resistance might be mediated in sea lice. As an example of such mechanisms, increased expression of P-gp is one of the causative factors for drug resistance in human cancer cells and avermectin resistance in nematode parasites such as Caenorhabditis elegans or Haemonchus contortus. Initial research involved screening for novel salmon lice P-gps that might contribute to EMB resistance. A novel P-gp, SL-PGY1, was discovered using a combined bioinformatic and molecular biological approach. The expression was compared in two well-characterised L. salmonis strains differing in their susceptibility to EMB (S = susceptible, R = resistant). Prior to EMB exposure, mRNA levels did not differ from each other, while, after 24 h exposure, a 2.9-fold increase in SL-PGY1 mRNA expression was observed in the R strain. SL-PGY1 appears not to be a major factor contributing to reduced EMB susceptibility, although it could play a role, as expression levels increased upon exposure to EMB. A further four additional drug transporters (ABC C subfamily) were also discovered showing high homology to multidrug-resistance proteins (MRP). The relative expression levels of each MRP was compared in the strains S and R, before and after exposure to EMB. No significant changes were found in their expression patterns. If ABC drug transporters mediate the efflux of EMB and thereby reduce the intracellular concentrations of the drug in exposed animals, the inhibition of those ABC drug transporters was expected to lead to higher intracellular levels of EMB. This could result in an enhanced toxic effect when EMB is co-administered with an inhibitor. Two known inhibitors of human P-gps and MRPs, cyclosporin A (CSA) and verapamil (VER), were co-administered with EMB. CSA increased the toxic effect of EMB in both tested strains, implying that the targets of CSA are expressed at comparable levels and that they may be part of the mechanism conferring EMB resistance. VER increased the toxic effect of EMB in the R strain, but had no significant effects on the S strain. This implies that the expression of factors inhibited by VER differs between the two L. salmonis strains. It is hypothesised that a number of ABC transporters with distinct, yet overlapping patterns of inhibitor specificity are affected by those inhibitors. The search for drug-resistance conferring genes was complemented with a systematic, genome-wide survey of ABC transporters in L. salmonis to find additional members of this important gene family. Next-generation high-throughput RNA sequencing (RNA-seq) was employed to assemble a reference transcriptome from pooled total RNA of salmon lice at different development stages. The transcriptome was assembled against the L. salmonis genome and annotated. Thirty-nine putative ABC transporters were found. Of further interest were transcripts of the subfamily B, C and G, as they contain drug-transporting ABC proteins. For the ABC B subfamily, one full (SL-PGY1) and three half transporter transcripts were found. Only full transporters are known to transport drugs and SL-PGY1 is apparently not a major factor contributing to EMB resistance. Fourteen ABCC sequences were found – 11 MRPs and 3 homologues to sulfonylurea receptors. Of interest are MRPs, as they contribute to drug detoxification in humans and invertebrates. Four MRPs had been identified previously and their expression ratios did not differ between S and R strain parasites. Seven sequences belonging to ABCG subfamily were found. However, none of the L. salmonis ABCG transcripts identified showed sufficient homology to known drug transporters in other species. With the currently limited understanding of the mechanisms conferring EMB resistance, monitoring the susceptibility of L. salmonis subpopulations is essential. Dose-response bioassays are currently widely used. Tests with pre-adult II or adult parasites requires relatively large numbers of parasites (~150) to conduct this type of bioassay, which may not always be available. Addressing this issue, we tested the feasibility of a single-dose bioassay (requiring fewer test animals than dose-response bioassays) to discriminate between L. salmonis strains with differing EMB susceptibility. This alternative approach uses time-course toxicity analysis, where the toxic effect of EMB is monitored over time. After clearly defining the effect criteria, we found that it is possible to discriminate between those L. salmonis strains. However, while requiring fewer test animals, time course toxicity analysis is more labour-intensive, but the alternative design can be suitable under certain circumstances. The work reported here has provided new knowledge concerning the mechanisms of EMB resistance in sea lice. Several novel putative drug transporters have been identified, an important first step toward unravelling the complex interactions of genes involved in EMB resistance in this commercially important parasite.

To combat the ever-growing clinical burden imposed by antibiotic-resistant pathogens, multiple-antibiotic treatments are increasingly being considered as promising treatment options. The impact of multiple-antibiotic treatments on the evolution of resistance is not well understood however, and debate is ongoing about the effectiveness of various multiple-antibiotic treatments. In this thesis, I investigate how aspects of multiple-antibiotic treatments impact the rate of evolution of antibiotic resistance in the opportunistic human pathogen Pseudomonas aeruginosa. In particular, I look at the impact of interactions between antibiotics in combination on the evolution of resistance, and how creating heterogeneity in the antibiotic environment by rotating the antibiotics used may change the rate of evolution of resistance. I characterise the interactions present in 120 combinations of antibiotics and find that the type of interaction can be predicted by the mechanism of action of the antibiotics involved. I investigate the effect of a subset of these combinations on the evolution of antibiotic resistance. My results refute the influential but poorly-evidenced hypothesis that synergistic combinations accelerate the evolution of resistance, even when synergistic combinations have the same inhibitory effect on sensitive bacteria as additive or antagonistic antibiotic combinations. I focus on a combination of the antibiotics ceftriaxone and sulfamethoxazole and test whether it is more effective in preventing the evolution of resistance than predicted by the inhibitory effect of the combination on sensitive bacteria. I do not find the combination to be more effective than predicted. Finally, I create heterogeneous antibiotic environments by rotating the antibiotic present at different rates. For the first time in a laboratory setting, I test how varying the rate of fluctuation in the antibiotics present in a heterogeneous antibiotic environment impacts the rate of evolution of resistance. Unexpectedly, I find the rate of evolution of resistance increases with increasing levels of antibiotic heterogeneity.

The synthesis, characterization, and evaluation of antifungal activity of peptides FQ15 and FG10 will be conducted to address the hypothesis. The hypothesis of this thesis is that a peptide with similar properties to AS15 would have similar activity against Cryptococcus neoformans. This study addresses the challenge of drug- resistant microbes by developing inhibitors targeting lipid flippase and exploring their potential as antimicrobial agents against Cryptococcus neoformans. Chapter 1 provides a historical context of the background on the development of antimicrobials and common antifungal treatments, as well as an introduction about C. neoformans and its resistance mechanism. Chapter 2 introduces the materials and the experimental methods used for the drug discovery of the antifungal peptide designed to target the fungus pathogen C. neoformans. Chapter 3 reviews the preliminary results and discussion. Lastly, Chapter 4 is the conclusion of this study. FQ15 and FG10 peptides demonstrated efficacy against C. neoformans in vitro. C. neoformans is a challenging and threatening fungal pathogen that is highly resistant to caspofungin drug, which poses a significant potential threat to cause severe meningitis in immunocompromised individuals like those with HIV/AIDS. This ongoing study aimed to investigate antifungal properties, focusing on exploring the elements within CDC50 loop region of the fungal pathogen C. neoformans to develop potential flippase inhibitors to design peptides as antifungal agents. Previous research has highlighted the essential role of Cryptococcus lipid translocase (flippase) in caspofungin resistance mechanism. 3B lab members focused on identifying peptides resembling the original AS15 peptide with similar biophysical characteristics, including hydrophobicity, net charge, isoelectric point, and molecular weight within the CDC50 loop sequence to demonstrate antifungal efficacy. Flippase inhibitors show promise in combating drug-resistant microbes. Solid phase peptide synthesis (SPPS) was utilized to synthesize 15 amino acid-long sequences, FQ15 and FQ15MA, and 10 amino- acid long sequences, FG10 and FG10MA. The utilization of Mass Spectroscopy (MS) and High-Performance Liquid Chromatography (HPLC) confirmed the detection and isolation of the synthesized peptides. HPLC played a role in analyzing peptide detection and fractions isolation. Adding myristic acid on the N- terminus of the same amino acid sequence was done to explore its potential as an antimicrobial agent, and it showed efficacy at higher concentrations. FQ15MA demonstrated an MIC of 16 μg/mL against H99 and FG10MA demonstrated an MIC of 8 μg/mL against H99 wild type and 64 μg/mL against ΔCDC50 mutant, and a fractional inhibitory concentration (FIC) index value of less than 0.5 when combined with caspofungin.

Caligid copepods, also called sea lice, are ectoparasites of marine fish, with Lepeophtheirus salmonis (Krøyer, 1837) emerging as a problem for mariculture of Atlantic salmon (Salmo salar Linnaeus, 1758) in the northern hemisphere. Annual costs of sea lice to global salmon farming was estimated to be in excess of €300 million in 2006, with the majority of this accounted for through expenses accrued from chemical treatments. Only a limited range of anti-sea louse drugs are available and licensed for the treatment of fish, and the continued use of only a few compounds creates a situation potentially favouring the development of drug resistance. Emamectin benzoate (EMB) is currently used as a salmon delousing agent, being employed as a 0.2 % in-feed pre-mix (SLICE®). Atlantic salmon farmers have reported increased incidence of reduced L. salmonis sensitivity to SLICE®, which has highlighted the requirement for further research into the molecular mechanisms controlling salmon louse resistance to EMB. Genomic and transcriptomic research concerning L. salmonis drug resistance mechanisms has not often been reported, with previous transcriptomic studies using candidate gene approaches and genetic studies focussing on population genetics. Drug resistance in ecdysozoan invertebrates is associated with a variety of molecular mechanisms including target site mutations and changes in the expression of components in drug detoxification pathways. The research reported in this thesis was aimed at the exploration of mechanisms employed by L. salmonis to reduce the toxicity of EMB exposure, following a transcriptomic approach that utilised custom oligonucleotide (oligo) microarrays and a genetic approach that utilised Restriction-site associated DNA sequencing (RAD-seq) to identify Single Nucleotide Polymorphism (SNP) markers. An EMB-resistant (PT) and drug-susceptible (S) L. salmonis laboratory-maintained strain were to be used as a model for this research, as these two strains differ in EMB susceptibility (~ 7-fold) and show stable susceptibility profiles through multiple generations, suggesting that this drug resistance phenotype may be a heritable trait. Sequence resources available for salmon lice are limited as an annotated L. salmonis genome is currently under construction. Therefore, a significant amount of this study involved creating new resources to facilitate the analysis of EMB susceptibility. Suppression subtractive hybridisation (SSH) was used to enrich for transcripts that were differentially expressed between strains PT and S, which provided sufficient target sequence for the development of 15K oligo microarrays when combined with sequences assembled from existing L. salmonis ESTs. Additionally, transcripts were generated through sequencing a pooled sample representing key developmental stages of the L. salmonis life cycle, which were later used in the construction of a 44K oligo microarray. The toxicity of EMB and other avermectins (AVMs) against ecdysozoan invertebrates is reported to be based mainly on their interaction with ligand-gated ion channels (LGIC), specifically glutamate-gated chloride channels (GluCl). However, -aminobutyric acid (GABA)-gated chloride channels (GABA-Cls) are also believed to be targeted by AVMs and neuronal acetylcholine receptors (nAChRs) can be allosterically modulated by the AVM compound ivermectin. Transcriptional responses in PT and S salmon lice were investigated using custom 15K L. salmonis oligo microarrays. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains. GABA-Cl and nAChR subunits showed significantly lower transcript levels in PT compared to S lice, which was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR using RT-qPCR, suggesting their involvement in AVM toxicity in caligids. Although, salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 µg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. RAD-seq analysis of both genders from L. salmonis strains S and PT identified 15 RAD-markers that show complete association with salmon louse strain, although these preliminary results will need further analysis to confirm marker association with reduced EMB susceptibility. Additionally, RAD marker Lsa101901 showed complete association with sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay, this SNP association pattern was further confirmed for three unrelated salmon louse strains. Marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. In conclusion, the identification of decreased transcript abundances for LGIC subunits in EMB-resistant salmon lice, and polymorphic SNP markers showing complete association with L. salmonis strains S or PT, provides suitable candidates for further investigation into their association with reduced EMB susceptibility. Further analysis will also be required to confirm whether EMB-induced mechanisms are not associated with reduced EMB susceptibility in L. salmonis. Additionally, the identification of sex-linked SNP Lsa101901 suggests that sex determination in the salmon louse is genetic and follows a female heterozygous system, with marker Lsa101901 providing a tool to determine the genetic sex of salmon lice. Improved knowledge of L. salmonis biology and the mechanisms potentially involved in EMB resistance, obtained during this study, may provide molecular markers that contribute to successful monitoring and management of this commercially important parasite of Atlantic salmon.

Antibiotic resistance in bacteria is a growing threat to global health. Many of the genes responsible for resistance are carried on mobile genetic elements which can be transferred laterally between strains and species. The most important of these are conjugative and mobilisable elements including plasmids and integrating and conjugating elements, ICEs. Haemophi/us influenzae is an important human pathogen, which was first identified as carrying antibiotic resistance genes in the 1970s. Much of this resistance is encoded by ICEHin1056, which is present in H. influenzae strains worldwide. The aims of this study were to describe features of the biology of ICEHin1056, with particular reference to the genetic site and control mechanisms responsible for instigating conjugative transfer. The origin of transfer has been localised to a sequence on ICEHin1056 and an environmental stressor initiating conjugative transfer, oxidative stress, has been identified. In addition, detailed phylogenetic analysis has demonstrated ICEHin1056 to be part of a much larger family of mobile genetic elements, widely distributed in proteobacteria and carrying accessory genes responsible for survival in adverse environments, virulence and antibiotic resistance. The ICEs in the family have conserved homology of gene content and synteny of gene arrangement over deep evolutionary time, challenging the accepted paradigm of modular mosaicism of mobile genetic elements. A key event in increasing dissemination of the ICE, acquisition of a phage type integrase gene has also been identified. The findings presented provide significant insight into the behaviour of ICEs and may in future allow predictions about the spread of virulence factors and antibiotic resistance genes, with important implications for human and animal health.

Resistance to antimalarial drugs continues to be a major obstacle in controlling and eradicating malaria. The identification of genetic markers of resistance is vital for disease management but they can be difficult to predict before resistance arises in the field. This thesis describes an alternative approach to gene identification, combining an in vivo experimental evolution model, Linkage Group Selection (LGS) and Solexa genome re-sequencing. Here this model was used to resolve the genetic basis of chloroquine and artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi. AS-30CQ is a parasite with high resistance to chloroquine and resistance to artemisinin. It was crossed with the genetically different drug-sensitive strain AJ. The resulting progeny were selected with drugs and backcrossed to the sensitive parent. Both crosses were treated with increasing concentrations of chloroquine and artemisinin. The frequency of markers from the sensitive parasite were analysed in order to characterize the signatures of drug selection. Three loci involved progressively in chloroquine resistance were identified on chromosomes 11, 3 and 2. One main locus on chromosome 2 was identified with artemisinin selection. The Solexa platform was used to re-sequence the genomes of both AS-30CQ and its sensitive progenitor, AS-sens. The differences between the two genomes were integrated with the LGS data to identify: 1) a strong candidate for the main CQresistance determinant - a putative amino acid transporter on chromosome 11 (aat1) 2) two candidates for high level chloroquine resistance on chromosome 3. and 3) a mutation in ubp1 gene on chromosome 2 that is likely to contribute to the highest level of chloroquine resistance and be main determinant of the artemisinin resistance phenotype. In addition the last section of this thesis describes two otherwise isogenic clones showing low- and high levels of chloroquine resistance were grown competitively to evaluate the effect of these mutations on parasite fitness. The highly resistant strain demonstrated a loss of fitness in relation to its more sensitive progenitor and was outcompeted in untreated and low-treated infections.

Drug resistance is a significant challenge in the fight against malaria. Importantly, reduced efficacy has been reported against artemether (ATM)/Lumefantrine (LM) (LM-ATM), amodiaquine (AQ)/artesunate (AS) (AQ-AS), two important combination treatment regimens in Africa, and against piperaquine (PQ), a drug which has been evaluated as a potential alternative in Africa, in combination with dihydroarteminisin (DHA). Chloroquine (CQ) resistance in P.falciparum is associated with two main transporters PfCRT and PfMDR1. I investigated the mechanisms of resistance to PQ, LM and AQ, with the overall goal of identifying molecular markers that can be used to track resistance. I used CQ as a reference. The key antimalarial drugs were highly active against clinical isolates from Kilifi, Kenya with median inhibitory concentrations (IC50s) of <5nM for DHA and <55 nM for CQ, AQ, PQ, LM and DEAQ (desethylamodiaquine, the active metabolite of AQ). pfcrt-76 and pfmdr1-86 mutations were associated with AQ, DEAQ and LM but not DHA or PQ activity. Interestingly, > 20% of analysed isolates had decreased susceptibility to LM (IC50 >100nM); these isolates were the most susceptible to CQ and carried wild type genotypes at pfcrt-76 and pfmdr1-86. I observed that CQ resistance had been declining in Kilifi since 1993 (prior to CQ withdrawal) to 2006 (7 years after its withdrawal), similar to observations in Malawi. My results support the hypothesis that susceptibility to antimalarial drugs returns when drug pressure is removed, and suggest that the use of LM-ATM may hasten the return of CQ susceptibility. Continued monitoring of drug susceptibility is crucial. pfcrt-76 and pfmdr1-86 may be useful molecular markers of LM-ATM efficacy in Kilifi and other African sites. Using a microarray approach, I identified additional genes (including various transporters) that may contribute to LM resistance. I recommend further studies to clarify the exact roles of the identified genes.

The genus Acinetobacter currently contains 34 species, the vast majority of which are not regularly implicated in causing infection. However, incidences of hospitalacquired infection with Acinetobacter species are increasing, mainly due to the rise in the number of infections caused by the species Acinetobacter baumannii in immunocompromised patients particularly in intensive care units (ICUs). Due to high levels of resistance in A. baumannii to many classes of antibiotic, the carbapenems have been portrayed as the ‘drugs of choice’ for treating infections with this species. However, the activity of the carbapenems against A. baumannii has come under threat with the identification of four groups of class D β-lactamases carried by members of the species. Of these, the OXA-51-like enzymes have been suggested to be ubiquitous and intrinsic enzymes within A. baumannii. This presents the worrying scenario of the potential for all A. baumannii to become carbapenem resistant, leaving few treatment options available for this species. This project aimed to investigate the epidemiological spread of the OXA-51-like β-lactamases, examine the diversity within these enzymes, and whether this diversity may have implications for their ability to confer resistance to the carbapenems. A functional map showing the amino-acid similarities between the OXA-51-like enzymes demonstrated that the enzymes fall into distinct closely-related groups, with notable clusters surrounding OXA-66, OXA-69 and OXA-98. PCR and sequencing analysis of a geographically diverse group of 64 A. baumannii isolates demonstrated that isolates forming specific sequence groups (SGs) as defined by Turton et al (2007) also contained the same or closely related blaOXA-51-like gene. Higher minimum inhibitory concentrations (MICs) of carbapenems were found in association with acquired carbapenemases, or with the presence of ISAba1 upstream of the blaOXA-51- like gene. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates did not demonstrate relatedness between isolates which formed the same sequence group. Multilocus sequence typing (MLST) of a subset of 44 isolates grouped isolates more consistently with the SGs and blaOXA-51-like alleles, indicating that PFGE is unreliable for use with A. baumannii unless studying a short time period, and that blaOXA-51-like alleles are a good epidemiological marker. Mutation studies using meropenem with five A. baumannii isolates encoding different OXA-51-like enzymes, while resulting in an increase in meropenem MICs of between 8- and 128-fold, did not result in a nucleotide substitution in the blaOXA- 51-like genes or a change in the upstream region of the genes in any isolate suggesting that the carbapenems may not be producing a strong selective pressure on the blaOXA- 51-like genes. Analysis of πN/πS ratios for the blaOXA-51-like genes, MLST genes and the TEM, SHV and CTX-M β-lactamase families showed the blaOXA-51-like genes to be under less positive selection than these other β-lactamases, though under less purifying selection than the MLST genes. Phylogenetic analysis of the MLST genes and the blaOXA-51-like genes indicates that the blaOXA-51-like genes have been evolving within A. baumannii since its speciation, and that different groups of blaOXA-51-like genes have been evolving at different rates corresponding to different rates of evolution within their parent lineages. Structural modelling studies based upon the published crystal structure for OXA-40 indicated that amino-acid variation at particular sites in the OXA-51-like enzymes are likely to have an effect of enzyme function. Alterations at amino-acid position 167 change the shape of the entrance to the active site which may affect hydrolysis by accommodating the antibiotic differently, or may affect the substrate profile of the enzyme. The substitution of glutamine for proline at position 194 may significantly alter the shape of the enzyme thereby affecting substrate hydrolysis. This project found that specific groups of blaOXA-51-like genes are associated with specific A. baumannii lineages and that these genes could serve as convenient epidemiological markers. Most of the diversity within the OXA-51-like enzymes is due to their continued evolution within A. baumannii since the species’ emergence. However, certain amino-acid changes may play a role in altering the rate of hydrolysis or substrate profile of these enzymes.

Drug resistance is a serious problem in health care in general, and in malaria treatment in particular, rendering many of our previously considered ‘wonder drugs’ useless. Recently, large sums of money have been allocated for the continuous development of new drugs to replace the failing ones. We seem to be one step behind the evolution of antimalarial resistance; is it possible to get one step ahead? Are interventions which slow down the evolution and spread of drug-resistant malaria parasites achievable? In this thesis, I address these issues with experimental data, using the well-established rodent malaria model Plasmodium chabaudi to understand the selective advantages and disadvantages drug-resistant parasites endure within a vertebrate host and the selective pressures various drug treatment regimes exert on these parasites. Competitive interactions between drug-resistant and drug-sensitive parasites were observed within the host, with resistant parasites having a competitive disadvantage in the absence of drug treatment. The frequency of resistant parasites at the start of the infection was an important determinant of the strength of selection: the lower their frequency, the stronger the competitive suppression in non-treated hosts and the greater their competitive release following drug treatment. Genetically similar genotypes, one resistant and one sensitive, showed similar dynamics following drug treatment. Multiplicity of infection did not have an effect on the within-host dynamics: a larger number of co-infecting susceptible genotypes did not lead to greater competitive suppression or release of resistant parasites. Lastly, various drug treatment regimes were compared. Conventional drug treatment resulted in the greatest selective advantage for drug-resistant parasites, while less aggressive treatments were equally as effective, or even better, at improving host health and reducing overall infectiousness. These studies demonstrate that altering the within-host ecology of drug-resistant parasites by administering drugs and hence removing the drug-sensitive competitors has a large influence on the transmission potential of drug-resistant parasites. Furthermore, this thesis provides proof of principle that other drug treatment regimes different from those currently in use could better control drug-resistant parasites, without compromising other treatment goals. In the case of malaria, less drugs may mean extending the useful lifespan of that drug.

Drug resistance is one of the most medically relevant forms of pathogen evolution. To date, vaccines have not failed with the same depressing regularity as drugs. Does that then make vaccines evolution-proof? In the face of vaccination, pathogens are thought to evolve in two ways: by evolving epitope changes at the antigenic target of vaccination (epitope evolution); or by evolving changes at other antigenic loci, some of which may involve virulence (virulence evolution). The fundamental difference between these two forms of evolution is that virulence evolution could lead to disease outcomes in unvaccinated people that are more severe than would have been seen prior to evolution. One of the theoretical assumptions of virulence evolution is that more virulent parasites will have a selective advantage over less virulent parasites in an immunized host, and are thus more likely to be transmitted. The assumption is that more virulent parasites may be competitively more superior in mixed infections, or may be better able to evade/modulate the host immune response. Thus, the aim of this thesis was to experimentally test whether more virulent parasites have a within-host selective advantage in an immunized host or whether vaccine efficacy is more likely to depend on genetic differences at the targeted sites of vaccination. I used clones (genotypes) of the rodent malaria Plasmodium chabaudi originally derived from wild-caught Thicket (Thamnomys rutilans) rats to infect laboratory mice and a rodent analogue of the candidate blood-stage malaria vaccine apical membrane antigen 1 (AMA-1). I found that within-host selection did not depend on parasite virulence, and that protective efficacy depended on genotype-specific differences at the vaccine target. Vaccine-induced protection was not enhanced by including a number of allelic variants. However, such genotype-specific responses were only observed when the vaccine was tested against genetically distinct P. chabaudi parasites. When one P. chabaudi genotype was serially passaged through naïve mice the derived line was more virulent and was subsequently less well controlled by vaccine-induced immunity. In other experiments I found within host competition not to be immune-mediated. Thus my results suggest that vaccination has the potential to select for more virulent parasites but that the selective advantage is likely to be independent of competition. The selective advantage may be attributable to the enhanced immune evasion of more virulent parasites. However, without genetic markers of virulence, the mechanisms that mediate this selection remain unknown. My thesis contributes towards a growing body of evidence that vaccines have the potential to differently alter the within-host parasite dynamics of particular pathogen genotypes and that the selection imposed is likely to be system specific, depending on the fine specificity of the vaccine-induced responses and the identity of infecting parasites. Although vaccine potency may not be enhanced by including more than one allelic variant of an antigen, multi-valent vaccines may be one of the best ways to avoid the inadvertent selection for more virulent malaria parasites.

The aim of this thesis was to isolate the compounds responsible for the antibacterial activity of cell extracts of the marine diatom, Phaeodactylum tricornutum. Marine microalgae are not only important primary producers but, due to their phylogenetic diversity, they are also a potential source of novel bioactive compounds. The marine diatom, P. tricornutum, was selected for study because its cell extracts are known to be antibacterial but the compounds responsible have not been isolated. In this thesis, the compounds responsible for the antibacterial activity are isolated from aqueous methanol P. tricornutum cell extracts by column chromatography and reverse phase high-performance liquid chromatography using a bioassay-guided approach. The compounds in three active fractions were identified by mass spectrometry and nuclear magnetic resonance spectroscopy as the unsaturated fatty acids (5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid, (9Z)-hexadecenoic acid and (6Z, 9Z, 12Z)-hexadecatrienoic acid. The fatty acids were found to be antibacterial against Staphylococcus aureus at micromolar concentrations. P. tricornutum exists in different cell morphs and, interestingly, extracts prepared from cultures in the fusiform morph were found to have greater antibacterial activity than extracts from oval cultures. This is explained by greater levels of the three antibacterial fatty acids in the fusiform cell extracts. The antibacterial fatty acids are proposed to be released by enzyme action when the diatom cells lose their integrity. The release of free fatty acids by diatoms is suggested to be a simple, very low cost population-level activated defence mechanism against potential pathogenic bacteria triggered when the cell loses its integrity. Further, this pathway may act against multiple threats to the microalga, including grazers, as fatty acids exhibit activity in diverse biological assays. Finally, whilst two of the fatty acids, (9Z)-hexadecenoic acid and (5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid, inhibited the growth of MRSA their usefulness as therapeutic compounds may be limited due to their instability and their broad biological activity.