Due to its selectivity in killing cancer cells, Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has provided a potential new agent for cancer treatment. However, despite promising pre-clinical results, it appears that TRAIL therapies will be most effective when used in combination with a sensitizing agent. In light of previous evidence suggesting that cell adhesion could influence sensitivity to Tumour necrosis factor family ligands, this thesis presents a study of the effects of disrupting matrix adhesion on the sensitivity of human MDA-MB-231 breast and 1205Lu melanoma cell lines to TRAIL-induced apoptosis. This was investigated using a number of models including i) culturing cells on normal and low attachment plates; ii) disrupting the transcription of genes involved in cell attachment and spreading in MDA-MB-231 cells using shRNA to Myocardin-related transcription factors-A and B (MRTF-A/B); iii) disrupting the integrin signalling pathway using inhibitors or siRNA to β integrin subunits, talin, integrin-linked kinase (ILK), focal adhesion kinase (FAK) and SRC. With the exception of ILK depletion, disruption of cell adhesion and spreading in all models resulted in sensitisation to TRAIL-induced apoptosis. Cells under these conditions also showed alterations in death receptor signalling and amplification of intrinsic apoptosis pathway signalling through caspase-9. Both MRTF-A/B depleted cells and those treated with the SRC family kinase inhibitor PP2 showed alterations in signalling through ERK1/2. When investigated in an experimental model of metastasis in mice, FAK and SRC inhibitors increased the clearance of MDA-MB-231 cells from the mouse lung when used in combination with recombinant human TRAIL therapy. By utilizing these models, the work in this thesis has shown that disrupting cell adhesion could provide a new combination strategy to sensitise tumour cells to TRAIL therapy.

Centriole and basal bodies are related nine-fold symmetric microtubule-based eukaryotic organelles central to the organisation of cilia/flagella and centrosomes. Mechanisms of eukaryotic centriole and basal body assembly are mainly based on studies in animal systems. To understand which centriolar proteins are the universally important ones in the assembly across eukaryotes, a bioinformatic survey presented here investigates the distribution of centriolar and cilia-associated proteins across a diverse range of eukaryotes. This analysis showed also that the basal body function is ancestral to eukaryotes, whereas centrosomal components are specific to Holozoa (which include animals). It also suggested that the ancestor of all eukaryotes possessed a cilium/cilia not only with motility function but also with a sensory role. The most frequently conserved proteins in extant ciliated eukaryotes found in this analysis included SAS-6, SAS-4 and WDR16. To test whether these proteins are also important for basal body assembly in distantly-related species to metazoan and other model organisms where the proteins have been studied to date, the proteins were investigated in Trypanosoma brucei. I used a combination of genetic tools and microscopy techniques to demonstrate that SAS-6 but not SAS-4 is essential for basal body assembly in T. brucei. I showed that WDR16 is a stably integrated component of the transition zone and axoneme but not the basal body. Furthermore, I identified a novel SAS-6 like protein which localises to a position consistent with the basal plate and has the capacity to form into filaments. This thesis provides new insights into the evolution of centrioles and basal bodies, and into the function of conserved centriole proteins in T. brucei, a distantly-related organism to animals.

The physics of the series Pr_yY_1-yBa₂Cu₃O_6+x, and ability of Pr to suppress superconductivity, has been a subject of frequent discussions in the literature for more than a decade. This thesis describes a polarised neutron diffraction (PND) experiment performed on PrBa₂Cu₃O_6.24 designed to find out something about the electron structure. This experiment pushed the limits of what can be done using the PND technique. The problem is one of a limited number of measured Fourier components that need to be inverted to form a real space image. To accomplish this inversion the maximum entropy technique has been employed. In some cases, the maximum entropy technique has the ability to increase the resolution of ‘inverted’ data immensely, but this ability is found to depend critically on the choice of constants used in the method. To investigate this a Bayesian robustness analysis of the maximum entropy method is carried out, resulting in an improvement of the maximum entropy technique for analysing PND data. Some results for nickel in the literature have been re-analysed and a comparison is made with different maximum entropy algorithms. Equipped with an improved data analysis technique and carefully measured PND data for PrBa₂Cu₃O_6.24 a number of new interesting features are observed, putting constraints on existing theoretical models of Pr_yY_1-yBa₂Cu₃O_6+x and leaving room for more questions to be answered.

The Protein kinase C (PKC) superfamily is a key regulator in platelet activation with individual isoforms playing distinct roles. This thesis focuses on the role of the novel PKC isoforms downstream of several agonists using both pharmacological and genetic approaches and human and mouse platelets. Quantification of the protein levels of PKC isoforms identified different levels of the five major PKC isoforms expressed in human platelets and also differences between levels of the same isoform in human and mouse platelets. Use of a selection of broad spectrum and isoform-specific inhibitors, identified both positive and negative novel roles for PKC in the regulation of human and mouse platelets. A net positive role for PKC was found in GPVI, Clec-2, and PAR receptor signalling, with classical isoforms of PKC playing a major role in aggregation and dense granule secretion. A novel negative regulatory role was also identified in the regulation of ADP-induced platelet activation for PKC~, and both PKCE and PKC~ in human and mouse platelets respectively. Gene knock-out mouse models confirmed a positive regulatory role for PKCe in allb~3 outside-in signalling but identified no other regulatory role for PKCe in agonist induced platelet activation. Despite this relatively minor role, functional redundancy was identified between PKCe and PKCE isoforms in haemostasis, as tail bleeding was significantly increased in mice deficient in both novel isoforms. The work presented here identifies key roles for the PKC superfamily in the complex regulation of platelet activation, with different isoforms supporting and limiting the process of thrombus formation and haemostasis.

During severe hypoxia (<0.01% oxygen) the protein folding machinery becomes dysfunctional, resulting in the accumulation of unfolded proteins with consequent endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) and autophagy, a process involved in the physiological turnover of cytoplasmic components. The link between the UPR and autophagy is not clearly defined. The aim of this thesis is to investigate the role of the induction of UPR under severe hypoxia in tumour survival and resistance to therapy. The results of this research suggest that the activating transcription factor 4 (ATF4), a component of the PKR-like ER kinase (PERK) pathway, fundamental in the UPR, is required for the ER-stress induced upregulation of autophagy. Mechanisms other than hypoxia for UPR induction were investigated, using the proteasome inhibitor bortezomib (BZ). BZ treatment increased ATF4 protein levels in MCF7 cells, even transfected with short-interference RNA (siRNA) against the classical UPR activator PERK, suggesting that the proteasomal stabilization is likely the main mechanism for ATF4 protein accumulation. The induction of autophagy by BZ is dependent upon the upregulation of the microtubule-associated protein 1 light chain 3B (LC3B), an autophagy marker, by ATF4 and acts as a survival mechanism. Hypoxia, UPR and autophagy markers (such as Pimonidazole, carbonic anhydrases IX (CAIX), C/EBP homologous protein (CHOP) and LC3B) were evaluated by immunohistochemical approach in spheroids, xenografts models and breast cancer samples. CHOP immunohistochemical staining was performed in breast cancer sections from a series of patients. CHOP was expressed in cells surrounding necrotic areas. No correlation were found with clinical outcome and further studies are needed.

Research detailed in this thesis investigated the generation of Nitric Oxide (NO) and its role in the pathogenesis of the rice blast fungus Magnaporthe oryzae. Two putative nitric oxide synthase genes and single copy nitrate and nitrite reductase genes were cloned as potential sources of NO in M. oryzae. Single and double gene disrupted mutants were generated and their phenotypes assessed. Detection of NO is problematic. Herein, a fluorescent plate reader assay was developed, exploiting the NO sensitive dye DAR-4M AM and the NO scavenger PTIO, to compare wildtype NO generation with the mutant strains. All strains were assessed for infection-related development on an artificial surface inductive to appressorium formation and maturation in the wildtype strain. Appressorium formation in the presence of PTIO and the NO donor DETANONOate was recorded for all strains on this surface. The pathogenicity of the wildtype and mutant strains were assessed, in terms of their ability to infect rice and barley plants. Finally, the capacity of each strain to metabolise nitrogen was evaluated to confirm the disruption of the nitrate and nitrite reductase genes. Collectively, the data demonstrate that the plate reader assay provides robust evidence for the generation of NO in M. oryzae. However, none of the various mutant strains showed a reduction in NO emission during germling morphogenesis. However, they exhibited significantly different infection-related development on an inductive artificial surface as compared with the wildtype strain. Moreover, exogenous application of PTIO to the wildtype strain provided evidence for NO and its involvement in germination and appressorium development. No significant differences in the infection of rice and barley leaves were observed between the wildtype and mutant strains, indicating their disrupted genes are dispensable for pathogenesis. The nitrate and nitrite reductase genes were found to be essential for nitrate assimilation. In summary, this work provides the most robust evidence for the generation of NO in fungi to-date, but the molecular mechanism underpinning the generation of NO in M. oryzae remains elusive.

Trypanosomatid protozoan parasites are the causative agents of a number of diseases responsible for the death of thousands of people in developing countries. There is currently little hope for the development of vaccines and existing treatment regimens are associated with high toxicity. Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of these parasites are intimately linked to their shape and form which are in turn derived from a highly ordered microtubule-based cytoskeleton. Here I have investigated some of the critical structures of the cytoskeleton in terms of their molecular composition with a view toward interrogating their functions. I have used a combined reverse genetics/comparative proteomics approach to identify over 20 novel components of the paraflagellar rod, an essential structure for the mammalian infective form of the parasite. I have iterated this approach to define interdependent sub-groups within the cohort which provide clues to the function of the paraflagellar rod. I next applied the same comparative proteomics techniques to investigate the differences between the protein composition of two life-cycle stages of the parasite. I have identified novel components of a unique mobile transmembrane junction called the flagella connector, and of the flagellum attachment zone, a structure that is essential for cell division. In addition I have defined a pair of paralogous cytoskeletal proteins that show life-cycle stage specificity. Finally, I have used electron tomography, reverse genetics and in situ protein tagging to define the morphology of the flagellar pocket collar, a critical structure required for parasite viability, and provide new insights into its molecular composition, function and biogenesis.

A good distance metric can improve the accuracy of a nearest neighbour classifier. Xing et al. (2002) proposed distance metric learning to find a linear transformation of the data so that observations of different classes are better separated. For high-dimensional problems where many un-informative variables are present, it is attractive to select a sparse distance metric, both to increase predictive accuracy but also to aid interpretation of the result. In this thesis, we investigate three different types of sparsity assumption for distance metric learning and show that sparse recovery is possible under each type of sparsity assumption with an appropriate choice of L1-type penalty. We show that a lasso penalty promotes learning a transformation matrix having lots of zero entries, a group lasso penalty recovers a transformation matrix having zero rows/columns and a trace norm penalty allows us to learn a low rank transformation matrix. The regularization allows us to consider a large number of covariates and we apply the technique to an expanded set of basis called rule ensemble to allow for a more flexible fit. Finally, we illustrate an application of the metric learning problem via a document retrieval example and discuss how similarity-based information can be applied to learn a classifier.

Induced regulatory T cells (iTreg) play an important role in the induction of tolerance to self and non-self antigens. Harnessing their suppressive potential has therapeutic implications for the treatment of autoimmune conditions and transplant rejection. Although the role of TGF&beta;-conditioned iTreg in natural and therapeutic tolerance is indisputable, their mechanism of action as well as factors that influence their function and stability in vivo remain unclear. Here it is shown that TGF&beta;-conditioning of T cells in the absence of any Foxp3 expression is insufficient for conferring a suppressive phenotype in vivo, whilst Foxp3 expression is sufficient to enable naïve T cells to become suppressive both in vitro and in vivo. Graft antigen was found to enhance the number of iTreg-derived Foxp3+ cells localising to the draining lymph nodes of recipients, and this was associated with histone modifications at the Foxp3 locus that suggested a stabilisation or 'affirmation' of Foxp3 expression. Finally, iTreg were shown to 'out-compete' naïve T cells in forming clusters with dendritic cells. Activated inflammatory T cells could also 'out-compete' naïve T cells. However, unlike activated T cells, iTreg did not activate interacting DCs to the same extent, and this may potentially be a mechanism of their action in vivo.

Mitosis is the process by which new cells are formed. Following accurate duplication of chromosomes, a cell must segregate its chromosomes into separate daughter cells with great accuracy. Failure to do so can result in genomic instability. Thus, entry into mitosis is tightly regulated via complex protein interactions. Microtubules (MTs) are versatile Tubulin polymers that constitute a considerable portion of the cytoskeleton, and it is the dramatic rearrangement of MTs upon mitotic entry that is required to build the mitotic spindle – the structure responsible for segregating the duplicated sister chromatids. MTs are modulated by MT-Associated Proteins (MAPs) that enact major MT rearrangements during mitosis. Identifying and understanding the role of MAPs is essential to the study of MT behaviour during mitosis. Recently, an RNAi screen for Drosophila MAPs identified two proteins that are the subject of this thesis; Msd1 and dTD-60. This thesis demonstrates that Msd1 is a novel MAP that is a component of the recently identified Augmin complex – the action of which is to generate a novel and redundant population of MTs within the mitotic spindle from existing MTs. Experiments described below demonstrate that Msd1 is required for Augmin-directed MT generation, and further investigate the role of this novel population of MTs within the developing fruit fly. Furthermore, a role for Msd1 in central spindle formation during anaphase in Drosophila is identified. dTD-60, the Drosophila homologue of human TD-60 (hTD-60), is the subject of another study described in this thesis. While hTD-60 has a role in metaphase progression through interaction with the Chromosomal Passenger Complex, a contrasting role for dTD-60 is investigated here. This thesis describes both a divergent localisation and phenotype of dTD-60, and further investigates the role of dTD-60 and its interactors in mitotic spindle formation.