The innate immune system is capable of responding to tissue injury by detecting the abnormal exposure of intracellular, often ubiquitously expressed, molecules referred to as damage-associated molecular patterns (DAMPs). DAMPs are normally sequestered inside healthy cells but become exposed to the extracellular environment upon loss of membrane integrity during cell death. Exposed DAMPs are then recognised by receptors of the innate immune system. One such DAMP receptor is DNGR-1 (CLEC9A), which is expressed on CD8+ DCs, a rare but specialised subset of DCs involved in regulating T cell responses. Loss of DNGR-1 on CD8+ DCs impairs cross-presentation of dead-cell associated antigens to CD8+ T-cells indicating that DNGR-1 couples DAMP recognition to the generation of cytotoxic T cell immune responses. Prior to the work presented in this thesis, the DAMP ligand for DNGR-1 had not been identified. Using a variety of experimental approaches, I demonstrated that this ligand corresponds to filamentous actin (F-actin), a component of the cytoskeleton of all cells. Given its extreme evolutionary conservation, abundance and ubiquitous expression, as well as its association with tissue damage in a range of inflammatory conditions, actin possesses ideal DAMP characteristics. Thus, I further hypothesised that actin may engage receptors other than DNGR-1 and act as a universal and evolutionarily ancient sign of cell damage that is more generally detected by metazoans as a means of inducing sterile inflammation and/or tissue repair. In order to test this hypothesis, I made use of the Drosophila melanogaster model system. I found that actin injection into flies stimulates strong activation of the stress-induced JAK/STAT pathway without triggering immune defence pathways. Given the conservation of innate defence mechanisms in invertebrates and vertebrates, it is tempting to speculate that understanding the recognition of actin in Drosophila melanogaster will provide useful insights into the induction of inflammation in mammals.

Live, attenuated measles virus (MV) has demonstrated tumour-specific replication and anti-tumour activity in murine models of hematological and non-hematological malignancy, leading to a number of published or on-going clinical trials. However, the potentially overriding issue of how to achieve successful administration of oncolytic measles virotherapy in patients with intact humoral immunity remains. I have examined mechanisms for optimizing delivery of oncolytic MV to acute lymphoblastic leukaemia (ALL) – a disseminated haematological malignancy. Anti MV antibody titres in adult patients with ALL were evaluated before and after leukaemia treatment regimens including high dose corticosteroids, cyclophosphamide and anti-B cell monoclonal antibodies. Antibody titres were unaffected by induction and intensification treatment protocols, suggesting the need for consideration of optimal virotherapy delivery strategies to achieve therapeutic success. Here, I demonstrate that human bone marrow-derived mesenchymal stromal cells (BM-MSCs) can be used effectively as virus delivery vehicles, permitting ex-vivo cellular virus loading and intracellular virus amplification, delivery of virus to distant sites of disease following systemic infusion, and virus hand-off to precursor B lineage ALL cell targets in the presence of pre-existing anti-MV antibodies. In vivo modelling using SCID mice bearing disseminated pre-B ALL xenografts demonstrated enhanced survival of passively immunized animals following IV treatment with BM-MSC-delivered MV versus naked MV or BM-MSCs alone. In order for vaccine MV to be safely and rationally utilized as a novel therapeutic for ALL, a detailed mechanistic understanding of how the virus exerts its oncolytic effect is paramount. In this thesis, I have utilized a previously characterized model of cellular transformation generated using human BM-MSCs to characterize the phenomenon of relative tumour cell specificity by oncolytic MV, in terms of infectivity, productivity and cell killing. Furthermore, this thesis begins to explore some of the potential mechanisms that confer vaccine MV its tumour-tropic and anti-cancer properties.

Immune reconstitution following conventional allogeneic transplantation is a major determinant of survival. A detailed investigation of T and B cell immune reconstitution and clinical outcome in 19 patients with myeloma undergoing reduced intensity stem cell transplantation using in vivo T cell-depletion with alemtuzumab was undertaken. The rate of recovery of lymphocyte numbers and function following transplant was studied using immunophenotyping with 3-colour flow cytometry and intracellular cytokine staining. In addition, T and B cell spectratyping were used to study the repertoire of immune recovery. The patients in this study experienced delayed T cell recovery and T cell receptor spectratype analysis showed a reduced repertoire diversity, which improved rapidly following the administration of DLI and subsequent conversion to full donor T cell chimerism. Post transplant recovery of B cells was also significantly delayed. Spectratype analysis of IgH CDR3 repertoire revealed a gradual normalisation in spectratype complexity by 6- 12 months post transplant. There was a high incidence of viral infection, particularly CMV reactivation but the regimen related mortality was low, perhaps due to the very low incidence of severe acute graft-versus-host disease (GVHD). A total of 10 patients experienced GVHD. Of these patients, 8 eventually demonstrated a disease response alongside clinical evidence of GVHD, demonstrating that the graft-versus-myeloma effect is frequently obtained at the expense of GVHD. Over 80% of all patients have relapsed at a median of 9 months following transplant, suggesting that the initially low rate of GVHD has been achieved at the expense of the desired graft-versus-myeloma effect.

Telomeres and centromeres have traditionally been considered to perform fundamentally distinct roles. A non-classical, but fundamental role of telomeres occurs in meiosis. Disruption of the fission yeast telomere bouquet (the conserved chromosomal configuration specific to meiotic prophase) causes a failure of the spindle pole body (SPB, the yeast centrosome equivalent) to accumulate the gamma-tubulin complex and nucleate spindles at the first meiotic division, resulting in defective meiotic SPB separation and spindle formation (Tomita and Cooper, 2007). Hence, the association of telomeres with the SPBs during meiotic prophase is crucial for subsequent SPB behaviour. Nonetheless, in 50% of bouquet deficient zygotes, spindles form properly. Here we show that bouquet deficient cells can successfully undergo meiosis using centromeres instead of telomeres to generate spindle formation. Moreover, forced association between centromeres and SPBs fully rescues SPB and spindle defects incurred by bouquet disruption - a rescue that is not induced to the same extent by non-centromeric regions. The rescue mediated by centromeres is independent of heterochromatin and outer kinetochore components. These observations demonstrate a shared ability of telomeres and centromeres to modify the SPB and ensure meiotic progression. Another interesting feature in those bouquet deficient cells displaying bipolar meiotic spindles is an increase in chromosome mis-segregation. With high temporal resolution the interaction between centromeres and telomeres in the early stages of meiosis was characterised in live cells and defined as the point at which the telomeres may communicate with the centromeres to ensure complete meiotic kinetochore assembly. These observations demonstrate a striking level of functional interchangeability between distinct chromosomal landmarks as well as an unprecedented level of communication between centromeres and telomeres, and between specialised chromosomal regions and the spindle assembly machinery. Experiments to determine whether the centromeres play a similar role in controlling mitotic spindle formation are also described.

Bendamustine has demonstrated clinical efficacy in the treatment of haematological malignancies and distinguish itself from other alkylating agents. The mechanistic and clinical differences associated with Bendamustine may be related to its structural features including a benzimidazole ring, although the mechanism of action is poorly understood. Understanding the molecular mechanism of Bendamustine could explain the therapeutic efficacy and identify potential biomarkers for response. The Bendamustine-DNA interaction in naked DNA, cytotoxicity, and ICL formation and repair (unhooking) in naked DNA or in cell lines and patient multiple myeloma cells by the single cell gel electrophoresis (comet) assay, were analyzed. DNA damage response (DDR) and potential mechanisms of acquired resistance to Bendamustine were also evaluated. Bendamustine alkylated DNA at guanine-N7 positions, produced ICLs in naked DNA and in cells, and demonstrated a cytotoxic effect comparable to conventional ICL drugs (Cisplatin, Melphalan). However, ICLs were not efficiently repaired (unhooked) in A549 cells, which could repair Cisplatin or Melphalan ICLs. In plasma cells from both Non-Melphalan Treated or Melphalan Treated patients, no evidence of efficient repair of Bendamustine ICLs was observed. Bendamustine DDR compared to Cisplatin or Melphalan gave a more selective pattern of expression of genes involved in DNA damage signaling pathways, cells defective in ERCC1, XPF, and homologous recombination repair showed less sensitivity to Bendamustine, there were differences in ɣH2AX and RAD51 foci formation and cell cycle distributions. In derived acquired resistant cell lines, resistance was associated with a reduced level of ICL at an equimolar drug dose compared to the parental lines. The molecular mechanism of Bendamustine is similar to conventional alkylating agents: DNA alkylation and ICL formation. However, ICL repair inefficiency and altered DDR are the differences between Bendamustine and conventional alkylating agents due to its benzimidazole ring that influences, showed by G1/S arrest of cell cycle population, its mechanism.

The formation of the lymphatic vasculature takes place in two main steps. The first step establishes the lymphatic endothelial cell fate and primitive vascular network during the early stages of development and is controlled by the transcription factor PROX-1. In the second step at late embryonic and postnatal stages the initial lymphatic plexus is remodelled into a hierarchical mature lymphatic network. A key regulator of this process is the transmembrane protein ephrin-B2, a ligand for the Eph family of receptor tyrosine kinases. With the research depicted in this thesis I have focused on understanding the role ephrin-B2 and its receptor EphB4 play during lymphatic development and the underlying molecular mechanisms. Ephrin-B2 and the signalling mediated via its PDZ-binding motif were previously described to be required for lymphatic development. Using constitutive and inducible tissue-specific loss-of-function mouse models I have shown that ephrin-B2 and its PDZ-related signalling are required not only to initiate the process of lymphatic remodelling but also for the maintenance of a mature lymphatic network. Loss of either of them results in immature lymphatic vessels that lack intraluminal valves. In vitro and in vivo experiments suggest that this is due to defects in the regulation of lymphatic endothelial cell cytoskeleton and cell-cell adhesion. Similarly, EphB4 is required for the initial steps of lymphatic remodelling; however, it is dispensable for the maintenance of adult lymphatic network. Finally, I demonstrated that ephrin-B2 and EphB4 are essential regulators of lymphatic sprouting, but this process is not dependent on ephrin-B2 PDZ-binding motif. These data show that ephrin-B2 signalling is required all through lymphatic development but the underlying molecular mechanisms differ between different developmental stages and processes. While the experimental evidence points towards a synergistic role of ephrin-B2 and EphB4 during lymphatic sprouting and initial remodelling, the role of ephrin-B2 in lymphatic maintenance is EphB4- independent. In addition, lymphatic sprouting is, to our knowledge, the first lymphangiogenic process where ephrin-B2 is required in a PDZ-independent manner. To study the lymphatic-specific deletion of ephrin-B2 and EphB4 I used a novel mouse line that drives Cre recombinase expression under the control of Prox1 promoter. While characterising the line, I described a previously unreported expression of Prox1 in the mesenteric veins. This expression was associated with the appearance of Prox1-positive sprouts from the veins and lympho-venous connections that I further analysed by micro computed tomography.

Graft versus Host Disease (GvHD) remains one of the main complications after haematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, CD4+CD25highFoxp3high regulatory T (Treg) cells have been proposed as a cellular therapy to prevent GvHD. However it has been shown that Treg cells can inhibit natural killer (NK) cell functions. NK cells are key effectors of the Graft versus Leukaemia (GvL) effect post-transplant; therefore, it is plausible that a Treg cell therapy may impact on NK cell function and differentiation from haematopoietic stem cells (HSC). This study sought to elucidate the effects of Treg cells on NK cell function and differentiation using umbilical cord blood (CB) as a cell source. Herein, it is confirmed that CB CD4+CD25highFoxp3high Treg cells are fully functional and upon TCR-stimulation express CTLA-4 and LAP, and secrete TGF-β and IL-10. Also, they express receptors associated with trafficking to lymphoid tissues and the bone marrow, which are potential NK cell/Treg cell interaction sites. Furthermore, it is shown that CB Treg cells can suppress CB NK cell functions after TCR-stimulation in steady state but not in the presence of exogenous cytokines. Lastly, in an in vitro model of NK cell differentiation, a 90% reduction in total NK cells was observed when TCR-stimulated Treg cells were added at the time when HSC commitment to the NK cell lineage occurs. Interestingly, the few NK cells that developed in these cultures showed normal phenotype, IFN-γ secretion and cytotoxicity. Notably, the addition of human recombinant TGF-β to HSC cultures caused a similar reduction in NK cell differentiation as shown when TCRstimulated Treg cells were added to HSC cultures. Moreover, the Treg cellmediated effect was contact-dependent and cytokine competition-independent. Collectively, these results demonstrate for the first time that TCR-stimulated CB Treg cells inhibit NK cell differentiation through TGF-β, providing information for optimisation of the time of delivery for an adoptive Treg cell therapy post-HSCT to prevent GvHD.

Cell polarity is a fundamental feature of most cells, and is required for a diverse range of cell functions. A conserved set of proteins is involved in the establishment and maintenance of cell polarity. Several of these polarity proteins in Drosophila are recognized as neoplastic tumour suppressors, and loss of cell polarity in general has been linked to cancer malignancy. Although much is known about the general role of the polarity proteins, many questions still remain about the details and extent of their functions. I have focused on the Lgl protein in Drosophila epithelia: a basolaterally-localized tumour suppressor. In mitosis, Lgl undergoes a dramatic relocalisation away from the plasma membrane into the cytoplasm. I have found that this is regulated by direct phosphorylation by the Aurora cell cycle kinases: this is in contrast to the regulation of Lgl in interphase polarity, which is mediated by aPKC. The mitotic relocalisation event appears to play an important role in the correct orientation of the mitotic spindle and cell division in epithelia, which may be necessary for maintaining tissue integrity. Thus in addition to the established role for Lgl in cell polarity, my work suggests a novel function for Lgl during mitosis.

CC-1065 and the duocarmycins are highly potent anticancer agents, exerting their biological activity through covalently reacting with adenine-N3 in the minor groove of AT-rich sequences. The alkylation properties and cytotoxicity of a series of novel chiral analogues are reported in this thesis. Structural modifications of established pharmacophores resulting in novel alkylating functionalities as well as variations of the DNA binding domain were introduced in the analogues considered, The sequence specificity of these compounds was assessed by a Taq Polymerase stop assay, identifying the sites of covalent modification on plasmid DNA and the purine-N3 adducts probed by a thermally-induced strand cleavage assay. The cytotoxic potency of the analogues was determined against human, chronic myeloid leukemia, K562, cells, using a MTT based growth inhibition assay. The importance of the chiral centre present in the natural products was subsequently investigated with a series of achiral analogues. The studies established that the chiral centre is not absolutely required for DNA interaction and cytotoxicity. This finding offers the possibility of a new platform for the design of novel, active CC-1065/duocarmycin analogues. A key chiral and an achiral analogue were selected for DNA repair studies. The sensitivity of yeast mutants deficient in specific DNA repair pathways was assessed in order to delineate the mechanisms involved in the repair of the relevant adenine-N3 adducts. Nucleotide excision repair (NER) and post replication repair mutants were the most sensitive to the two analogues. Single-strand ligation PCR was employed to follow the induction and repair of the lesions at nucleotide resolution. Adduct elimination of both agents was by transcription-coupled NER, and dependent upon functional Radl8. Finally, the involvement of NER as the predominant excision pathway was further confirmed in mammalian DNA repair mutant cells.

BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen family. BORIS is normally present in the testes; however, it is aberrantly expressed in various tumours and cell lines. The main aim of this study was to investigate BORIS expression in prostate cell lines and tumours, and the importance of BORIS in the regulation of genes in prostate cells, in particular, the androgen receptor CAR) gene, associated with the development of more aggressive prostate tumours.