This thesis explores the role of small RNAs as regulators of early vertebrate development using the frog species Xenopus laevis and Xenopus tropicalis as experimental systems. Firstly, I investigate spatial control of Nodal signalling, which is critical for organisation of the vertebrate body plan during gastrulation, by uncovering the functional relevance and mechanism of spatial regulation of the Nodal coreceptor Cripto-1 in the early X. laevis embryo. Xenopus Cripto-1 (XCR1) mRNA is ubiquitous in the early embryo, but the protein is absent in the prospective endoderm, from which Nodal signalling originates. Cripto-1 is a stem cell marker and upregulated in many cancers and Nodal signalling is reactivated in melanomas and prostate cancer. Therefore understanding how control of Nodal signalling and XCR1 regulation is achieved may have implications for its misregulation in cancer. I show that spatial regulation of XCR1 is critical for the location, timing and magnitude of Nodal signalling in the early Xenopus embryo. Spatial regulation occurs at the level of translational repression, is dependent on the 3’ UTR and the microRNA-processing enzyme Dicer and is abolished by mutating four nucleotides in the minimal regulatory region of the 3’ UTR. I show that XCR1 is negatively regulated by xla-miR-427 in a 3’ UTR-independent mechanism, adding to recent research in Xenopus and zebrafish showing microRNA control of Nodal signalling at the ligand, receptor and antagonist level. Whilst studying Xenopus Cripto-1, it became clear that relatively little was known about microRNAs in early vertebrate development. To fill this void, I performed genome-wide sequencing of all small RNAs expressed in the early X. tropicalis embryo at three stages of development. This revealed dynamic and localised expression of hundreds of microRNAs in the early embryo and the presence of Piwi-interacting RNA sequences. Extensive validation of the small RNA-seq dataset is presented, and novel microRNAs and a novel class of intronic small RNAs are uncovered within the 95 % of the early vertebrate embryo small RNAome that was previously uncharacterised.

Pyrrole (P)-Imidazole (I) containing polyamides are small synthetic molecules which can target predetermined DNA sequences with high affinity and modulate gene expression by interfering with the binding of transcription factors to DNA. Hairpin and H-pin analogues show enhanced DNA binding affinity and selectivity compared to monomers, but are associated with poor cellular uptake. To address this problem, a new class of unlinked polyamide precursors containing appropriately designed pendant reactive and biocompatible functional groups is presented in this thesis. The ability of these agents to interact in situ and potentially form either H-pin or Hairpin conjugates was tested with DNase I footprinting analysis. The effects of modification on the formamido-imidazole-pyrrole-imidazole (f-IPI) polyamide and related analogues, by the addition of an amino group, on DNA binding affinity and sequence selectivity were also evaluated. Dbf4 is the regulatory subunit of Cdc7 kinase, which is essential for the initiation of DNA replication. Human Cdc7/Dbf4 (HuCdc7/Dbf4) kinase activity is critical for cell proliferation and high expression levels occur in multiple solid human malignancies. F-IPI can target the MIuI Cell cycle Box (MCB) sequence 5’-ACGCGT-3’which is the critical site for transcription factor binding and activation of expression of the HuCdc7/Dbf4 core gene in mammalian cells. DNA sequence selective binding of f-IPI to the MCB sequence was shown in DNase I footrpinting experiments. A f-IPI-induced, dose-dependent inhibition of protein binding to the MCB was demonstrated in an Electrophoretic Mobility Shift Assay (EMSA). RT-PCR and immunoblotting analysis confirmed the inhibitory effects of f-IPI on HuDbf4 in MDA-MB231 cells. Small interfering RNA-mediated depletion of HuDbf4 decreased cell survival and proliferation, and induced G1 arrest in MDA-MB231 cells. F-IPI treatments also showed a dose-dependent reduction of cell survival and proliferation and induced G1 arrest. Although the f-IPI H-pin analogue exhibited better DNA binding affinity and protein inhibition, no marked effects at mRNA and protein levels were detected in cells, likely attributed to its large size. The unlinked f-IPI analogues containing reactive groups [i.e f-IP(C3NH2)I and f-IP(C3Cl)I] able to interact in situ and form H-pin were tested in the Dbf4 promoter model system. f-IP(C3NH2)I presented the same DNA binding affinity and protein inhibition to the MCB as the combination of f-IP(C3NH2)I and f-IP(C3Cl)I (potential hybrid) but it produced much higher effects than f-IP(C3Cl)I. RT-PCR and immunoblotting analysis showed that the potential hybrid produced greater reduction of HuDbf4 mRNA and protein levels than either f-IP(C3NH2)I or f-IP(C3Cl)I. In addition, the potential hybrid demonstrated greater G1 arrest than each polyamide alone. Finally, a series of fluorescent f-IPI analogues are presented, providing an intrinsic probe for monitoring the cellular uptake of polyamides. Aza-Hx-PI was shown to target the same sequence as f-IPI (DNase I footprinting), inhibit protein binding (EMSA) and reduce the levels of HuDbf4 in cells. Time and dose-dependent nuclear localisation was detected by confocal microscopy. Overall the data presented in this thesis highlight the potential of small molecule polyamides as modulators of gene expression.

Superparamagnetic iron oxide nanoparticles (SPION) are used clinically to improve the sensitivity of magnetic resonance imaging (MRI). A less exploited property of SPION is their ability to generate heat when subjected to an alternating magnetic field, a process called magnetic alternating current hyperthermia (MACH). Hyperthermia has been shown to be a cancer effective treatment modality in the clinic when given together with radio/chemotherapy. However, delivery of sufficient heat to damage tumours without harming healthy tissue remains challenging. The central hypothesis for this thesis is that MACH activated SPION can be used to generate hyperthermia in situ and therefore will have potential to achieve localised hyperthermic cancer treatments. The aim of the thesis was to evaluate the potential of SPION to deliver localised hyperthermia by: (1) Characterization and comparison of SPION to select a lead candidate for clinical application. (2) Developing conjugation methods to confer SPION with cancer-binding properties by attachment of single chain Fv antibodies (scFv). (3) Evaluating the localisation and heating potential in vivo. SPION were characterized with regard to their hydrodynamic diameter, core size, magnetic properties, atomic iron content and heating potential for hyperthermia application. Different chemistries were evaluated to functionalize the most promising candidate using shMFEm, an scFv targeting the carcinoembryonic antigen (CEA). A CEA-non-binding scFv variant, shNFEm, was used as a negative control. Functionality of the scFv-SPIONs was assessed using quartz crystal microbalance. In vivo heating potential of the SPION was tested in a xenograft tumour model in vivo, using bespoke MACH apparatus. The results established Ferucarbotran (FX), unformulated Resovist®, an MRI contrast agent, as the most suitable candidate for hyperthermia application. Cyanogen bromide chemistry was selected to functionalise Ferucarbotran with the scFvs shMFEm. The FX-scFv conjugates were purified and analysed. Functionality was confirmed by quartz crystal microbalance, enabling the first visualisation of the interaction between a SPION-scFv conjugate and cognate antigen in real-time. The in vivo assessment of Ferucarbotran and the FX-scFv conjugates confirmed the in vitro heating potential of Ferucarbotran. In vivo analysis of heating showed that localised hyperthermia was achievable with intratumoral injection followed by MACH. Histological analysis of the tumours revealed an uneven distribution of particles within the tumours and an accumulation of the particles within the surrounding stroma indicating the future work should include study of innovative tumour delivery methods. These results support the hypothesis of a therapeutic potential for targeted magnetic nanoparticle hyperthermia and indicates the challenges that have be addressed to enable clinical application of this treatment modality.

The mixed lineage leukaemia (MLL) gene is frequently the target of chromosomal translocation in infant leukaemia. Translocation results in an in-frame chimeric fusion gene which is implicated in both ALL and AML, with particularly poor prognoses. It is widely accepted that MLL has a crucial role in regulating haematopoiesis. Our lab has previously developed a murine model for conditional expression of MLL-fusions to establish a list of transcriptional target genes using Affymetrix GeneChip analysis. In order to study the role of MLL-fusion target genes in human leukaemia cells, we generated four independent immortalised myeloid cell lines from human cord blood, using the MLL-AF9 fusion, by means of lentiviral transduction. The transduced cells proliferated exponentially in liquid culture and were found to cause leukaemia upon xenotransplantation into immunodeficient mice. One of the target genes up-regulated by the MLL-fusions, RUVBL2, encodes an ATPase belonging to AAA+ family that has multiple roles in telomerase and chromatin-remodelling complexes. In this study, we demonstrate that RUVBL2 is also up-regulated by MLL-AF9 in human immortalised myeloid cells. shRNA knock down of RUVBL2 expression in these cells, and in the human leukaemia cell line THP-1, results in decreased cell proliferation and clonogenic potential, accompanied by an increase in apoptosis and differentiation, as judged by CD15 expression. Furthermore, inhibition of RUVBL2 expression in THP-1 cells leads to a reduction in hTERT mRNA expression and telomerase activity. Together, these data demonstrate the requirement of RUVBL2 to mediate MLL-fusion induced telomerase activity in human cells, and suggest the possibility of targeting RUVBL2 as a potential therapeutic strategy for MLL-fusion associated leukaemia.

Neuroendocrine tumours(NETs) are heterogeneous with respect to biological behavior. Consequently, prognosis is variable and biomarkers predicting survival or tumour progression are required to inform clinical management. The best available biomarker, histological grade, is assigned using Ki-67 or mitotic count. Agreement between these two indices is implied but analysis of 131 pancreatic and 136 midgut NETs suggested discordances of 44% and 38% respectively. Ki-67 was the superior prognostic marker, making the additional value of mitotic count questionable. Detection of Circulating Tumour Cells(CTCs) using the Cellsearch™ platform requires expression of epithelial cell adhesion molecule(EpCAM). I demonstrated EpCAM expression by immunohistochemistry and detected CTCs in patients with metastatic NETs. In 175 patients, ≥1 CTC was detected in 51%(midgut) and 36%(pancreatic). ≥1 CTC was an independent poor prognostic factor, offering better prognostic value than grade or chromogranin A(CgA). Changes in CTCs 3-5 weeks after commencing therapy were predictive of response and survival, suggesting CTCs could provide an early assessment. Using chip-based capillary-electrophoresis, higher concentrations of circulating free DNA(cfDNA) were found in 88 patients with NETs compared to healthy controls with a correlation between cfDNA quantity and CTCs. Since cfDNA was detected in 25% of cases, more sensitive methods of detection are required before studies are conducted to validate cfDNA as a biomarker and to analyse mutations. The hypervascular nature of NETs suggested that circulating endothelial cells(CECs) might be informative. Using immunomagnetic separation and CD105 phenotyping, CECs were demonstrated in 55 patients. Although not significantly elevated, there was a wider range of CECs in NETs compared to controls. Further studies investigating changes with anti-angiogenic therapy could prove valuable. My research suggests circulating biomarkers, specifically CTCs, provide additional and better prognostic information than grade. Furthermore, detection of CTCs and cfDNA in NETs may allow future studies into molecular analysis, which may enhance understanding of NET pathogenesis.

Synthetic antisense oligoribonucleotides can be used to modulate gene splicing by masking key motifs on the pre-mRNA required for spliceosome assembly. Yet, intracellular expression of oligoribonucleotides generates only a transitory effect whereas stable delivery of antisense sequences can be achieved by linking them to chimeric small RNAs delivered and expressed by viral vectors. In the murine model of Duchenne Muscular Dystrophy a chimeric U7 snRNA (U7Dtex23) induces skipping of the mutated exon 23 and restores the Dystrophin mRNA reading frame. The main limitation of this approach remains the large amount of snRNA vector needed to be produced and administered to patients. To optimize this system we used self-complementary AAV vectors (scAAV) to express the U7snRNA shuttles. ScAAV vectors were tested in mouse myoblast cultures and we observed an increase in U7Dtex23 expression and in dystrophin exon 23 skipping compared to single-stranded AAV, highlighting the potential for this strategy to reduce the vector dose. Alternatively, we have used a muscle and heart-specific enhancer (MHCK) to drive the expression of U7Dtex23 cassettes delivered with AAV vectors and our results showed that MHCK improves chimeric U7snRNA expression and increases dystrophin exon 23 skipping in vitro and in vivo. However, additional U7snRNA species were produced following gene transfer, pointing at a possible limitation of the cellular processing machinery capability with saturating levels of U7 shuttles. We have also explored the possibility of using small nucleolar (sno) RNAs as novel molecular platforms for antisense delivery. We replaced the original antisense of MBII-52 snoRNA with the Dtex23 sequence and observed low levels of exon 23 skipping in AAV-transduced myotubes. While our observation validates the approach, the efficiency of skipping is still considerably lower than with the U7snRNA cassette. As a last approach, we engineered the human C/D box U24snoRNA to specifically target the methylation of an adenosine branch point in a luciferase reporter pre-mRNA in order to induce the skipping of the downstream exon. We were not able to observe any modulation of splicing using this strategy.

While telomerase-mediated DNA extension is the most commonly used mode of eukaryotic chromosome end maintenance, the requirement of stable chromosome ends for genomic stability has selected for the evolution of alternative strategies. Telomerase deletion leads to progressive loss of telomeric DNA and loss of viability for the majority of cells. However, populations of survivors arise. In fission yeast, these survivors either maintain their telomeres via recombination or survive complete telomere loss by undergoing intra-chromosomal fusion (circularisation) of their chromosomes. Previous work in the lab led to the identification of a novel class of survivors that appear to lack telomeric DNA, but do not circularise their chromosomes. Intriguingly, these novel survivors have amplified either of two classes of repetitive sequences at their chromosome ends, the rDNA or the subtelomeric elements; in the latter case, these elements have also spread throughout the genome. This thesis involves the characterisation of these survivors, named HAATI survivors. The amplified repetitive sequences in HAATI are associated with heterochromatin and we find that the heterochromatin machinery is crucial for HAATI survival. Furthermore, chromosome linearity in HAATI relies on Pot1, a canonical chromosome end protection factor whose recruitment to chromosome ends had been thought to rely on telomere sequences. Our data suggest that Pot1 localises to HAATI chromosome ends via interactions with the heterochromatin machinery and non-telomeric single strand overhangs at the chromosome termini. This discovery not only reveals a previously unrecognised mode by which cancer cells could escape the requirement for telomerase activation, but also provides a tool for studying genomes that sustain unusually high levels of heterochromatinization.

Genetic testing for high-penetrance cancer predisposing mutations is currently only accessible to individuals with a strong family-history (FH). This misses a number of at-risk individuals. Limitations to this approach could be overcome by population-based testing. This research evaluates the feasibility and impact of population-screening (PS) for inheritable cancer by comparing it with the standard FH-based approach. The Ashkenazi Jewish (AJ) population is used as a 'population model' and BRCA1/2 mutations as a 'disease model'. The study design involves a randomised trial called: GCaPPS. Inclusion criteria: (a) AJ ethnicity, (b) age 18 years. Exclusion criteria: (a) first-degree-relative BRCA-carriers (b) prior BRCA testing. Outcomes: (a) BRCA carriers, (b) acceptability, (c) psychological, quality-of-life impact (d) uptake of screening/preventive options (e) health-economics. All participants underwent pre-test genetic counselling (DVD-based/ traditional face-to-face). All PS- arm participants and those fulfilling standard criteria in FH-arm underwent genetic testing. This thesis describes the development, design, recruitment and outcome of the trial to 3-months follow-up. 1034 participants were randomised to FH(504)/ PS(530) arms. Their mean/median age was 54.3(SD14.66)/56(IQR43,65) years; 33.2% were men and 66.8% women. 72% of registered people attended genetic-counselling, 40% attendees indicated clear intention-to-test and 89% consented to genetic-testing. 13-carriers were identified in PS (7BRCA1, 6BRCA2), 9 in FH (5BRCA1, 4BRCA2) arms (p=0.522) and a further 5-carriers in FH-negative participants in FH-arm following study completion. 56% carriers in the entire cohort were missed by FH compared to PS. FH had poor ability to predict (positive likelihood-ratio=3.86) or rule out (negative likelihood-ratio=0.63) a mutation. BRCA1/2 prevalence is 2.45%(CI:1.31%,4.16%): 1.16%(C1:0.60°/0,2.02%) for FH-positive and 1.89%(CI:0.91%,3.44%) for FH-negative carriers. There was no difference at 7-days/ 3-months for outcomes of anxiety, depression, quality-of-life, health-anxiety, distress and uncertainty between population-based and FH-based testing. DVD-based counselling halved counselling time (p<0.0005), led to cost minimisation of £14/volunteer, without affecting counselling satisfaction (p=0.468) or increase in knowledge (p=0.174) compared to standard counselling. Probabilistic-sensitivity-analysis indicated PS is highly cost- effective in 92% simulations, with an incremental cost-effectiveness-ratio of £499/QALY, equating to 21days gain in life- expectancy, and 0.37% reduction in ovarian cancer incidence. These findings provide strong arguments for further exploration of a new paradigm of population-based testing for BRCA1/2 and other germ line mutations.

Ovarian cancer (OC) is the leading cause of gynaecological cancer mortality in developed countries. There is concern that ultrasound (first/second line test in all screening strategies) might be better at detecting Type I OC than the aggressive Type II OC. If ultrasound is to be part of population screening, there is a need for quality assurance (QA), given the subjective nature of transvaginal scan (TVS). In the United Kingdom Collaborative Trial of Ovarian Cancer Screening, 48230 women had initial scan. Prospective cohort studies of women with ultrasound detected inclusion cysts (IC) and abnormal adnexal morphology (unilocular, multilocular, unilocular solid, multilocular solid cysts and solid masses) were undertaken. On median follow up of 6.13 years, no increased OC risk was associated with IC (Relative Risk 2.32, 95%CI 0.86-6.28). The absolute risk (AR) of overall OC associated with abnormal adnexal morphology at three years was 1.08%, 0.73% for Type I OC and 0.34% for Type II OC. At one year from first scan, sensitivity for Type II OC was highest for multilocular solid cyst at 27.8% with a PPV of 1.5%. TVS visualisation of normal ovary was the chosen metric to assess impact of the QA programme. 43867 scans were analysed to assess the effect of non-subjective factors on ovarian visualisation. Previous hysterectomy, tubal ligation, increasing age, unilateral oophorectomy and rising BMI decreased visualisation whereas increasing age at menopause and infertility increased visualisation. These factors were included in a Generalised Estimating Equation model and comparison undertaken of observed versus adjusted visualisation rate (VR) between individual sonographers/trial centres showing that while VR should be adjusted for non-subjective factors, the magnitude of differences between observed and adjusted VR was small. An accreditation programme for scanning postmenopausal ovaries was developed. Sonographers were assessed on knowledge of ultrasound protocol, three monthly VR, practical scanning technique, central training day attendance and submitted scan images. A 48-item questionnaire study (150 women/centre) on scan experience (including pain/discomfort) was conducted to assess the acceptability/ability to deliver ultrasound screening. TVS was well accepted with only 3.5% women reporting moderate/severe pain. This thesis provides accurate risk estimates for overall OC, Type I and Type II OC associated with ultrasound detected adnexal morphology in an asymptomatic postmenopausal population. In addition, it describes the development of QA, training/accreditation of sonographers and delivery of large scale ultrasound screening.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus that is the etiologic agent of Kaposi sarcoma (KS). KS is an angioproliferative neoplasm composed of cells of endothelial origin. For the work described in this thesis, KSHV infection of endothelial cells was used as a tractable model to study the role of microRNAs (miRNAs) in endothelial cell biology. Previous work in the laboratory had identified miRNAs which are either upregulated or downregulated upon KSHV infection of lymphatic endothelial cells (LEC). Target prediction analysis of these miRNAs and cross-comparison of predicted targets with expression levels of pro- and anti-angiogenic genes following KSHV infection, revealed the predicted targeting of Delta-like 4 (DLL4) by the miR-30 family. DLL4 is significantly upregulated in KSHV-infected lymphatic endothelial cells (KLEC) whereas the miR-30 family is significantly downregulated. DLL4 is a membrane-bound ligand belonging to the Notch signalling family that plays a fundamental role in vascular development and angiogenesis. Targeting of DLL4 by miR-30b and miR-30c was confirmed by examining mRNA and protein expression following transfection of endothelial cells with miR-30 mimics and inhibitors or infection with miR-30-expressing lentiviruses. The exact target site within the DLL4 3’UTR was identified using a luciferase reporter assay and site-directed mutagenesis. Overexpression of miR-30b in endothelial cells led to increased vessel number and length in an in vitro model of sprouting angiogenesis. Microinjection of miR-30 into zebrafish embryos resulted in suppression of dll4 and subsequent excessive sprouting of intersegmental vessels and reduction in dorsal aorta diameter. Use of a target protector against the miR-30 site within the dll4 3’UTR upregulated dll4 and synergised with Vegfa signalling knockdown to inhibit angiogenesis. Furthermore, restoration of miR-30b or miR-30c expression during KSHV infection attenuated viral induction of DLL4. Overall, the work presented in this thesis demonstrates that the miR-30 family targets DLL4 to regulate angiogenesis.