MicroRNAs are small non-coding RNA molecules that regulate mRNA translation by binding to complementary sequences usually within the 3’ un-translated region (UTR). By inserting four perfectly complementary binding sites for the hepatic specific microRNA mir122 into the 3’ UTR of adenovirus wild type 5 (Ad5 WT) E1A mRNA I show that the acute liver toxicity caused by Ad5 WT in mice can be significantly reduced. This virus, termed Ad5-mir122, is a promising virotherapy candidate and causes no obvious liver pathology whilst maintaining Ad5 WT replication in mir122 negative cells. Data shows that repeat intravenous administration of Ad5-mir122 (2x1010vp) to HepG2 tumour bearing mice mediated significant anti-cancer efficacy. RT-QPCR for E1A mRNA demonstrated a 29-fold reduction when compared to Ad5 WT in murine liver whilst western blot confirmed that all E1A protein variants were knocked down. Viral genomic replication was also reduced in mouse liver by 25-fold compared to Ad5 WT. This control of virus activity reduced alanine and aspartate transaminase release by >15-fold and histological analysis showed little to no pathology in Ad5-mir122 infected livers. Measurement of mature mir122 levels in Ad5-mir122 infected livers by RT-QPCR showed that the quantity of mir122 remained unaffected at therapeutic doses. Complete genome mRNA array profiling of infected livers showed that the transcript levels of >3900 different mRNAs were changed more than 2-fold following Ad5 WT infection whilst less than 600 were changed by Ad5-mir122. A non-replicating control adenovirus vector altered >550 mRNAs. No known mir122 target mRNAs were affected following infection with Ad5-mir122. Western blot analysis of a known mir122 regulated target (Aldolase A) confirmed these results, demonstrating no change in protein level despite infection with Ad5-mir122. These data combined demonstrate that the exploitation of microRNA mir122 regulation to control adenovirus replication is a safe method of control and does not alter the endogenous level or activity of the microRNA or its endogenous mRNA targets. Ad5-mir122 is a potent anti-cancer agent that replicates to wild-type levels in microRNA mir122 negative cells but is specifically and safely attenuated in hepatocytes.

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<p>Based on the concept that the tactile hypersensitivity and the central sensitization observed in animal models of peripheral neuropathy are maintained by peripheral drive from primary sensory neurons, the present project measured the changes in electrophysiological, immunohistochemical, and pharmacological properties of the dorsal root ganglia (DRG) neurons induced by a peripheral neuropathy. The aim of this study was to make a systematic survey and a unique understanding of changes that occur in primary sensory neurons that can sustain peripheral drive in this model. The data of this study indicate a prominent role of large diameter Aβ-fibers, including low threshold mechanoreceptors in peripheral neuropathy.</p> / Doctor of Philosophy (Medical Science)

<p>The focus of this thesis was the role of fibrocytes in chronic lung disease. These bone marrow derived cells have been identified in the lung and the circulation in patient samples and animal models of lung injury. However, the precise mechanistic role of the fibrocyte is still to be elucidated.</p> <p>Live assessment of lung changes in animal models of chronic lung disease allows for real time observation of changes, and gives a readout which can be translated to humans who undergo similar tests. In this thesis, we adapted an existing model of lung injury, and delivered a discrete treatment to a single lung lobe while monitoring its successful delivery.</p> <p>I also developed a robust system to examine the relationship between fibrocyte response, and cytokine expression previously identified in chronic lung disease. We found a connection between cytokine expression and fibrocyte mobilisation. Our model showed that fibrocyte mobilisation in the presence of existing lung injury does not improve and rather can worsen existing lung injury. This was a significant finding as it confirms the role of the fibrocyte as a participator or conductor in fibrogenesis and it suggests that this cell may play a role in the development of chronic lung diseases.</p> <p>Finally, we contributed to the ongoing characterisation of the fibrocyte as a prospective biomarker. We confirmed the cell’s identity by characterising it by its known markers and biological characteristics. We also identified the presence of this cell in chronic lung disease and linked its presence to disease progression.</p> / Doctor of Philosophy (Medical Science)

<p>IL-15 is a cytokine that has effects on both innate and adaptive immune cells, including NK and CD8 T cells. The involvement of these cell types in tumor immunosurveillance and eradication has led to interest in IL-15 as an immunotherapy. Its role in spontaneous solid cancers has not been studied thoroughly. Here, we have shown for the first time that IL-15 overexpression in a spontaneous breast cancer model, MMTV-polyoma Middle T antigen (MT), leads to increased survival, tumor destruction, and decreased metastasis (IL-15 TG/MT). In contrast, lack of IL-15 led to decreased survival (IL-15 KO/MT) and increased metastasis. Protection in IL-15 TG/MT mice was dependent upon the presence of highly activated NK1.1+ cells, but not dependent upon CD8 T cells. The cytokine environment in IL-15 TG/MT tumors was capable of activating human NK cells to kill human triple negative breast cancer cells. In a model of metastasis, we found that overexpression of IL-15 protected from metastasis in a NK cell dependent manner. Lack of IL-15 promoted the polarization of CD4 T cells to a Th2 phenotype and they influenced polarization of macrophages to an M2 phenotype. M2 macrophages help establish tumors at the metastatic site. Here we found that M1 polarized macrophages could prevent engrafted breast tumor formation, whereas M2 macrophages promoted it. Overall, IL-15 is an extremely promising immunotherapy that has more anti-tumor effects on the immune system than were previously appreciated. Additionally, our data argues that it could generate immune responses against both primary breast cancer and metastasis.</p> / Doctor of Philosophy (Medical Science)

<p>Cytochrome b5 heme binding domain 2 (CYB5D2) is a heme binding protein that was initially identified for its ability to attenuate the function of the PTEN tumor suppressor gene. CYB5D2 sustains ectopic PTEN expression in U87 cells, and can also confer survival from serum starvation in NIH3T3 cells. An antibody was generated to the carboxyl terminus of CYB5D2 to detect endogenous protein expression. The highest expression of CYB5D2 protein is in neural cancer cell lines. CYB5D2 is weakly expressed in breast and kidney cancer cell lines, and moderately expressed in prostate cancer cell lines. To investigate the role of the heme binding domain in CYB5D2, a conserved aspartic acid (D86) within this domain was mutated to glycine, and this was characterized as being unable to bind heme. CYB5D2(D86G) displayed a loss of function compared to wild-type CYB5D2. To study the loss of expression of CYB5D2, stable CYB5D2 shRNA was achieved in HeLa and Huh7 cells. While ectopic CYB5D2 inhibited HeLa cell proliferation and growth in soft agar, CYB5D2(D86G) expression and CYB5D2 shRNA increased cell proliferation and soft agar growth. While ectopic CYB5D2 conferred survival from chemotherapeutic drugs in HeLa cells, CYB5D2(D86G) and CYB5D2 shRNA cells were susceptible to drug treatments. CYB5D2 inhibits SREBP signalling, which requires its heme binding ability. Using cyclohexamide treatments, CYB5D2 stabilized ectopic Insig1, while CYB5D2(D86G) destabilized ectopic Insig1. CYB5D2 shRNA reduced endogeneous CYP51A1 (lanosterol demethylase) and Insig1 protein levels, and increased the susceptibility of HeLa cells to mevalonate treatments. Furthermore, CYB5D2 shRNA HeLa cells displayed reduced CYP3A4 activity, a cytochrome P450 enzyme involved in drug metabolism. CYB5D2 binds to cytochrome P450 reductase (POR), while CYB5D2(D86G) cannot. CYB5D2 co-immunoprecipitates with endogenous POR under serum-free conditions in HeLa and Huh7 cells, while CYB5D2(D86G) cannot. Collectively, CYB5D2 is a POR interacting protein, which regulates CYP51A1 and CYP3A4 activity.</p> / Doctor of Philosophy (Medical Science)

<p>Virulence in <em>Staphylococcus aureus </em>is mediated by the <em>accessory gene regulator </em>(agr) quorum sensing system. This regulatory system is activated by a secreted thiolactone peptide termed autoinducing peptide (AIP) and its receptor histidine kinase, AgrC. Interaction of extracellular AIP with a cognate AgrC receptor generates an intracellular signal that is transduced by conformational changes and phosphorylation events in a two-component sensor histidine kinase system. At the heart of the <em>agr</em> quorum-sensing cascade lies the two-component histidine kinase, AgrC, and the response regulator protein, AgrA. Interaction of AgrC and AgrA, and the resulting phosphotransfer event results in expression from the divergent promoters P2 and P3, inducing expression of the master quorum-sensing regulator RNAIII and upregulating the <em>agr</em> operon respectively. Signal transduction systems function as intracellular information-processing pathways that link sensation of external stimuli to specific adaptive processes. In <em>S. aureus</em> , these include the up-regulation of virulence factors and hemolysis production, biofilm formation, and colonization-based regulation of surface proteins and adhesion factors. As such, the interactions of these systems have become key targets in the design of small inhibitor compounds.</p> <p>Through the creation of a protein truncation series, we proposed the development of a small protein for the inhibition of key protein-protein interactions involved in <em>S. aureus</em> <em>agr</em> two-component signaling. Herein, we demonstrate the efficacy of these protein truncations as dominant negative inhibitors of AgrC:AgrA interactions, likely acting as a dominant phosphoacceptor in place of endogenous AgrA. We provide evidence of this function through <em>in vitro </em>hemolysis assays and phosphate-detection based gel electrophoresis.</p> / Master of Science (MSc)

Type 2 diabetes (T2D) has a genetic component which is only partially understood. The majority of genetic variance in disease susceptibility is unaccounted for, whilst the precise transcripts and molecular mechanisms through which most risk variants exert their effect is unclear. A complete understanding of T2D susceptibility mechanisms could have benefits in risk prediction, and in drug discovery through the identification of novel therapeutic targets. Work presented in this thesis aims to define relevant transcripts and disease mechanisms at known susceptibility loci, and to identify disease association with classes of genetic variation other than common single nucleotide polymorphisms (SNPs). KCNQ1 contains intronic variants associated with T2D susceptibility and β-cell dysfunction, but only maternally-inherited alleles confer increased disease risk. It maps within an imprinted domain with an established role in congenital and islet-specific growth phenotypes. Using human adult islet and foetal pancreas samples, I refined the transcripts and developmental stage at which T2D susceptibility must be conferred by demonstrating developmentally plastic monoallelic and biallelic expression. I identified a potential risk mechanism through the effect of T2D risk alleles upon DNA methylation. The disease-associated regions identified through genome-wide association (GWA) studies often contain multiple transcripts. I performed mRNA expression profiling of genes within loci associated with raised proinsulin/insulin ratios in human islets and metabolically relevant tissues. Some genes (notably CT62) were not expressed and therefore excluded from consideration for a risk effect, whilst others (for example C2CD4A) were highlighted as good regional candidates due to specific expression in relevant tissues. GWA studies for T2D risk have focused predominantly upon common single nucleotide polymorphisms. As part of a consortium conducing GWA analysis for copy number variation (CNV) and T2D risk, I optimised and compared alternative methods of CNV genotyping, before using this information to validate two signals of disease association. I genotyped three rare single nucleotide variants emerging from an association study with T2D risk based on imputed data, providing an indication of imputation accuracy and more powerful disease association analysis. These data underscore the challenge of translating association signals to causal mechanisms, and of identifying alternative forms of genomic variation which contribute to T2D risk. My work highlights candidates for functional analysis around proinsulin-associated loci, and makes significant progress towards uncovering risk mechanisms at the KCNQ1 locus.

Since the initial description of cancer stem cells (CSCs) as a self-renewing subpopulation of malignant cells with tumor-initiating capacity, a growing body of evidence has supported the existence of CSCs in virtually every tumor type. Our previous work in colorectal cancer has identified the transcription factor CDX1 as a key regulator of colorectal CSC differentiation. CDX1 expression is frequently lost in colorectal cancer, resulting in more aggressive, poorly differentiated tumors with higher proportions of CSCs. Many miRNAs have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in colorectal cancer, remain poorly understood. We began by identifying miRNAs downstream of CDX1 by using high-throughput small-RNA sequencing to profile miRNA expression in two pairs of colorectal cancer cell lines with stable CDX1 overexpression or knockdown. Validation of candidates identified by RNAseq in a larger cell line panel revealed miR-215 to be most significantly correlated with CDX1 expression. ChIP-qPCR and promoter reporter assays confirmed that CDX1 directly transactivates miR-215 transcription. MiR-215 is depleted in FACS-enriched CSCs compared to unsorted samples. Overexpression of miR-215 in poorly-differentiated, highly clonogenic cell lines causes growth arrest and a dramatic decrease in colony formation. miR-215 knockdown using a miRNA sponge causes an increase in clonogenicity and impairs differentiation in CDX1-high cell lines. Indeed, the effects of CDX1 expression on both gene expression and colony morphology can be attenuated by miR-215 inhibition, indicating that miR-215 is a functional mediator of CDX1. Microarray studies following miR-215 overexpression indicate that miR-215 induces terminal differentiation-associated growth arrest, due in part to direct silencing of BMI1 expression and de-repression of BMI1 target genes including CDKN1A. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in colorectal cancer. We further characterize another miRNA-transcription factor axis in colorectal cancer, and we identify the novel miR-3189-3p as a potent effector of cell death with potential therapeutic implications.