The optimal route for clinical delivery of oncolytic viruses (OVs) is thought to be intravenous (IV) injection; however, the immune system is armed with several highly efficient mechanisms to remove pathogens from the circulatory system. To overcome the challenges in trying to deliver OVs IV, cell carriers have been investigated to determine their suitability as delivery vehicles for systemic administration of OVs. Here we demonstrate the utility of a Drosophila melanogaster cell platform for the production and in vivo delivery of multi-gene biotherapeutic systems. We show that cultured Drosophila S2 cell carriers can stably propagate OV therapeutics that are highly cytotoxic for mammalian cancer cells without adverse effects on insect cell viability or cellular gene expression. Drosophila cell carriers administered systemically to immunocompetent animals trafficked to tumours to deliver multiple biotherapeutics with little apparent off-target tissue homing or toxicity, resulting in a therapeutic effect. S2 cells provide a genetically tractable platform supporting the integration of complex, multi-gene biotherapies while avoiding many of the barriers to systemic administration of mammalian cell carriers. Once OVs are delivered to tumour beds, they initiate replication in tumour cells, which often possess defects in antiviral pathways and are thus susceptible to infection. However, not all tumours have defects in their antiviral defenses and thus virus replication in these tumours is rather limited. Identifying and modulating host factors that regulate virus replication in OV-resistant cancer cells, but not normal cells, could lead to increased replication in these tumours and potentially improve therapeutic outcomes. We therefore conducted an RNA interference screen using Sindbis virus (SINV) in order to identify host factors that modulate OV replication in tumour cells. Specifically, serial passage of a SINV- artificial microRNA (amiRNA) library in a tumour cell line followed by deep sequencing of ii the selected virus populations led to the identification of several amiRNA sequences that were enriched. Furthermore, the identified amiRNA sequences increased the replication of various OVs both in vitro and in vivo, ultimately resulting in an enhanced therapeutic effect. Overall, the work presented here highlights strategies in which both the systemic delivery and tumour-specific replication of OVs can be improved.

Vector control is an essential and effective approach for controlling transmission of vector-borne diseases. However, increasing resistance to insecticide and drugs suggests that new strategies to control vector-borne diseases are needed. One possible strategy involves replacing mosquito populations with disease-resistant transgenic mosquitoes. Transposable elements (TEs) are an important component in this new strategy due to their ability to integrate exogenous DNA into chromosomes. They could potentially be useful tools in assisting the spread of disease-resistant genes in mosquito populations. This research focuses on two related subjects, TEs and their regulation. The first subject is on a Long Terminal Repeat (LTR) retrotransposon in the African malaria mosquito, Anopheles gambiae, namely Belly. The second subject focuses on the characterization of piwi genes in the dengue and yellow fever mosquito, Aedes aegypti. For the first subject we characterized Belly by identifying the two identical LTRs and one intact open reading frame. We also defined the target site duplications and boundaries of the full-length Belly element. This novel retrotransposon has nine full-length copies in the An. gambiae sequenced genome and their nucleotide similarity suggests that there has been fairly recent retrotransposon. We have shown that Belly is transcribed and translated in An. gambiae. Single LTR circles were recovered from An. gambiae cells, which is consistent with active transposition of Belly. The second subject focuses on the piwi genes of Ae. aegypti. We found nine potential piwi genes in Ae. aegypti and two in An. gambiae. Phylogenetic analysis suggests that these piwis formed two subgroups and gene duplication within each group occurred after the divergence between the two mosquito species. RT-PCR and transcriptome analysis showed Ago3 as well as all the seven tested piwi genes were expressed either in germline tissues or developing embryos. Differential expression patterns were observed. While most piwis were transcribed in the ovaries, testis, and embryos, two piwis appear to have a zygotic expression. Three piwi genes (piwi 3, piwi 4, and Ago3) were also detected in adult somatic tissues of Ae. aegypti. The expansion of the number of piwi genes in Ae. aegypti compared to An. gambiae and D. melanogaster may be correlated with a larger genome size and greater amount of TEs. The finding of piwi expression in adult somatic tissues is intriguing. It is possible that these piwi genes were expressed in the adult stem cells. It is also possible that they may be involved with anti-viral defense. Both of these hypotheses require further testing. / Master of Science

No description available.

An analysis of Glycine max homologs of the symbiosis genes DOES NOT MAKE INFECTIONS (DMI) DMI1, DMI2 and DMI3 was carried out as it relates to the defense response to Heterodera glycines parasitism. Transgenic analyses of the DMI1, DMI2 and DMI3 for overexpression showed decreased H. glycines parasitism while the analyses for RNAi showed increased H. glycines parasitism. The combination of decreased parasitism in the H. glycines-susceptible genotype G. max [Williams 82/PI 518671] and increased parasitism in the H. glycines resistant genotype G. max [Peking/PI 548402] is taken as the genes function in the defense process at some level. Prior analyses have shown that mitogen activated protein kinases (MAPKs) function in the defense response that has to H. glycines. A preliminary RNA seq analysis of MAPK3-1 and MAPK3-2 overexpressing roots reveal increased relative transcript abundance of DMI3, but only in the MAPK3-1 overexpressing roots. Additionally, examination of the expression profiles of two G. max MAPK3-1 and MAPK3-2 showed that their relative transcript abundances in some cases are influenced by DMI1, DMI2 and DMI3 expression. Taken together, the results show that the G. max DMI1, DMI2 and DMI3 function in the defense response to H. glycines and appear to involve MAPKs.

We have explored correlations between the measured efficiency of the RNAi process and several computed signatures that characterize equilibrium secondary structure of the participating mRNA, siRNA, and their complexes. A previously published data set of 609 experimental points was used for the analysis. While virtually no correlation with the computed structural signatures are observed for individual data points, several clear trends emerge when the data is averaged over 10 bins of N ~ 60 data points per bin. The strongest trend is a positive linear (r² = 0.87) correlation between ln(remaining mRNA) and ΔG_ms, the combined free energy cost of unraveling the siRNA and creating the break in the mRNA secondary structure at the complementary target strand region. At the same time, the free energy change ΔG_total of the entire process mRNA + siRNA → (mRNA – siRNA)_complex is not correlated with RNAi efficiency, even after averaging. These general findings appear to be robust to details of the computational protocols. The correlation between computed ΔG_ms and experimentally observed RNAi efficiency can be used to enhance the ability of a machine learning algorithm based on a support vector machine (SVM) to predict effective siRNA sequences for a given target mRNA. Specifically, we observe modest, 3 to 7%, but consistent improvement in the positive predictive value (PPV) when the SVM training set is pre- or post-filtered according to a ΔG_ms threshold. / Master of Science

Cilia are small microtubule based structures found on the surface of almost all mammalian cells, enclosed in a highly specialised extension of the cell membrane. Components of several key developmental signalling pathways, in particular Hedgehog (Hh) signalling, are enriched in cilia and cells with mutations in cilia structure show aberrant signalling, suggesting cilia act as “antennae” to focus these signalling cascades. A spectrum of human diseases, termed ciliopathies, are caused by problems in cilia formation or cilia function, which display wide ranging phenotypes from embryonic lethality to retinal degeneration, polydactyly to cystic kidneys. Despite recent advances in the understanding of the essential roles cilia play in mammalian development, exactly how these complex structures are put together, how they carry out their diverse functions, and how they are regulated is not well understood. In this thesis, I describe a screen for genes involved in cilia formation and function. While optimising ciliogenesis and immunofluorescence protocols for the screen, the phenotypes of two ciliary mutant cell lines were analysed. Wdr35yet/yet and Dync2h1pol/pol mouse lines were identified in an ENU screen for genes involved in early development, and shown to have gross phenotypes similar to other ciliary mutants (Mill et al. 2011). Intraflagellar transport (IFT) is the active transport of proteins up and down the ciliary axoneme. Dync2h1 is a retrograde IFT motor component, whereas Wdr35 is part of the retrograde IFT-A complex. In this thesis, the cellular phenotypes of mouse embryonic fibroblasts derived from these mutants are described, showing that despite the fact both genes are thought to be involved in retrograde IFT, they show distinct ciliary phenotypes, suggesting novel roles for Wdr35 in mouse ciliogenesis. An siRNA screen was carried out in mouse fibroblasts to identify genes involved in (i) cilia formation, assayed by immunofluorescence for ciliary markers, and (ii) cilia function, assayed by activity of a Hh responsive luciferase transgene as an indirect readout of ciliary function. Although scalable, I initially screened a small test set of thirty-six putative cilia candidates, identified by cross species transcriptomic analysis. We identified several possible hits, many of which were in the ciliome database but also importantly, several genes with no known link to ciliogenesis. Repeats, correlation of phenotype to knockdown efficiencies and localisation studies validated two hits, Ccdc63 and Azi1. Ccdc63 is a novel coiled-coil gene with no previous link to ciliogenesis; the phenotype for this gene was analysed in real time using fluorescently tagged ciliary markers. A second hit, Azi1, was followed up in more detail. The reduction in ciliogenesis upon Azi1 knockdown was confirmed with separate siRNAs, and was rescued by overexpressing siRNA insensitive Azi1-GFP, confirming the phenotype is not due to off-target effects of the siRNAs. Azi1 gene trap mutant mice were generated and confirmed to be null mutations. Surprisingly, the mice survive, showing Azi1 is not essential for mammalian ciliogenesis. However, mutant males are infertile, with highly reduced sperm count and sperm abnormalities indicative of an arrest at Stage IX of spermiogenesis, when the flagellum, a highly specialised motile cilium, forms. The small number of sperm that do get to the epididymus are immotile. We suggest Azi1 is essential to in the formation of the sperm flagella and male fertility.

Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase which activates, upon EGFR binding, a number of signaling pathways including the mitogenic protein kinase pathway (MAPK) and phosphatidylinositol 3-kinase cascade (PI3K). Over expression of EGFR is a common feature in variety of human cancers including lung, colorectal, breast, pancreatic and oesophageal cancers and results in autonomous cell growth, enhanced metastatic potential, tissue invasion and increased resistance to current cancer therapeutics. Thus EGFR has been identified as a potential target in cancer therapeutics. Using the RNA interference (RNAi) pathway, the aim was to specifically knockdown expression levels of endogeneous EGFR in human oesophageal squamous carcinoma cell (HOSCC) lines. The RNAi pathway was initiated through the transfection of three specifically designed short hairpin RNAs (shRNAs) against human EGFR. The shRNAs were specifically designed using bioinformatics tools and their individual knockdown efficacy determined through the introduction of an exogeneous based target reporter systems, psiCHECK and pcieGFP. Expression levels of EGFR were determined using Western blot analysis followed by densitometry. Knockdown of EGFR was achieved by all three EGFR shRNAs in the three HOSCC cell lines (WHCO1, WHCO5 and WHCO6) despite low transfection levels of ~10%. Greastest knockdown of EGFR (85%) was achieved by EGFR sh2 in WHCO5. EGFR sh2 and sh1 resulted in average knockdown of EGFR of ~ 65% in WHCO1 and WHCO5 respectively. Weakest knockdown of EGFR (~ 20%) was obtained by all three EGFR shRNAs following transfection of WHCO6. RNAi-based approaches therefore show substantial potential for the specific and efficient targeting of EGFR in human cancer cells.

No description available.

Phosphoinositides (PIs) are pivotal lipid molecules with both scaffolding and signalling functions regulating key aspects of cellular physiology. For example, phosphatidylinositol (3,4,5)-trisphosphate, generated by phosphoinositide 3-kinase (PI3K), is an essential mediator of the PI3K/AKT signalling pathway, which is crucial for cell proliferation, survival and apoptosis. Constitutive activation of this signalling cascade has been identified in acute myeloid leukaemia (AML), the most common haematopoietic malignancy in adults, and experimental deletion of the PI3K antagonists PTEN and SHIP cause leukaemia in mice. However, little is known regarding the role of other PI modulator proteins in AML. Thus, in this thesis, a lentivirally delivered small hairpin RNA (shRNA) library targeting 103 genes (345 pLKO knockdown constructs) with presumed or established roles in PI metabolism was utilised to screen for genes required for AML blast cell viability/proliferation and differentiation. First, knockdown constructs were tested for their impact on proliferation/viability in seven human AML cell lines by measuring fold change in fluorescence of the cell viability dye alamarBlue relative to controls (cells transduced with a non-targeting control hairpin) over three days. This identified 13 candidate genes selected with the criterion that two or more knockdown constructs per gene reduce cell viability/proliferation relative to control by greater than or equal to50 % across all cell lines. From these candidate genes, PIP4K2A, INPP5B and IMPAD1 were selected for downstream validation experiments, which reproduced the observation from the primary screen. For INPP5B and IMPAD1, knockdown constructs also reduced clonogenic potential of primary human AML samples but only showed a modest effect on normal CD34+ haematopoietic stem or progenitor cells (HSPCs) in a methylcellulose based assay. This could be recapitulated in a murine setting where knockdown constructs targeting both genes reduced clonogenic potential of murine MLL- AF9 AML cells with little effect on normal KIT+ HSPCs. In line with this, Inpp5b knockout KIT+ BM cells either failed to immortalise or weakly immortalised, following forced expression of the powerful MLL-AF9 oncogene. A further screen was performed to identify regulators of THP-1 blast cell differentiation, by seeding knockdown construct transduced cells into methylcellulose based semisolid media. After ten days of incubation the degree of macrophage differentiation was evaluated by light microscopy and an arbitrary differentiation score was given. With the criterion that greater than or equal to2 knockdown constructs per gene received the highest differentiation score, reflecting terminal macrophage differentiation of all seeded cells, SBF2 was identified as the top-scoring hit. Validation experiments have confirmed macrophage differentiation based on cytospin preparations of SBF2 knockdown THP-1 cells. Moreover, xenograft assays have shown that knockdown constructs targeting PIP4K2A and SBF2 delayed or abrogated in vivo leukaemogenesis. Thus this work has identified novel roles for PI modulator genes in human AML with possible therapeutic potential.

Macrophages are present in virtually all tissues and account for approximately 10% of all body mass. Although classically credited as the scavenger cells of innate immune system, ridding a host of pathogenic material and cellular debris though their phagocytic function, macrophages also play a crucial role in embryogenesis, homeostasis, and inflammation. De-regulation of macrophage function is therefore implicated in the progression of many disease states including cancer, arthritis, and atherosclerosis to name just a few. The diverse range of activities of this cell can be attributed to its exceptional phenotypic plasticity i.e. it is capable of adapting its physiology depending on its environment; for instance in response to different types of pathogens, or specific cocktail of cytokines detected. This plasticity is exemplified by the macrophages capacity to adjust rapidly its transcriptional profile in response to a given stimulus. This includes interferons which are a group of cytokines capable of activating the macrophage by interacting with their cognate receptors on the cell. The different classes of interferons activate downstream signalling cascades, eventually leading to the expression (as well as repression) of hundreds of genes. To begin to fully understand the properties of a dynamic cell such as the macrophage arguably requires a holistic appreciation of its constituents and their interactions. Systems biology investigations aim to escape from a gene-centric view of biological systems. As such this necessitates the development of better ways to order, display, mine and analyse biological information, from our knowledge of protein interactions and the systems they form, to the output of high throughput technologies. The primary objectives of this research were to further characterise the signalling mechanisms driving macrophages activation, especially in response to type-I and type- II interferons, as well as lipopolysaccharide (LPS), using a ‘systems-level’ approach to data analysis and modelling. In order to achieve this end I have explored and developed methods for the executing a ‘systems-level’ analysis. Specifically the questions addressed included: (a) How does one begin to formalise and model the existing knowledge of signalling pathways in the macrophage? (b) What are the similarities and differences between the macrophage response to different types of interferon (namely interferon-β (IFN-β) and interferon-γ (IFN-γ))? (c) How is the macrophage transcriptome affected by siRNA targeting of key regulators of the interferon pathway? (d) To what extent does a model of macrophage signalling aid interpretation of the data generated from functional genomics screens? There is general agreement amongst biologists about the need for high-quality pathway diagrams and a method to formalize the way biological pathways are depicted. In an effort to better understand the molecular networks that underpin macrophage activation an in-silico model or ‘map’ of relevant pathways was constructed by extracting information from published literature describing the interactions of individual constituents of this cell and the processes they modulate (Chapter-2). During its construction process many challenges of converting pathway knowledge into computationally-tractable yet ‘understandable’ diagrams, were to be addressed. The final model comprised 2,170 components connected by 2,553 edges, and is to date the most comprehensive formalised model of macrophage signalling. Nevertheless this still represents just a modest body of knowledge on the cell. Related to the pathway modelling efforts was the need for standardising the graphical depiction of biology in order to achieve these ends. The methods for implementing this and agreeing a ‘standard’ has been the subject of some debate. Described herein (in Chapter-3) is the development of one graphical notation system for biology the modified Edinburgh Pathway Notation (mEPN). By constructing the model of macrophage signalling it has been possible to test and extensively refine the original notation into an intuitive, yet flexible scheme capable of describing a range of biological concepts. The hope is that the mEPN development work will contribute to the on-going community effort to develop and agree a standard for depicting pathways and the published version will provide a coherent guide to those planning to construct pathway diagrams of their biological systems of interest. With a desire to better understand the transcriptional response of primary mouse macrophages to interferon stimulation, genome wide expression profiling was performed and an explorative-network based method applied for analysing the data generated (Chapter-4). Although transcriptomics data pertaining to interferon stimulation of macrophages is not entirely novel, the network based analysis of it provided an alternative approach to visualise, mine and interpret the output. The analysis revealed overlap in the transcriptional targets of the two classes of interferon, as well as processes preferentially induced by either cytokine; for example MHC-Class II antigen processing and presentation by IFN-γ, and an anti-proliferative signature by IFN-β. To further investigate the contribution of individual proteins towards generating the type-I (IFN-β) response, short interfering RNA (siRNA) were employed to repress the expression of selected target genes. However in macrophages and other cells equipped with pathogen detection systems the act of siRNA trasfection can itself induce a type-I interferon response. It was therefore necessary to contend with this autocrine production of IFN-β and optimise an in vitro assay for studying the contribution of siRNA induced gene-knock downs to the interferon response (described in Chapter-5). The final assay design incorporated LPS stimulation of the macrophages, as a means of inducing IFN-β autonomously of the transfection induced type-I response. However genome-wide expression analysis indicated the targeted gene knock-downs did not perturb the LPS response in macrophages on this occasion. The optimisation process underscored the complexities of performing siRNA gene knockdown studies in primary macrophages. Furthermore a more thorough understanding of the transcriptional response of macrophages to stimulation by interferon or by LPS was required. Therefore the final investigations of this thesis (Chapter-6) explore the transcriptional changes over a 24 hour time-course of macrophage activation by IFN-β, IFN-γ, or LPS and the contribution of the macrophage pathway model in interpreting the response to the three stimuli. Taken together the work described in this thesis highlight the advances to be made from a systems-based approach to visualisation, modelling and analysis of macrophage signalling.