Human papillomavirus (HPV) infections play a crucial role in human carcinogenesis. Greater than 96% of all cervical carcinomas are positive for high-risk HPV infections; especially types 16 and 18. High-risk HPV onco-proteins, E6 and E7, are consistently expressed in such cancers and function by inactivating p53 and pRb tumor suppressors, respectively. The presence of high-risk HPVs is also correlated with anogenital cancers. In this study, we examined the effect of high-risk HPV type 16 E6 and E7 oncoproteins in two normal human colorectal epithelial cell lines, NCE1 and NCE5. We report that the expression of E6/E7 proteins, alone, induced cellular transformation of both cell lines; consequently, NCE1-E6/E7 and NCE5-E6/E7 form colonies in soft agar with respect to their wild type cells. This is accompanied by cell cycle deregulation, as is demonstrated by the over-expression of cyclin dependant kinases (cdks) and their respective cyclins. Furthermore, we demonstrate that E6/E7 oncoprotein transduction induces migration of colorectal epithelial cells. More still, well analyzed Id gene expression, a family member of the helix-loop-helix (HLH) transcription factors involved in the regulation of cell invasion and metastasis of human cancer cells. In parallel, using tissue microarray analysis we found that the four members of the Id protein family are correlated with the presence of HPV type 16 and 18 in human colon cancer tissues. Our data suggests that high-risk HPV infections are sufficient to induce cellular transformation of normal human colorectal cells, in vitro. Furthermore, the correlation with the Id family of proteins may present a novel set of markers associated with HPV induced colorectal carcinogenesis. Our results may suggest a new approach to detect and prevent colorectal cancer.

The introduction of next-generation sequencing (NGS) technologies has dramatically changed the field of virology, with many significant discoveries of novel circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses. Traditionally, most research into CRESS DNA viruses has often focused on investigating plant and animal pathogens that are of significant economic importance. This research has led to the discovery and establishment of three different CRESS DNA families including Geminiviridae, Nanoviridae and Circoviridae, which infect eukaryotes. CRESS DNA viruses can have single or multicomponent genomes, with the latter requiring all components for infection. CRESS DNA viruses have circular single-stranded DNA (ssDNA) genomes with at least one protein encoding a Rep which is responsible for viral replication. It has been shown that CRESS DNA viruses are able to evolve rapidly with nucleotide substitution rates that are similar to those observed in RNA viruses. The Rep gene has conserved regions known as motifs which are often used to determine relatedness between CRESS DNA virus. NGS has expanded our knowledge on the diversity of novel CRESS DNA viruses. Viral genomes are now routinely recovered from different sample types without any prior knowledge of the viral sequence. This has led to the development of the field of viral ecology. This field places an emphasis on viruses being one of the most abundant organisms on earth, and are therefore likely to play a major role in ecosystems. Environmental metagenomic studies have isolated CRESS DNA viruses from sea water, freshwater, faecal matter from various animals, soil, the atmosphere, sediments and sewage; dramatically increasing the known CRESS DNA viral genomes in the public domain. These studies are shedding light on the distribution of CRESS DNA viruses, as well as providing baseline data for future studies to examine virus-host interactions, community structure and ultimately viral evolution. Vector enable metagenomics (VEM) is another novel approach utilising NGS techniques for discovering CRESS DNA viruses. As many plant-infecting CRESS DNA viruses such as geminiviruses and nanoviruses are vectored by insects, this approach exploits this mechanism by using insect vectors as a surveillance tool to monitor and survey these viruses circulating in ecosystems. Recent studies have used these methods to identify known viral plant pathogens as well as novel viruses circulating in insect vectors such as whiteflies and other higher order insects such a mosquitoes and dragonflies. These approaches successfully demonstrated that VEM can be used as a unique method, with the first mastrevirus discovered in the new world being recovered from dragonfly species Erythrodiplax fusca using this approach. The research in this thesis uses metagenomics to survey CRESS DNA viral diversity in different organisms and environments. Two hundred and sixty eight novel CRESS DNA viruses were recovered and verified in this study from a range of sample types (adult Odonata, Odonata larvae, Mollusca, benthic sediment, water, Oligochaeta and Chironomidae) collected in the United States of America, Australia and New Zealand. All viral genomes isolated had two major proteins encoding for a putative Rep and coat protein (CP), with major Rep motifs identified in most Reps. Phylogenetic analysis of the Reps encoded by the viral genomes highlighted that most were extremely diverse falling outside of the previously described ssDNA viral families. A top-down approach was implemented to recover CRESS DNA viruses and possible viral pathogens from Odonata and their larvae. Thirty six viral genomes were recovered from terrestrial adult dragonflies as well as the twenty four from aquatic larvae. Dragonfly cycloviruses were isolated from the some adult Odonata species which were closely related to the isolates previously described by Rosario et al. (2012). The viruses isolated in the aquatic and terrestrial ecosystems differed substantially indicating that different CRESS DNA viromes exist in both land and water. The diversity of CRESS DNA viruses in seven different mollusc species (Amphibola crenata, Austrolvenus stutchburyi, Paphies subtriangulata, Musculium novazelandiae, Potamopyrgus antipodarum, Physella acuta and Echyridella menziesi) from Lake Sarah and the Avon-Heathcote estuary both in New Zealand, were also investigated. One hundred and forty nine novel viral genomes were recovered. Two CRESS DNA genomes were recovered from molluscs which have Rep-like sequences most closely related to those found in some bacterial genomes. Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) was originally isolated from fungal species Sclerotinia sclerotiorum in china and was later found in benthic sediments in New Zealand. As part of this study, SsHADV-1 was recovered from dragonflies (Erythemis simplicicollis, Ischnura ramburii and Pantala hymenaea) collected in Arizona and Oklahoma, USA suggesting a larger distribution of these viruses and not surprising given the near global distribution of S. sclerotiorum. Dragonfly larvae-associated circular DNA viruses (DflaCVs) that were originally isolated in Odonata larvae samples from three New Zealand lakes were later recovered from water, benthic sediment, worms and molluscs from one of the lakes initially sampled, suggesting that these viruses are ubiquitous in freshwater environments. This study has attempted to generate baseline data of CRESS DNA viruses in certain environments using NGS-informed approaches. This data was used to try and establish whether viral distribution in different samples types can potentially be explained by the food web interactions between different samples types. Although the analysis did not show any significant relationships between sample type interactions and viral distribution a few common associations between Odonata larvae and benthic sediment were evident. This was expected as the larvae live within the sediment so it could be assumed that they potentially have similar CRESS DNA viral distribution. Although the distribution of viruses varied across sample types, molluscs proved the best sampling tool for isolating largest numbers of CRESS DNA viruses in an ecosystem with extensive diversity. Overall, this research demonstrates the applications of NGS for investigating the diversity of CRESS DNA viruses. It demonstrates that some sample types such as Odonata in terrestrial systems and molluscs in aquatic environments, can be used as effective sampling tool to determine the diversity of CRESS DNA viruses in different environments as well as detecting previously isolated viruses. The CRESS DNA viruses isolated in this body of work provides baseline data that can potentially be used in future research to investigate hosts of these viruses and their interactions with hosts and potential flow in their environments.

No description available.

The “connection generation” craves interaction with and connection to vast social networks through the sharing of information, photos, opinions, entertainment and news. This sharing comes in the form of electronic word-of-mouth or eWOM, and provides marketing and communication managers with an unparalleled opportunity to reach a large number of consumers quickly. With the ever increasing growth of the internet and the rise of social media and social network sites, viral marketing has cemented itself in the marketing and corporate agenda. However, while there has been a shift in marketing budgets towards online and social media, little is known about how to successfully leverage viral marketing. Consequently, understanding why some videos go viral and others do not is becoming an increasingly popular focus of academic research. This study aimed to answer the following research question: What are the factors that drive the virality of online content?   In an attempt to answer this exploratory research question, four papers were used to look at its constituent parts. In the first paper, the role of emotion in the sharing of online content was investigated. Rime’s social sharing of emotion theory was used to explain why emotion could drive the spread of content online. We suggested that people’s propensity to share viral content was a function of the intensity, sociality and complexity of the emotion elicited by the viral content.   The following two papers further investigated the role of emotion in viral marketing by looking at the relationship between content and emotion. Paper 2 used interviews in a qualitative research design to propose a decision-tree of the interplay between content and emotion in viral marketing. This paper showed that the relevance of the content has an influence on viewers’ emotional response. Paper 3 took a closer look at the relationship between content and emotion by using a two-stage design: First, content analysis was done on the comments of selected YouTube videos. Second, an experiment was used to test the emotions that these videos elicited in respondents, the valence of those emotions, the intensity with which they were felt, as well as various content-related factors (e.g. the creativity and humor used in the videos). This paper looked specifically at the use of political communication in viral marketing and showed that creativity, valence and the intensity of the emotions elicited by the content are key drivers of viral success.   The final and fourth paper culminates in a model for the sharing of content online. This paper built on the findings from the previous papers, but also made use of interviews, and the analysis of a longitudinal dataset to propose a comprehensive model for the spread of content online. The longitudinal dataset was compiled using the top 10 posts from Reddit.com, a viral aggregator website, over the period of 25 days. The comprehensive model shows that there are external, intrapersonal and interpersonal drivers of viral content. The external drivers of viral content are the viral videos themselves (content) and its popularity. The content construct refers to various aspects related to the content itself, for example how informative, creative, humorous etc. the content is. Its popularity, on the other hand, was driven by both WOM and mainstream media reports. The intrapersonal drivers of viral content refer to the emotions that the content elicited in viewers. Viewers’ emotional response to the content was influenced by its relevance, but also by the valence and intensity of the emotion that they felt. Even though some content elicited intense emotions in viewers, some viewers did not share the content and interpersonal drivers of viral content was introduced to the model. These drivers recognise the social aspect of social media, and that content gets shared with large social networks. The model contends that people share viral content with their social networks as a form of online gift giving, out of altruism, or simply to build their own reputation. Finally, we contend that, in this content à emotion à social sharing chain, people share viral content both online and offline, as many respondents simply told their friends about the content (thus prompting them to go and watch the content themselves) or showed them the content themselves. This online and offline sharing of content increased the popularity of the content and a self-reinforcing chain was created, increasing the exponential growth typically associated with viral content.   As consumers are exposed to an increasing amount of marketing messages, and marketing budgets shrink, marketing managers could greatly benefit from better understanding how to more effectively make social media part of their marketing strategy. Viral marketing allows for a low-cost way of communicating marketing messages with great potential for impacting the market. This study ultimately shows what marketing managers can do to increase their chances of viral success, and ends off with a list of managerial recommendations to leverage the external, intrapersonal and interpersonal factors present in viral campaigns. / <p>QC 20140911</p>

Glaucoma is a group of optic nerve degenerative diseases, which leads to gradual and permanent vision loss. Recent developments in the field of gene therapy have proposed increasingly promising treatments for glaucoma, in the form of delivery of neuro-protective or neuro-regenerative genes to the retina. Despite these developments, there are concerns related to the biocompatibility and invasiveness of common gene delivery systems, since they are commonly mediated by viral gene carriers and invasive administration methods. Non-viral gene delivery systems offer a safe and increasingly efficient alternative to deliver therapeutic genes to the retina. An example of these systems is gemini-phospholipid nanoparticles (GL-NPs), which have been successfully used to deliver genes in similarly challenging anatomical settings, such as the skin. The objective of this thesis is to demonstrate the potential of GL-NPs, as candidate gene delivery vehicles for topically administered genes, targeted to the retina. The dicationic gemini surfactant, 12-7NH-12 was used, along with the helper lipids, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), to prepare various types of GL-NPs, and assess their transfection efficiency in the rat retinal ganglion cell line (RGC-5). The transfection efficiency was evaluated using flow cytometry, as a function of several physical and chemical parameters of GL-NPs. These include a range of charge ratios (5:1 to 15:1 ????), helper lipid composition (several DOPE: DPPC ratios), order of assembly (plasmid-gemini + lipid versus gemini-lipid + plasmid), and manufacturing method of helper lipid vesicles (thin film versus high pressure homogenization method). Size and zeta (??) potential characterization of GL-NPs was carried out in parallel, using dynamic light scattering, to relate the physical parameters of GL-NPs to their respective transfection efficiency. A comprehensive toxicological evaluation was undertaken to assess the extent of GL-NP???s toxicity in RGC-5 cells, using the resazurin-based PrestoblueTM cell toxicity assay. Optimized GL-NPs were used to induce expression of the brain derived neurotrophic factor (BDNF) in RGC-5 cells, and were assessed in terms of their capacity to induce neurite outgrowth. Quantification of neurite outgrowth was carried out by measuring average neurite length in RGC-5 cells, by confocal microscopic imaging of immunostained neurites. Furthermore, confocal microscopic studies were carried out to assess the extent of GL-NP???s corneal permeation in a 3-D human corneal epithelial (HCE) model. A parallel toxicological evaluation was completed to ensure GL-NP???s biocompatibility with the corneal epithelial cells. Finally, GL-NP biodistribution pattern and gene transfer capacity was assessed in a mouse model, following topical and intravitreal administration. The transfection efficiency in RGC-5 cells, which ranged between 2.1 ?? 0.3% and 14.5 ?? 1.4%, was highly dependent on GL-NP???s charge ratio, helper lipid composition, order of assembly, and manufacturing technique. GL-NPs at 10:1 ???? charge ratio, assembled with homogenized DOPE (25%)-DPPC (75%) helper lipid vesicles, in the plasmid-gemini + lipid order, mediated the highest transfection efficiency in RGC-5 cells. These GL-NPs had a size of 222.8 ?? 4.2 nm and a ?? potential of +33.5??2.9 mV. Optimized GL-NPs were highly biocompatible with both RGC-5 and HCE model cells, with viability values ranging between 94.8 ?? 6 % to 100 ?? 3.4 %. Assessment of corneal permeation showed that GL-NPs were able to bind to the corneal epithelial surface and achieve a moderate permeation depth (35-40 ??m), following topical application in the HCE model. Intravitreal injection of the non-viral GL-NPs in mice has successfully led to their localization within the nerve fiber layer (NFL) of the retina. Finally, GL-NPs were non-invasively delivered to several anterior chamber tissues, including the limbus, the iris and conjunctiva, following topical administration. GL-NPs offer several advantageous features critical to topical and intravitreal ocular administration of gene carriers, including in vitro corneal binding and effective biodistribution following in vivo topical and intravitreal administration, high biocompatibility, and a highly tunable transfection efficiency. The current data presents 12-7NH-12 GL-NPs as a promising candidate for ocular gene therapy applications.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV8), has been been identified in all epidemiological forms of KS as well as in tissue obtained from primary effusion lymphoma (PEL) and Multicentric Castleman's disease (MCD). The KSHV genome contains several putative oncogenes, suggesting that viral infection may induce cellular transformation and tumorgenesis. Herpesviruses encode a number of different surface glycoproteins, which are involved in virus-host interactions. Studies have shown that the viral glycoproteins H and L form a complex that plays an essential role in viral attachment and cell to cell fusion. Both glycoproteins have been identified in KSHV and were expressed in mammalian cells. Expression studies revealed that KSHV gH and gL exhibit similar features to those seen in other herpesviruses. However, KSI-IV gL appears to traffic independently and may function in cell to cell fusion processes even when expressed alone. KSI-IV de novo infections are rare and the lack of a reliable cell culture system has delayed pathogenesis studies. As part of this thesis the hepatoma cell line HepG2 has been shown to allow limited KSHV infection, as judged by nested PCR. Studies have shown that infection leads to increased apoptosis, although viral replication could not be detected. Furthermore, Epstein Barr Virus (EBV) appeared to modulate the ability of KSHV to infect HepG2 cells. Finally, a microtitre plate assay has been established for the quantification of the KSHV genome. A comparison of plasma and serum samples obtained at the same time point showed that plasma is more reliable in testing for KSHV, the DNA copy number in serum samples being reduced up to 10 fold. In conclusion, this new assay is a potentially useful tool for both diagnostic proposes and research studies.

Herein we describe a novel APC population in mice, designated LAPCs. LAPCs are BM-derived myeloid leukocytes, distinctive from other immune cells. As APCs, LAPCs respond to various virus infections including VACV, CBV3 and influenza A virus. Notably, influenza virus-activated LAPCs capture Ag in the lungs, and migrate into the DLN and spleen with delayed kinetics compared to DCs. In the DLN, influenza virus-activated LAPCs co-localize with T cells and selectively induce Th2 effector cell polarization by cell-cell contact-mediated modulation of GATA-3 expression. In support of a role for LAPCs in anti-influenza T2 immunity, adoptive transfer experiments revealed that influenza virus-activated LAPCs selectively augmented Th2 effector T cell responses in the DLN, increased production of anti-influenza immunoglobulin (Ig) including IgE in peripheral blood and increased levels of IL-5 and eotaxin in BAL fluid in recipient influenza infected mice. LAPC recipient mice exhibited exacerbated pulmonary pathology, with delayed viral clearance and enhanced pulmonary eosinophilia. Collectively, these results highlight the importance of LAPCs as novel immuno-modulators of T2 immunity during influenza A virus infection, which is implicated in both immunoprotection and immunopathology. Subsequently, we examined the immuno-modulatory effect of type-I IFN, specifically IFN-on the immune response against pulmonary influenza virus infection. We have provided evidence that a single dose of IFN- (1×105U) augmented DC migration but inhibited LAPC migration into the DLN. mIFN- treatment skewed the immune balance toward T1 immunity, identified as enhanced T1 effector T cell responses (Th1 and CTL) but diminished T2 effector T cell responses (Th2) in influenza virus infected mice. Finally, IFN- treated mice showed accelerated viral clearance and diminished pulmonary eosinophilia in lung tissue compared to control mice. Taken together, these results suggest that anti-influenza T1 and T2 immunity may be modulated differently by DCs and LAPCs, respectively. Furthermore, these results support the therapeutic potential of type I IFNs, especially IFN-, as an alternative antiviral to control both viral replication and immunopathology induced by influenza A virus infection in humans.

Evolution of viral myocarditis to dilated cardiomyopathy (DCM)represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. RESULTS: IRF9-/- mice showed dramatically increased mortality compared to the wildtype littermates (0% WT vs 72% IRF-9-/- on day 14, P<0.0001). On day 42, there was less cardiac hypertrophy and inflammation in IRF-9-/- mice compared to WT controls (p<0.05). Onn day 42 there was a dramatic increase in the number of cytotoxic and helper T-Cells in the wild-type mice that was not observed in the IRF-9-/- spleens (p<0.05). CONCLUSIONS: These data suggest a novel dual role of IRF-9 in not only regulating interferon in acute stage of viral infection in myocarditis, but also late acquired immunity activation, including CD4/8 populations, contributing to the development of chronic cardiomyopathy.

Includes bibliography (10 unnumbered leaves) / 66 leaves, [20] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1984

Copies of author's previously published articles contained in back cover pocket. / Bibliography: leaves 129-152. / xiv, 152, [60] leaves, [28] leaves of plates : ill. (some col.) (some folded) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines hepadravirus and HDV replication and gene expression with particular emphasis on the block(s) preventing HBV infection invitro, the extent of the helper function provided by HDV by HBV and the mechanism of HDV RNA replication. / Thesis (Ph.D.)--University of Adelaide, Depts. of Microbiology and Immunology, 1992