Epstein-Barr virus (EBV) is one of the most successful human pathogens in the world and establishes a lifelong persistent infection in 95% of adult population worldwide. It is associated with a number of malignancies including Burkett’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma(NPC) and gastric carcinoma. EBV was the first virus reported to produce microRNAs (miRNAs) and it encodes 44 mature miRNAs from 2 viral transcripts, BART and BHRF1. The BART transcript is abundantly expressed in all latently infected cells, particularly in epithelial cells. The BART miRNAs (miR-BARTs) were shown to be involved in apoptosis inhibition, immune evasion, metastasis, viral and cellular transcripts regulation. The high expression profile and the diverse functions of miR-BARTs suggest that they may play a critical role in the development of EBV-associated NPC. In order to understand the importance of miR-BARTs in NPC development, in this thesis, I conducted a study on the miR-BARTs function in nasopharyngeal carcinogenesis. In the first part, I characterized the cellular target and function of an abundantly expressed miR-BART in NPC. In the second part, I established a novel recombinant EBV construction system for genetic studies of miR-BARTs in nasopharyngeal epithelial (NP) cells. In the first part of my study, I characterized the cellular target and function of miR-BART3* in NPC. As predicted by bioinformatics, tumor suppressor protein DICE1 was a cellular target of miR-BART3*. The specific targeting between miR-BART3* and DICE1 3’UTR was validated by luciferase assays and the downregulation of both endogenous DICE1 protein and mRNA was observed in EBV+epithelial cells and miR-BART3* expressing cells. In addition, restoration of DICE1 protein expression by inhibition of miR-BART3* was also demonstrated in EBV+epithelial cells. Moreover, miR-BART3* was shown to promote cell proliferation via suppression of DICE1. Analysis of22 human nasopharyngeal(NP)biopsy samples demonstrated the inverse correlation between miR-BART3* and DICE1 expression. Taken together, miR-BART3* downregulates the tumor suppressor DICE1 protein to promote cell proliferation and transformation in NPC. Besides the candidate approach, genetic studies can provide a systematic view of the functions of all miR-BARTsand shed light on the importance of miR-BARTs in NPC under a more physiological condition. At present, bacterial artificial chromosome (BAC) technology is commonly used for recombinant EBV construction. However, the intrinsic disadvantages of BAC prevent its use in NP epithelial cells. Therefore in the second part of my study, I established a novel CRISPR/Cas9-mediated recombinant EBV construction system and constructed a miR-BART deleted recombinant EBV. The CRISPR/Cas9 system was demonstrated to be effective in EBV genome editing and Akata cells were infected by the recovered recombinant mutant virus. Infected Akata cells served as the source for NP cell infection through co-culture. The new CRISPR/Cas9 system have many advantages over the conventional EBV BAC method. My work reported in this thesis not only illustrated the importance of miR-BARTs in NPC, but also provide a new technology platform for further study of miR-BARTs in NP epithelial cells. (An / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The etiology of nasopharyngeal carcinoma (NPC) is based on intricate interactions among environmental factors, genetic susceptibility and Epstein-Barr virus (EBV) infection. Information concerning the role of EBV infection, particularly during the early stage of NPC development is poorly understood. Our laboratory has shown that stable infection of EBV could be achieved in immortalized epithelial cell lines which harbor genetic alterations and altered cell signaling pathway. In this study, these cell models were used to elucidate early events involved in EBV infection in premalignant nasopharyngeal epithelial cell models and their implications on development and progression of nasopharyngeal carcinoma. The response of EBV-infected cells to a stromal inflammatory cytokine, interleukin-6 (IL-6), was examined. EBV infection and long-term propagation of EBV-infected nasopharyngeal epithelial cells confer enhanced sensitivity to STAT3 activation induced by IL-6. IL-6-induced STAT3 activation reinforced their malignant properties in nasopharyngeal epithelial cells and may play a role in the development of nasopharyngeal carcinoma. Furthermore, constitutive STAT3 activation was demonstrated to facilitate malignant transformation of EBV-infected premalignant nasopharyngeal epithelial cells to cancer cells, suggesting that EBV infection and STAT3 activation might synergistically promote the development of NPC. This study also provides support for the existence of a positive feedback loop of IL-6/STAT3/LMP in NP460hTert-EBV cells, which enhanced STAT3 activation in EBV-infected cells. Elevated levels of IL-6Rα expression were observed in EBV-infected NP460hTert cells compared with uninfected cells and were largely responsible for the enhanced sensitivity of IL-6-induced STAT3 activation in these cells. High expression level of IL-6Rα could amplify IL-6 signaling in nasopharyngeal epithelial cells to promote growth proliferation in NP460hTert cells and increase the growth rate of xenografted NPC cells in immune-suppressed animals, suggesting that IL-6Rα overexpression may play a role of contributing to the development of nasopharyngeal carcinoma. The serum concentrations of both IL-6 and sIL-6R were also higher in NPC patients than healthy individuals and may have prognostic values to predict clinical outcome and disease progression in NPC patients. In conclusion, these data support the hypothesis that EBV infection under inflammatory environment may activate aberrant gene expressions and cell signaling to facilitate malignant transformation. The inflammatory cytokine, IL-6, may mediate the role of EBV infection in the development of NPC. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

En aquesta tesi s'han dut a terme dos tipus d'estudis diferents. L'objectiu del primer era la preservació del semen de porcí a 15ºC i el del segon eren els co-cultius homòlegs de cèl·lules epitelials de l'oviducte i espermatozoides de porcí. Pel que fa al primer estudi, s'ha observat que l'addició de la prostaglandina F2α i àcid hialurònic a les dosis seminals no malmena la qualitat espermàtica i que la tolerància dels espermatozoides als canvis d'osmolalitat del medi es pot correlacionar proves de fertilitat i prolificitat..Respecte el segon, s'ha determinat que les cèl·lules oviductals afecten els paràmetres espermàtics i que la presència d'espermatozoides sobreexpressa els gens que codifiquen per les proteïnes de xoc tèrmic. Així, se suggereix que aquestes proteïnes tenen algun paper en els processos reproductius que tenen lloc a l'oviducte, malgrat que s'hagi observat, mitjançant la tècnica de la interferència de l'RNA, que la HSP90AA1 no està implicada en el perllongament de la viabilitat espermàtica. / In this thesis, two different studies have been conducted. The aim of the first experimental chapter was boar sperm preservation at 15ºC, the second dealing with in vitro homologous co-culture of oviductal epithelial cells (OEC) and spermatozoa. Regarding the first, it has been observed that the addition of prostaglandin F2α and hyaluronic acid do not cause any harm on sperm quality, and the osmotic tolerance of spermatozoa can be correlated with fertility and prolificacy rates of a given ejaculate.As far as the second study is concerned, OEC specifically affect sperm functional parameters and the presence of spermatozoa upregulates the expression of some genes encoding for heat shock proteins. Some role in the reproductive processes taking place in the oviduct is therefore suggested for this protein family, even though it has been observed, by means of RNA interference, that HSP90AA1 is not the protein involved in prolonging sperm survival.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

Copies of author's previously published articles inserted. / Bibliography: leaves 152-176. / xiv, 177 leaves, [4] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Explores aspects of the relationship between metabolism and proliferation of colonic epithelial cells from rats and humans. Emphasis is placed on developing and integrating in vivo and in vitro models for both metabolic and proliferative studies. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1990

Content removed due to copyright restriction: Appendix 6 Cooper, C. V., Stelwagen, K., Singh, K., Farr, V. C., Prosser, C. G., and Davis, S. R. (2004): Expression of the tight junction protein zonula occludens-1 during mammary engorgement. Proceedings of the New Zealand Society of Animal Production 64,43-47. Singh, K., Dobson, J., Phyn, C. V. C., Davis, S. R., Farr, V. C., Molenaar, A. J., and Stelwagen, K. (2005): Milk accumulation decreases expression of genes involved in cell-extracellular matrix communication and is associated with induction of apoptosis in the bovine mammary gland. Livestock Production Science 98,67-78. Appendix 7 McMahon, C.D., Farr, V.C., Singh, K., Wheeler, T.T. and Davis, S.R. (2004). Decreased expression of ß1-integrin and focal adhesion kinase in epithelial cells may initiate involution of mammary glands. Journal of Cellular Physiology 200, 318-325 / Extended periods of milk accumulation result in loss of secretory activity, increased apoptosis and eventually, involution of mammary glands. This process is associated with increased permeability of the tight junction (TJ) complexes between adjacent mammary epithelial cells (MECs). The change in cell shape during mammary engorgement from a cuboidal to a flattened morphology may initiate changes in protein and gene expression (mechanotransduction) that trigger these processes. Therefore, this study examined the regulation of the major TJ protein components during mammary engorgement, and in particular the role of physical distension of the mammary epithelium in the regulatory process. Expression of the integral transmembrane TJ proteins, occludin and claudin-1, and the cytoplasmic TJ protein, ZO-1, were down-regulated in both bovine and rat mammary glands during the early stages of mammary apoptosis and involution following the abrupt cessation of milk removal. In the rat, these responses were locally regulated as they occurred only in teat-sealed glands in a hemi-suckled model. Furthermore, the down-regulation of TJ proteins is consistent with a loss of TJ integrity during mammary engorgement. Induced physical distension of rat mammary glands in vivo transiently up-regulated the expression levels of occludin protein and mRNA, and ZO-1 mRNA, followed by an accelerated decrease in expression compared with the effects of milk accumulation alone. This was associated with the initiation of apoptosis, the up-regulation of the pro-apoptotic factor pSTAT3, and the down-regulation of the cell-ECM survival factor βl-integrin. An in vitro model was also developed to stretch MECs, mimicking the flattening in cell shape during mammary engorgement in vivo. While stretching MECs in vitro did not conclusively alter TJ protein expression, the overall results of this project support further investigation into the role of the TJ complex in mechanotransduction pathways. In addition, the results point to crosstalk between cell-ECM survival signalling and STAT3 death signalling as a candidate for regulation by physical distension of the mammary epithelium. In conclusion, this study supports the hypothesis that physical distension during engorgement of the mammary glands with milk is a primary trigger initiating apoptosis of MECs through changes in the regulation of gene pathways controlling cell survival and death, and the disruption of TJ function.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 120-147).

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.