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Paraquat (PQ) is a cationic non-selective bipyridyl herbicide widely used in agriculture to control weeds and grasses. Epidemiologic studies indicate that exposure to pesticides can be a risk factor in the incidence of Parkinson`s disease (PD). A strong correlation has been reported between exposure to paraquat and PD incidence in Canada, Taiwan, and United States. This correlation is supported by animal studies showing that paraquat produces toxicity in dopaminergic neurons of the rat and mouse brain. However, it is unclear how paraquat triggers toxicity in dopaminergic neurons. Based on the previous reports, it was hypothesized that paraquat may induce oxidative stress and proteasomal dysfunction-mediated toxicity in dopaminergic neurons. To explore this possibility, dopaminergic SH-SY5Y human neuroblastoma cells were treated with paraquat, and several biomarkers of oxidative stress or proteasomal dysfunction were investigated. First, a specific dopamine transporter inhibitor GBR12909 significantly protected SY5Y cells against the toxicity of paraquat, indicating that paraquat exerts its toxicity by a mechanism involving the dopamine transporter (DAT). Second, paraquat increased the levels of reactive oxygen species (ROS) in SY5Y cells, but decreased the levels of glutathione. Third, paraquat inhibited glutathione peroxidase activity, but did not affect glutathione reductase activity. On the other hand, paraquat increased GST activity by 24 hr, after which GST activity returned to the control value at 48 hr. Fourth, paraquat decreased mitochondrial transmembrane potential (MTP). Fifth, paraquat produced the increases in malondialdehyde (MDA) and protein carbonyls, as well as DNA fragmentation, indicating oxidative damage to major cellular components. Sixth, paraquat decreased proteasomal activity, the activities of mitochondrial complex I and V, and intracellular ATP levels, but increased the activities of caspase 3 and 9, indicating that proteasomal inhibition is linked to mitochondrial dysfunction accompanied by the activation of apoptotic signaling pathway. Seventh, paraquat increased the protein levels of heme oxygenase-1 (HO-1), p53, Bax, α-synuclein and ubiquitinated proteins. Eighth, paraquat induced nuclear condensation. Taken together, these findings support the hypothesis that paraquat produces oxidative stress and proteasomal dysfunctionmediated toxicity in SY5Y cells. Thus, current findings suggest that paraquat may induce the pathogenesis of dopaminergic neurons through oxidative stress and proteasomal dysfunction.

Viral hepatitis infection represents a significant epidemiological and economic burden on society. Following infection, some patients mount a blunted immune response to the virus, which ultimately can result in chronic infection. FGL2, a member of the fibrinogen-related protein superfamily, has been implicated in vitro in suppressing both innate and adaptive immune responses. In a murine model of acute viral hepatitis caused by Lymphocytic Choriomeningitis Virus strain WE, we demonstrate that FGL2 expressed by reticuloendothelial cells limits viral spread. When expressed by Treg cells FGL2 binds to FCγRIIB and prevents DC maturation and suppresses virus-specific T and B cell responses. We provide compelling evidence to suggest that hepatitis viruses utilize the FGL2-FCγRIIB pathway to evade immune detection. Inhibition of this pathway restores effective cellular and humoral antiviral immune responses towards hepatitis viruses.

Viral hepatitis infection represents a significant epidemiological and economic burden on society. Following infection, some patients mount a blunted immune response to the virus, which ultimately can result in chronic infection. FGL2, a member of the fibrinogen-related protein superfamily, has been implicated in vitro in suppressing both innate and adaptive immune responses. In a murine model of acute viral hepatitis caused by Lymphocytic Choriomeningitis Virus strain WE, we demonstrate that FGL2 expressed by reticuloendothelial cells limits viral spread. When expressed by Treg cells FGL2 binds to FCγRIIB and prevents DC maturation and suppresses virus-specific T and B cell responses. We provide compelling evidence to suggest that hepatitis viruses utilize the FGL2-FCγRIIB pathway to evade immune detection. Inhibition of this pathway restores effective cellular and humoral antiviral immune responses towards hepatitis viruses.

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus (SLE), and is characterized by the production of anti-dsDNA antibodies. It is an important cause of renal failure. The mechanism through which anti-dsDNA antibodies bind to tissues and mediate kidney injury remains to be fully elucidated. Emerging evidence suggests that anti-dsDNA antibodies can bind to cells and extracellular antigens directly through cross-reactivity, independent of bridging chromatin material. Mesangial cells play an important role in normal kidney structure and functions, and its pathophysiology. Mesangial abnormalities in lupus nephritis precede more severe injuries such as lesions in the glomerular capillary loop. We previously demonstrated that the binding of human anti-dsDNA antibodies to mesangial cells (HMC) correlated with disease activity and induced inflammatory as well as fibrotic pathways. The aim of this project is to identify the cross-reactive antigen(s) on the mesangial cell surface that mediates anti-dsDNA antibody binding and the alterations in cell functions that result from this interaction. HMC plasma membrane proteins were purified. Using proteomic and biochemical approaches, we identified annexin II as the predominant cross-reactive antigen on the HMC surface that mediated human polyclonal anti-dsDNA antibody binding. Following this interaction, anti-dsDNA antibodies were internalized in a time- and temperature-dependent manner, and translocated to both the cytoplasm and nucleus within 30 min. This resulted in induction of annexin II synthesis, IL-6 secretion and cell proliferation, which was mediated through the activation of p38 MAPK, JNK and AKT. The binding activity to annexin II in the serum immunoglobulin fraction correlated with the titre of anti-dsDNA antibody. Binding activity of anti-dsDNA antibodies to annexin II correlated with clinical disease activity and circulating anti-dsDNA antibody levels. These correlations were more prominent in male patients with lupus nephritis. Glomerular annexin II expression was increased in patients with active lupus nephritis and co-localized with IgG and C3 deposition. Gene silencing of annexin II in HMC reduced anti-dsDNA antibody binding, which was accompanied by reduced IL-6 secretion and cell proliferation. Using female NZB/W F1 mice, an established murine model of lupus nephritis, we demonstrated that intra-glomerular annexin II expression increased with disease progression and was accompanied by an increase in the expression of p11, its cellular protein ligand. Our data suggest that annexin II may exist in the kidney as a heterotetramer and is involved in disease pathogenesis. At the ultrastructural level, annexin II was detected in the mesangial matrix, amongst electron dense deposits in the glomerular basement membrane, on the foot processes in podocytes and within the Bowman’s capsule. In conclusion, our data demonstrated that annexin II is the major cell surface antigen on HMC that mediates the cross-reactive binding of human anti-DNA antibodies. Through this interaction, cellular processes are triggered that contribute to the pathogenesis of lupus nephritis. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Background: Breast cancer is the commonest female cancer. DNA double-strand breaks (DSBs) associated proteins such as BRCA1 have been shown to be involved in tumourigenesis of breast tissue. One of the key regulators of DSBs, the RING Finger Protein 168 (RNF168), controls DNA damage responses (including the manipulation of homologous recombinant and non-homologous end-joining repair) which are responsible for correction of errors that occur during DSBs in order to maintain genomic stability. The nature of this protein suggests that RNF168 may play an important role in development of breast cancer. Material and methods: This study investigated the relationship of RNF168 expression in breast cancer by immunohistochemistry staining of 118 breast cancer samples in tissue microarray. The nuclear stain and cytoplasmic stain of the sections were assessed. Nuclear localization score was obtained and correlated with clinico-pathological features of the patients. Results: Immunohistological staining of RNF168 was successful in 99 cases of the tested breast cancer specimens. The expression of RNF168 was found to be significantly correlated with the occurrence of breast cancer metastasis (p=0.032). Strong expression of the protein was also found to be significantly associated with poorer breast cancer prognosis (p=0.033). In addition, correlation analysis also showed marginal correlation between nuclear localization of RNF168 with the age of patients at their first disease diagnosis (p=0.061). Conclusion: RNF168 might play a critical role in promoting breast cancer metastasis during the advanced stage of breast cancer, which results in poor disease prognosis. Detailed mechanism involved in metastasis promotion remained to be revealed in further study. / published_or_final_version / Pathology / Master / Master of Medical Sciences

abstract / Pathology / Doctoral / Doctor of Philosophy