Chan, Wing Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 129-152). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / 摘要 --- p.5 / Table of Contents --- p.7 / List of Abbreviations and symbols --- p.13 / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemics and pandemics of influenza virus --- p.17 / Chapter 1.2 --- Biology of influenza virus --- p.19 / Chapter 1.2.1 --- Types of influenza virus --- p.19 / Chapter 1.2.2 --- Viral structure and viral proteins --- p.20 / Chapter 1.2.3 --- Life cycle of influenza virus --- p.21 / Chapter 1.2.4 --- An ever-changing virus --- p.22 / Chapter 1.3 --- Pathogenesis and immunology of influenza virus --- p.24 / Chapter 1.3.1 --- Diseases and symptoms caused by influenza virus infection --- p.24 / Chapter 1.3.2 --- Production of cytokines during influenza virus infection --- p.25 / Chapter 1.3.3 --- Immune responses in the hosts --- p.27 / Chapter 1.4 --- Non-structural protein 1 (NS1) --- p.28 / Chapter 1.4.1 --- Overview of NS1 --- p.28 / Chapter 1.4.2 --- Roles of NS1 in influenza virus infection --- p.29 / Chapter 1.5 --- Aims of study --- p.33 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Chemical reagents --- p.34 / Chapter 2.1.2 --- Buffers --- p.37 / Chapter 2.1.2.1 --- Preparation of buffers --- p.37 / Chapter 2.1.2.2 --- Commonly used buffers --- p.37 / Chapter 2.1.3 --- Strains and plasmids --- p.40 / Chapter 2.1.4 --- Primer list --- p.40 / Chapter 2.2 --- Methods --- p.42 / Chapter 2.2.1 --- Preparation of competent cells --- p.42 / Chapter 2.2.2 --- Molecular cloning --- p.43 / Chapter 2.2.2.1 --- Amplification of the target genes by PCR --- p.43 / Chapter 2.2.2.2 --- Agarose gel electrophoresis --- p.43 / Chapter 2.2.2.3 --- Extraction and purification of DNA from agarose gels --- p.44 / Chapter 2.2.2.4 --- Restriction digestion of DNA --- p.45 / Chapter 2.2.2.5 --- Ligation of digested insert and expression vector --- p.45 / Chapter 2.2.2.6 --- Transformation and plating out transformants --- p.46 / Chapter 2.2.2.7 --- Verification of insert by PCR --- p.46 / Chapter 2.2.2.8 --- Mini-preparation of plasmid DNA --- p.47 / Chapter 2.2.2.9 --- Confirmation of insertion in the miniprep DNA by restriction digestion --- p.48 / Chapter 2.2.2.10 --- Sequencing of the plasmid DNA --- p.48 / Chapter 2.2.3 --- Cell culture --- p.53 / Chapter 2.2.3.1 --- Cultivation of human lung epithelial NCI-H292 cells --- p.53 / Chapter 2.2.3.2 --- Transfection of cell culture --- p.53 / Chapter 2.2.4 --- Western blot analysis --- p.54 / Chapter 2.2.4.1 --- Protein extraction --- p.54 / Chapter 2.2.4.2 --- Determination of protein concentration --- p.54 / Chapter 2.2.4.3 --- Protein Blotting --- p.55 / Chapter 2.2.4.4 --- Membrane blocking and antibody incubations --- p.56 / Chapter 2.2.4.5 --- Detection of proteins --- p.57 / Chapter 2.2.5 --- Total RNA extraction --- p.58 / Chapter 2.2.5.1 --- Preparation of cell culture for total RNA extraction --- p.58 / Chapter 2.2.5.2 --- Spectrophotometric analysis of total RNA --- p.58 / Chapter 2.2.5.3 --- Agarose gel electrophoresis of total RNA --- p.59 / Chapter 2.2.6 --- DNA Microarray --- p.60 / Chapter 2.2.6.1 --- Preparation of biotin-labeled antisense cRNA --- p.60 / Chapter 2.2.6.2 --- "Hybridization, washing and scanning of DNA microarray chips" --- p.60 / Chapter 2.2.6.3 --- Data processing and analysis --- p.61 / Chapter 2.2.7 --- Quantitative real-time PCR (QRT-PCR) --- p.62 / Chapter 2.2.7.1 --- Preparation of cDNA --- p.62 / Chapter 2.2.7.2 --- Analysis of mRNA gene expression by QRT-PCR --- p.62 / Chapter Chapter 3 --- Roles of NS1A and NS1B on cellular gene expression / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results --- p.67 / Chapter 3.2.1 --- NS1 protein expression in transfected NCI-H292 cells --- p.67 / Chapter 3.2.2 --- Purity and integrity of total RNA extracted --- p.67 / Chapter 3.2.3 --- Microarray data processing and analysis --- p.70 / Chapter 3.2.3.1 --- Genes perturbed by NS1A --- p.88 / Chapter 3.2.3.1.1 --- Effect of NS1A on antiviral gene expression --- p.91 / Chapter 3.2.3.1.2 --- Regulation of JAK-STAT pathway by NS1A --- p.92 / Chapter 3.2.3.2 --- Genes perturbed by NS1B --- p.93 / Chapter 3.2.3.2.1 --- Effects of NS1B on IFN-stimulated gene expression --- p.96 / Chapter 3.2.3.3 --- Genes perturbed by both NS1A and NS1B --- p.96 / Chapter 3.2.4 --- Verification of differentially expressed genes --- p.98 / Chapter 3.3 --- Discussion --- p.100 / Chapter 3.3.1 --- Human lung epithelial cell line as a model --- p.100 / Chapter 3.3.2 --- DNA microarray technology --- p.101 / Chapter 3.3.3 --- Different actions of NS1A and NS1B on host cell gene expression --- p.102 / Chapter 3.3.4 --- Novel roles of NSIA --- p.103 / Chapter 3.3.5 --- Novel role of NSIB --- p.107 / Chapter 3.3.6 --- Implications --- p.108 / Chapter Chapter 4 --- "Roles of NSIA, NS1B and NS1C on cytokine expression" / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.2 --- Results --- p.113 / Chapter 4.2.1 --- NS1 protein expression in transfected NCI-H292 cells --- p.113 / Chapter 4.2.2 --- Purity and integrity of total RNA extracted --- p.113 / Chapter 4.2.3 --- QRT-PCR --- p.116 / Chapter 4.3 --- Discussion --- p.119 / Chapter 4.3.1 --- Human lung epithelial cell line as a model for cytokine study --- p.119 / Chapter 4.3.2 --- Implications of different cytokine patterns induced by different NS1 proteins --- p.120 / Chapter Chapter 5 --- General Discussion and Future Perspectives --- p.125 / References --- p.129

Abstract not available

Mucositis refers to the widespread damage of mucosal surfaces throughout the length of the alimentary tract (AT) that can occur during cancer treatment. Its development is an important clinical problem that complicates and limits treatment options as well as adversely affecting the quality of life and treatment outcomes for patients. Recent studies directed at determining the pathobiology of mucositis have indicated increasing evidence for the role of transcription factors, such as nuclear factor-κB (NF-κB), and certain pro-inflammatory cytokines, for example tumour necrosis factor (TNF), interleukin-1β (IL-1β) and interleukin- 6 (IL-6), in its development. This thesis developed from an initial clinical investigation in which the expression of NF-κB and COX-2 in oral mucosa was investigated in patients undergoing chemotherapy. Increased levels of NF-κB were demonstrated in the buccal mucosa following chemotherapy. It is well established that mucositis occurs in different sites of the AT. The aims of this research, therefore, were to compare and contrast the changes that do occur at different sites of the AT following chemotherapy in an established animal model (Dark Agouti (DA) rat). Furthermore, the studies were conducted to determine whether changes in tissue and serum levels of NF-κB and pro-inflammatory cytokines occurred following chemotherapy and, with respect to tissue levels, identify whether there were differences in expression at different sites throughout the AT. The final aim was to examine whether the histological changes and changes in pro-inflammatory cytokines were affected by the type of chemotherapy drug used. The effects of three chemotherapy drugs with different mechanisms of action (irinotecan, methotrexate and 5-fluorouracil) were investigated, all of which can cause mucositis in the clinical setting. The thesis is divided into a Literature Review (Chapter 1) followed by 4 research papers: Chapter 2 – “Nuclear factor- κB (NF- κB) and cyclooxygenase-2 (COX-2) expression in the oral mucosa following cancer chemotherapy” Chapter 3 -“Characterisation of mucosal changes in the alimentary tract following administration of irinotecan: Implications for the pathobiology of mucositis” Chapter 4 – “Is the pathobiology of chemotherapy-induced alimentary tract mucositis influenced by the type of mucotoxic drug administered?”, Chapter 5 – “Serum levels of NF-κB and pro-inflammatory cytokines following administration of mucotoxic drugs”. Chapter 6 provides an overall summary and discussion of the results. Previous research has indicated that following administration of chemotherapeutic agents there may be subclinical changes occurring in the mucosa prior to obvious clinical manifestations. The results presented in this thesis also demonstrate this in both humans and animals following administration of chemotherapy. Immunohistochemical analysis of tissue taken from the oral cavity, jejunum and colon from the DA rats following chemotherapy demonstrated that changes in NF-κB and the pro-inflammatory cytokines, TNF, IL-1β and IL- 6, occurred at all sites over a 72 hour time period. This was evident before severe histological evidence of mucositis were observed such as epithelial atrophy in the oral mucosa, atrophy, blunting and fusion of the villi in the jejunum and crypt ablation in the jejunum and colon. Furthermore, each of the three drugs caused different patterns of NF-κB and pro-inflammatory cytokine expression in the tissues; in spite of this, however, histological features of damage were similar. With respect to serum levels of NF-κB and pro-inflammatory cytokines, differences were observed between the serum and tissue levels. Generally, serum changes followed initial histological changes in the tissues, or occurred simultaneously with histological changes. The mechanisms behind this are unclear; however it may be that elevated cytokines in the tissues “overflow” into the serum as tissue damage increases. Furthermore, the use of serum cytokine level measurement to predict mucosal damage is limited because of the differences in timing and short time intervals between changes in the serum and tissues. This thesis has provided additional important information on mucositis pathobiology and highlights its complexity. In particular, it has provided new evidence supporting the notion that mucositis is not restricted to the oral cavity and that other sites of the AT are also affected. Furthermore, these results confirm previous data indicating that subclinical changes occur in the mucosa prior to the development of obvious histological damage or clinical manifestations of mucositis. Contrary to previous reports, these studies have indicated that, although the clinical and histological changes may be similar, the alterations in NF-κB and pro-inflammatory cytokines in the tissues are affected by the type of drug used. This has important implications in the management and prevention of mucositis in the clinical setting particularly when multi-drug or chemotherapy-radiotherapy regimens are used. A common pathway that leads to mucosal damage is yet to be determined. The fact that serum levels appear to reflect the “global” nature of the effects of chemotherapy, highlights the fact that ongoing research needs to be directed, not necessarily at specific side effects, but rather how side effects of chemotherapy are interrelated so that better patient management can be achieved and ultimately provide optimum treatment and better survival for patients with cancer. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1321557 / Thesis (Ph.D.) -- University of Adelaide, School of Dentistry, 2008

Chronic Kidney Disease (CKD) is a progressive condition that in the majority of cases leads to End Stage Renal Failure (ESRD) and the need for dialysis, with the only cure being renal transplant. CKD affects both adults and children; however the underlying causes of the disease are different. CKD in adults is most commonly secondary to diabetes and/or hypertension while CKD in children is usually caused by congenital structural abnormalities that result directly in renal dysfunction. There have been numerous reports of inflammatory and immunological disturbances in adult CKD that involve both the cellular and humoral immune systems. Consequences of these include an increased rate of cardiovascular disease (CVD), decreased response to vaccinations, as well as increased rates of infection, anaemia and malnutrition. Children with CKD display many of the clinical complications seen in adult kidney disease that are associated with inflammatory and immunological changes. In adults however, many of the primary conditions associated with CKD are inherently pro-inflammatory; therefore it is not clear whether the inflammatory changes observed in adults with CKD are due to pre-existing inflammatory conditions, renal disease per se or a combination of both. The majority of CKD in children is caused by conditions that are not inflammatory in nature. This presents a unique opportunity to study the inflammatory consequences of CKD alone, without the added complication of underlying inflammatory disorders. Despite this, there has been little investigation of the inflammatory and immunological status of children with CKD. Some very recent studies have shown that children with CKD have an increased systemic inflammatory state[1-3], however the nature of these immunological and inflammatory changes remains poorly defined. Identification of the specific inflammatory processes that occur in CKD may provide new treatment targets and the opportunity to develop urgently needed new therapies. The purpose of this thesis is to investigate the presence of immunological changes associated with inflammation in children with CKD. This is the first study to include children with very mild disease, and the significant changes that are present in the early stages of the disease are of particular note. I have shown that CKD in children is an intrinsically inflammatory condition, with increased accumulation of markers of oxidative stress and production of pro-inflammatory cytokines. The inflammatory markers identified in this study may be applied as a foundation for more sensitive diagnostic markers of disease progression as well as provide a basis for novel treatment strategies in this group of patients. Early identification of increased inflammation is a prerequisite for the application of preventive strategies. In addition, a better understanding of the level and mechanisms of systemic inflammation in children with CKD may enable a more accurate assessment of their risk of other inflammatory conditions such as CVD, anaemia, muscle wasting, and malnutrition. Future research that specifically focuses on the reasons and mechanisms for different rates of disease progression may emerge as a result of this study. Importantly, the findings of this study may have implications in the long term treatment of disease and may allow identification of new treatment strategies to achieve better patient outcomes. The outcomes of the study are: • Better definition of inflammatory profiles in paediatric CKD and correlation with disease severity and progression, which should contribute to improved management strategies. • Identification of new treatment targets to reduce the damage caused by chronic systemic inflammation. • Mechanistic understanding of the relationship of the inflammatory profile in regard to source leucocytes or other contributing cell types. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1330366 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

The production of viable germ cells during human embryonic development determines adult reproductive success. This is particularly true for females, as development of germ cells (GCs) into primordial follicles before birth is imperative for future fertility. During fetal development GCs migrate to the genital ridge to form the gonad, after which several tightly regulated events, including proliferation, differentiation, and association with somatic cells, must occur to form a functional gonad. In the ovary these processes also include the initiation and subsequent arrest of meiosis. These developmental processes are orchestrated by local autocrine and paracrine factors, many of which remain to be identified in the human. In order to decipher further the pathways by which the gonad and GCs develop, potential regulators including prostaglandin (PG) E2, the interleukin (IL)6-type cytokines, and the prokinetecins (PROKs), were examined in the human fetal ovary and PROKs in the human fetal testis. Patterns of gene expression, protein localisation, function, and interaction of the potential mediators throughout human development (8-20 weeks gestation) were determined. Primary fetal tissue was investigated, in addition to immortalized GCs (T-Cam2 cells) and a murine model of fetal ovarian development. PGE2 interacts with known regulators of GC development in non-reproductive organs. It was postulated PGE2 may regulate GC progression by modulating these factors. Examination of PGE2 receptors and precursor enzymes in the fetal ovary revealed that all were present and some were developmentally regulated, with mRNA expression increasing with gestation. These developmentally regulated components were localised to the GCs. The PGE2 receptors were among those differentially expressed, with one localised solely to mature GCs. Culture of human fetal ovary confirmed that PGE2 regulates known regulators of GC development, increasing expression of survival and anti-apoptotic factors. To test the hypothesis that PGE2 is necessary for female GC development, paracetamol, an inhibitor of PGE2 precursor enzymes, was utilised in a murine model of fetal exposure. Fetal ovaries from this experiment displayed disruption of normal development. The IL6-type cytokines are also postulated to be involved in early gonad development, and are known to regulate proliferation and differentiation of mouse embryonic stem and GCs in vitro. A significant increase in transcript levels of the shared receptor components was determined in second trimester human ovaries, as well as developmental increases of several of the IL6-type ligands. Both common receptor components were located specifically in the GCs identifying them as the target of IL6 action in the human fetal ovary. The PROKs regulate cell migration, proliferation and differentiation, and modulate secretion of PGE2 and expression of some IL6-type cytokines. To-date, PROKs have not been examined in the human fetal gonad. Transcript levels were higher in the fetal testis compared to the ovary, with receptor and ligand components increasing with gestation. Most components also increased with gestation in the ovary. However, location of PROK components was strikingly different between the two tissues, with GCs being the primary target of PROK action in the fetal ovary, and Leydig and interstitial cells being the target in the testis. PROKs interaction with other regulators of gonad development was examined utilising a GC line in the case of the ovary and primary interstitial cell cultures in the case of the testis. These studies have identified new factors involved in human fetal gonad development, and how they interact with known regulatory pathways of development.

Chronic Kidney Disease (CKD) is a progressive condition that in the majority of cases leads to End Stage Renal Failure (ESRD) and the need for dialysis, with the only cure being renal transplant. CKD affects both adults and children; however the underlying causes of the disease are different. CKD in adults is most commonly secondary to diabetes and/or hypertension while CKD in children is usually caused by congenital structural abnormalities that result directly in renal dysfunction. There have been numerous reports of inflammatory and immunological disturbances in adult CKD that involve both the cellular and humoral immune systems. Consequences of these include an increased rate of cardiovascular disease (CVD), decreased response to vaccinations, as well as increased rates of infection, anaemia and malnutrition. Children with CKD display many of the clinical complications seen in adult kidney disease that are associated with inflammatory and immunological changes. In adults however, many of the primary conditions associated with CKD are inherently pro-inflammatory; therefore it is not clear whether the inflammatory changes observed in adults with CKD are due to pre-existing inflammatory conditions, renal disease per se or a combination of both. The majority of CKD in children is caused by conditions that are not inflammatory in nature. This presents a unique opportunity to study the inflammatory consequences of CKD alone, without the added complication of underlying inflammatory disorders. Despite this, there has been little investigation of the inflammatory and immunological status of children with CKD. Some very recent studies have shown that children with CKD have an increased systemic inflammatory state[1-3], however the nature of these immunological and inflammatory changes remains poorly defined. Identification of the specific inflammatory processes that occur in CKD may provide new treatment targets and the opportunity to develop urgently needed new therapies. The purpose of this thesis is to investigate the presence of immunological changes associated with inflammation in children with CKD. This is the first study to include children with very mild disease, and the significant changes that are present in the early stages of the disease are of particular note. I have shown that CKD in children is an intrinsically inflammatory condition, with increased accumulation of markers of oxidative stress and production of pro-inflammatory cytokines. The inflammatory markers identified in this study may be applied as a foundation for more sensitive diagnostic markers of disease progression as well as provide a basis for novel treatment strategies in this group of patients. Early identification of increased inflammation is a prerequisite for the application of preventive strategies. In addition, a better understanding of the level and mechanisms of systemic inflammation in children with CKD may enable a more accurate assessment of their risk of other inflammatory conditions such as CVD, anaemia, muscle wasting, and malnutrition. Future research that specifically focuses on the reasons and mechanisms for different rates of disease progression may emerge as a result of this study. Importantly, the findings of this study may have implications in the long term treatment of disease and may allow identification of new treatment strategies to achieve better patient outcomes. The outcomes of the study are: • Better definition of inflammatory profiles in paediatric CKD and correlation with disease severity and progression, which should contribute to improved management strategies. • Identification of new treatment targets to reduce the damage caused by chronic systemic inflammation. • Mechanistic understanding of the relationship of the inflammatory profile in regard to source leucocytes or other contributing cell types. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1330366 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

[Truncated abstract] Bone remodeling is intricately regulated by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The elevation in osteoclast number and/or activity is a major hallmark of several common pathological bone disorders including post-menopausal osteoporosis, osteoarthritis, Paget's disease, and tumour-mediated osteolysis. Receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine for osteoclast differentiation and activation. The association of RANKL to its cognate receptor, RANK, which is expressed on osteoclast precursors and mature osteoclasts, is essential for osteoclast formation and activation. The intimate interaction between RANKL and RANK triggers the activation of a cascade of signal transduction pathways including NF-κB, NFAT, MAPK and PI3 kinase. Although osteoclast signaling pathways have been intensively studied, the precise molecules and signaling events which underlie osteoclast differentiation and function remain unclear. In order to dissect the molecular mechanism(s) regulating osteoclast differentiation and activity, this thesis herein explores the key RANKL/RANK-mediated signaling pathways. Four truncation mutants within the TNF-like domain of RANKL [(aa160-302), (aa160-268), (aa239-318) and (aa246-318)] were generated to investigate their potential binding to RANK and the activation to RANK-signal transduction pathways. All were found to differentially impair osteoclastogenesis and bone resorption as compared to the wild-type RANKL. The impaired function of the truncation mutants of RANKL on osteoclast formation and function correlates with their reduced ability to activate crucial RANK signaling including NF-κB, IκBα, ERK and JNK. Further analysis revealed that the truncation mutants of RANKL exhibited differentially affinity to RANK as observed by in vitro pull-down assays. ... It is possible that Bryostatin 1 acts via upregulation of a fusion mechanism as the RANKL-induced OCLs are morphologically enlarged, exhibiting increased nuclei number expressing high level of DC-Stamp. Furthermore, Rottlerin was shown to inhibit NF-κB activity, whereas Bryostatin 1 showed the opposing effects. Both inhibitor and activator were also found to modulate other key osteoclastic signaling pathways including NFAT and total c-SRC. These findings implicate, for the first time, Protein Kinase C delta signaling pathways in the modulation of RANKL-induced osteoclast differentiation and activity. Taken together, the studies presented in this thesis provide compelling molecular, biochemical and morphological evidence to suggest that: (1) RANKL mutants might potentially serve as peptide mimic to inhibit RANKL-induced signaling, osteoclastogenesis and bone resorption. (2) A cross talk mechanism between extracellular Ca2+ and RANKL exist to regulate on osteoclast survival. (3) TPA suppressed RANKL-induced osteoclastogenesis predominantly during the early stage of osteoclast differentiation via modulation of NF-κB. (4) Selective inhibition of Protein Kinase C signaling pathways involved in osteoclastogenesis might be a potential treatment method for osteoclast-related bone diseases. (5) Protein Kinase C delta signaling pathways play a key role in regulating osteoclast formation and function.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.