published_or_final_version / Community Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

SARS疫情風暴，於台灣民國九二年時在極短的時間內奪去多條人命，台灣抗煞歷經數個月的搏鬥，除了當時被世界衛生組織（WHO）點名成為全球ＳＡＲＳ疫情擴散速度最快的地區外，也在九二年五六月份付出沈重的代價。台灣快速陷入疫情的主因，來自於急診室的傳染病管制漏洞，以及公衛體系的年久失修的監測系統。台灣各級政府機構紛紛啟動危機管理系統，以應變SARS疫情的遽增惡化。自這是因為SARS雖是危機，但有別於一般危機管理所定義的危機。一般危機管理所指的危機，是指事情發生後，在很短的時間內，如果不能有效的處理，就會形成更大的問題。而當問題愈來愈嚴重時，甚至於會危及政權的穩定與國家安全。 如今事過境遷，本文將檢視二００三年自始的SARS侵襲過程，以臺北市政府處理方式為個案，並對六位抗煞政策參與者進行質化訪談，輔以都市危機管理的模式，評估其應變政策的適當性與及時性。此外，亦藉由SARS疫情的案例，檢討我國醫療體系的缺失以及一般大眾對公衛資源利用的錯誤印象，以提供往後應變相關案例之參考。

La transfusion sanguine est une thérapeutique indispensable pour laquelle il n’existe pas actuellement de substitut. La transfusion de produits sanguins labiles est dans la grande majorité des cas très bien tolérée mais elle peut être à l’origine d’effets indésirables chez les receveurs (EIR) notamment de type inflammatoire. Ceci dépend de facteurs liés aux produits eux-mêmes et/ou aux receveurs de par leur prédisposition génétique et de leur état clinique. Les concentrés plaquettaires (CP) sont la principale source de manifestations inflammatoires et/ou allergiques. Ceci est notamment dû, en partie, à la capacité des plaquettes à sécréter une multitude de molécules ayant une activité inflammatoire. De plus, les processus de collecte, de préparation et de conservation induisent un stress vis-à-vis des cellules, qui peut activer les plaquettes et donc induire la production de produits inflammatoires dans les CP. Le but de ce travail de thèse a été dans un premier temps d’identifier les molécules les plus impliquées dans les manifestations inflammatoires. Le sCD40L en particulier est identifié comme étant largement impliqué dans les EIR après transfusion de CP, mais pas systématiquement. Aussi, la composante inflammatoire de ces réactions est multifactorielle. De plus, nous avons évalué le potentiel inflammatoire des CP sur l’endothélium vasculaire. Des différences d’activation des cellules endothéliales, dans un modèle in vitro, ont été observées lorsqu’elles sont en présence de surnageants de CP ayant induits un EIR. Ce travail de thèse poursuit l’effort entrepris par notre équipe de recherche, en vue de prédire la survenue d’EIR et de préciser les mécanismes qui influencent la physiopathologie plaquettaire transfusionnelle ; un corollaire de ces travaux est ainsi d’optimiser les processus de production et de conditionnement des CP transfusés afin de réduire ces réactions inflammatoires. / Blood transfusion is an indispensable therapy for which there is currently no substitute. Transfusion of blood products is in the great majority of cases very well tolerated but it can be at the origin of serious adverse reactions (SARs), notably of inflammatory reactions. This depends on the factors related to the products themselves and/or to the recipients, their genetic predisposition and clinical condition. Platelet concentrates (PCs) are the main source of inflammatory and/or allergic manifestations. This is due, in part, to the ability of platelets to secrete a multitude of molecules with inflammatory activity. In addition, the collection, processing and storage conditions induce stress on cells, which can activate platelets and thus induce the production of inflammatory products in PCs. The purpose of this work is to identify the molecules involved in inflammatory manifestations. sCD40L was identified as being involved in SARs after PCs transfusion, but not systematically. Also, the inflammatory component of these reactions is multifactorial. In addition, we evaluated the inflammatory potential of PCs on the vascular endothelium. Differences in endothelial cell activation, in an in vitro model, were observed when they were in the presence of PC supernatants involved in SARs. This thesis work continues the effort undertaken by our research team to predict the occurrence of SARs and to clarify the mechanisms that influence transfusional platelet physiopathology; a corollary of this work is to optimize the production and conditioning process of PCs transfused in order to reduce these inflammatory reactions.

by Chan Wai Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 170-185). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of Content --- p.vii / Abbreviations --- p.xii / for Nucleotides --- p.xii / for Amino acids --- p.xii / for Standard genetic codes --- p.xiii / for Units --- p.xiii / for Prefixes of units --- p.xiv / for Terms commonly used in the report --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of the Severe Acute Respiratory Syndrome --- p.1 / Chapter 1.2 --- The SARS Coronavirus --- p.3 / Chapter 1.3 --- Cell Biology of Coronavirus Infection and Replication and the Role of Nucleocapsid Protein --- p.9 / Chapter 1.4 --- Recent Advances in the SARS-CoV Nucleocapsid Protein --- p.16 / Chapter 1.5 --- The Sumoylation System --- p.24 / Chapter 1.6 --- Objectives of the Present Study --- p.28 / Chapter Chapter II --- SARS-CoV N protein and Fragment Purification --- p.29 / Chapter 2.1 --- INTRODUCTION --- p.29 / Chapter 2.2 --- METHODOLOGY --- p.31 / Materials --- p.31 / Methods --- p.39 / Chapter 2.2.1 --- Construction of the pMAL-c2P vector --- p.39 / Chapter 2.2.2 --- Sub-cloning of the N protein into expression vectors --- p.42 / Chapter 2.2.2.1 --- Design of primers for the cloning of N protein --- p.43 / Chapter 2.2.2.2 --- DNA amplification using Polymerase Chain Reaction (PCR) --- p.44 / Chapter 2.2.2.3 --- DNA extraction from agarose gel --- p.45 / Chapter 2.2.2.4 --- Restriction digestion of purified PCR product and vectors --- p.46 / Chapter 2.2.2.5 --- Ligation of N protein into expression vectors --- p.47 / Chapter 2.2.2.6 --- Preparation of competent cells --- p.48 / Chapter 2.2.2.7 --- Transformation of plasmids into competent Escherichia coli --- p.49 / Chapter 2.2.2.8 --- Preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.1 --- Mini-preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.2 --- Midi-preparation of plasmid DNA --- p.51 / Chapter 2.2.3 --- Expression of tagged and untagged N protein --- p.53 / Chapter 2.2.3.1 --- Preparation of E. coli competent cells for protein expression --- p.53 / Chapter 2.2.3.2 --- Expression of N protein --- p.53 / Chapter 2.2.3.3 --- Solubility tests on the fusion proteins expressed --- p.54 / Chapter 2.2.4 --- Purification of N protein Chromatographic methods --- p.55 / Chapter 2.2.4.1 --- Affinity chromatography --- p.55 / Chapter 2.2.4.1.1 --- Ni-NTA affinity chromatography --- p.55 / Chapter 2.2.4.1.2 --- Glutathione affinity chromatography --- p.56 / Chapter 2.2.4.1.3 --- Amylose affinity chromatography --- p.56 / Chapter 2.2.4.2 --- Ion exchange chromatography --- p.57 / Chapter 2.2.4.2.1 --- Cation exchange chromatography --- p.57 / Chapter 2.2.4.2.2 --- Anion exchange chromatography --- p.58 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.58 / Chapter 2.2.4.4 --- Size exclusion chromatography Purification strategies --- p.60 / Chapter 2.2.4.5 --- Purification of His6-tagged N proteins --- p.60 / Chapter 2.2.4.6 --- Purification of MBP-tagged N proteins --- p.60 / Chapter 2.2.4.7 --- Purification of GST-tagged N proteins --- p.61 / Chapter 2.2.4.8 --- Purification of untagged N proteins --- p.61 / Chapter 2.2.5 --- Trypsin digestion assay for the design of stable fragment --- p.64 / Chapter 2.2.6 --- Partial purification of the N protein amino acid residue 214-422 fragment --- p.65 / Chapter 2.2.7 --- Sumoylation of the SARS-CoV N protein --- p.67 / Chapter 2.2.7.1 --- In vitro sumoylation assay --- p.67 / Chapter 2.2.7.2 --- Sample preparation for mass spectrometric analysis --- p.68 / Chapter 2.3 --- RESULTS --- p.70 / Chapter 2.3.1 --- Construction of the vector pMAL-c2P --- p.70 / Chapter 2.3.2 --- "Construction of recombinant N protein-pAC28m, N-protein- pGEX-6P-l,N protein-pMAL-c2E and N protein-pMAL-c2P plasmids" --- p.72 / Chapter 2.3.3 --- Optimization of expression conditions --- p.79 / Chapter 2.3.4 --- Screening of purification strategies --- p.82 / Chapter 2.3.4.1 --- Purification of His6-N protein --- p.82 / Chapter 2.3.4.2 --- Purification of MBP-N protein --- p.84 / Chapter 2.3.4.3 --- Purification of GST-N protein --- p.85 / Chapter 2.3.4.4 --- Purification of untagged N protein --- p.87 / Chapter 2.3.5 --- Limited trypsinolysis for the determination of discrete structural unit --- p.91 / Chapter 2.3.6 --- Partial purification of the N protein 214-422 fragment --- p.94 / Chapter 2.3.7 --- Sumoylation of N protein --- p.97 / Chapter 2.2.7.1 --- Sumoylation site prediction --- p.97 / Chapter 2.2.7.2 --- In vitro sumoylation assay --- p.99 / Chapter 2.2.7.3 --- Mass spectrometric identification of sumoylated SARS-CoV N protein --- p.103 / Chapter 2.4 --- DISCUSSION --- p.109 / Chapter Chapter III --- Characterization of the Nucleic Acid Binding Ability of N protein --- p.119 / Chapter 3.1 --- INTRODUCTION --- p.119 / Chapter 3.2 --- METHODOLOGY --- p.120 / Materials --- p.120 / Methods --- p.124 / Chapter 3.2.1 --- Spectrophotometric Measurement of ratio OD260/ OD280 --- p.124 / Chapter 3.2.2 --- Native gel electrophoresis --- p.124 / Chapter 3.2.3 --- Quantitative determination of nucleic acids content --- p.125 / Chapter 3.2.3.1 --- Dische assay - quantitative determination of DNA content --- p.125 / Chapter 3.2.3.2 --- Orcinol assay - quantitative determination of RNA content --- p.126 / Chapter 3.2.4 --- RNase digestion of the N protein-bound RNA --- p.128 / Chapter 3.2.5 --- Isolation of RNA from purified GST-N proteins --- p.128 / Chapter 3.2.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.129 / Chapter 3.2.7 --- Vero E6 cell line maintenance and total RNA extraction --- p.131 / Chapter 3.2.8 --- Electrophoretic mobility shift assay (EMSA) --- p.131 / Chapter 3.3 --- RESULTS --- p.133 / Chapter 3.3.1 --- Detection of nucleic acids in the purified N proteins byspectrophotometric Measurement of ratio OD260/ OD280 --- p.133 / Chapter 3.3.2 --- Native gel electrophoresis --- p.135 / Chapter 3.3.3 --- Quantitative determination of nucleic acids content in purified GST-N proteins --- p.136 / Chapter 3.3.3.1 --- Dische assay for the determination of DNA --- p.136 / Chapter 3.3.3.2 --- Orcinol assay for the determination of RNA --- p.138 / Chapter 3.3.4 --- RNase digestion treatment --- p.139 / Chapter 3.3.5 --- Extraction of RNA from GST-N proteins --- p.140 / Chapter 3.3.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.142 / Chapter 3.3.7 --- Electrophoretic mobility shift assay (EMSA) --- p.144 / Chapter 3.4 --- DISCUSSION --- p.147 / Chapter Chapter IV --- Discussion --- p.154 / Chapter 4.1 --- "Purity, Aggregation and RNA Binding Property of the SARS-CoV Nucleocapsid Protein" --- p.154 / Chapter 4.2 --- Future perspectives --- p.156 / Chapter 4.2.1 --- Structural study of the SARS-CoV N protein through x-ray crystallography --- p.156 / Chapter 4.2.2 --- Mapping the RNA binding domain in the SARS-CoV N protein --- p.156 / Chapter 4.2.3 --- Determination of aggregation state by lateral turbidimetry analysis --- p.156 / Chapter 4.2.4 --- Exploring protein interacting partners that enhance RNA binding specificity --- p.157 / Appendix --- p.159 / Chapter I. --- Sequence of the SARS-CoV N protein --- p.159 / Chapter II. --- Sequence of the SARS-CoV genome fragment used for RNA binding assay in section 3.37.1 --- p.161 / Chapter III. --- Vector maps --- p.161 / Chapter a) --- Vector map of pACYC177 --- p.161 / Chapter b) --- Vector map and MCS of pET28a --- p.163 / Chapter c) --- Vector map and MCS of pAC28 --- p.164 / Chapter d) --- Vector map and MCS of pGEX-6P-1 / Chapter e) --- Vector map of pMAL-c2X and MCS of pMAL-c2E / Chapter IV. --- Electrophoresis markers --- p.166 / Chapter V. --- SDS-PAGE gel parathion protocol --- p.169 / References --- p.170

Background. Medical imaging has played an important role in the diagnosis, progress monitoring and follow-up of most disease entities, in particular chest infections. The emergence of a novel chest disease poses an immediate challenge to the pillars of imaging, namely chest radiography and computed tomography. The characteristic imaging appearances, differential diagnoses and diagnostic pitfalls need to be established for correct diagnosis and appropriate management. The sensitivity and utility of the different imaging modalities will also need to be addressed. / In the event of an outbreak or epidemic, these challenges are made more difficult by an overwhelming number of patients and limited resources. In March 2003, we were faced with such a situation in our institution and the disease was later termed Severe Acute Respiratory Syndrome (SARS). / Patients in Hong Kong were treated with a combination of an antiviral agent and corticosteroids in addition to respiratory support. The majority of patients improved with treatment, although between 20--36% required treatment in an intensive care unit. / Problems and importance. This novel disease of high infectivity, morbidity and mortality posed a major threat to public health and a challenge to health authorities both locally and internationally. With regard to medical imaging, the following research questions were identified: (1) What are the imaging signs of this new disease? (2) Does chest imaging provide a high degree of sensitivity for diagnosing the infection? (3) Are the imaging signs disease-specific or are they similar to other pathology? (4) Does the progressive evolution of the imaging appearance correlate with the clinical status of the patient? (5) Could the imaging appearance be useful for predicting the final outcome? (6) Are there complications that require detection by imaging? / The lung parenchyma is the main site of infection and the resultant microscopic pathology included: pulmonary exudate, sequestration of macrophages, diffuse alveolar damage, proliferation of epithelial cells and hyaline membrane formation. Macroscopic features include alveolar consolidation in the early stages and later, organizing pneumonia or bronchiolitis obliterans organizing pneumonia. / These answers to these questions are essential to our understanding of the disease and to increase our diagnostic ability. (Abstract shortened by UMI.) / This newly emerged disease is a respiratory infection with a high morbidity/mortality and was found to be caused by a coronavirus (SARS CoV). By the end of the outbreak a total of 8098 probable cases of infection were reported worldwide, with a mortality rate of 9.6% (774 deaths). Hong Kong was one of the hardest hit regions, totaling 1755 probable cases of infection and 299 deaths by the end of the outbreak. / Antonio Gregory Ernest. / "September 2005." / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3745. / Thesis (M.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 245-258). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

abstract: Understanding the consequences of changes in social networks is an important an- thropological research goal. This dissertation looks at the role of data-driven social networks on infectious disease transmission and evolution. The dissertation has two projects. The first project is an examination of the effects of the superspreading phenomenon, wherein a relatively few individuals are responsible for a dispropor- tionate number of secondary cases, on the patterns of an infectious disease. The second project examines the timing of the initial introduction of tuberculosis (TB) to the human population. The results suggest that TB has a long evolutionary history with hunter-gatherers. Both of these projects demonstrate the consequences of social networks for infectious disease transmission and evolution. The introductory chapter provides a review of social network-based studies in an- thropology and epidemiology. Particular emphasis is paid to the concept and models of superspreading and why to consider it, as this is central to the discussion in chapter 2. The introductory chapter also reviews relevant epidemic mathematical modeling studies. In chapter 2, social networks are connected with superspreading events, followed by an investigation of how social networks can provide greater understanding of in- fectious disease transmission through mathematical models. Using the example of SARS, the research shows how heterogeneity in transmission rate impacts super- spreading which, in turn, can change epidemiological inference on model parameters for an epidemic. Chapter 3 uses a different mathematical model to investigate the evolution of TB in hunter-gatherers. The underlying question is the timing of the introduction of TB to the human population. Chapter 3 finds that TB’s long latent period is consistent with the evolutionary pressure which would be exerted by transmission on a hunter- igatherer social network. Evidence of a long coevolution with humans indicates an early introduction of TB to the human population. Both of the projects in this dissertation are demonstrations of the impact of var- ious characteristics and types of social networks on infectious disease transmission dynamics. The projects together force epidemiologists to think about networks and their context in nontraditional ways. / Dissertation/Thesis / Doctoral Dissertation Anthropology 2019

Prostaglandins (PG) are ubiquitous lipid mediators that play key roles in pathophysiological responses to infections. They are considered to have both pro and anti-inflammatory roles depending upon the time of inflammation, the receptors that they bind to and the tissues that they act upon. Hence given their pleiotropic effects, a perfect balance between the pro and anti-inflammatory functions of PGs are required to ensure that a controlled timely immune response is elicited to mediate protection and to avoid immunopathology. PGD2 is one such PG that was reported to increase with age in the lungs of mice and to mediate an anti-inflammatory effect thereby blunting the immune response following Severe Acute Respiratory Syndrome – Coronavirus (SARS-CoV). Increase in PGD2 with age incapacitates respiratory dendritic cells (rDC) to migrate from lungs to the draining lymph node following SARS-CoV infection due to down regulation of CCR7 (a receptor for chemokines CCL19/21). Migration of rDCs to draining lymph nodes requires high expression of CCR7 and it's binding to CCL19/21, a chemokine that mediates migration of dendritic cells along its gradient. Although increase in levels of PGD2 might prove beneficial in high inflammatory conditions, it should be noted that high levels of such a potent anti-inflammatory mediator during the initiation of an immune response could prove detrimental. In chapter II of this thesis I show that age-related increases in oxidative stress result in the upregulation of a single phospholipase (PLA2) group II D (G2D) (PLA2G2D) with anti-inflammatory roles. PLA2G2D functions by releasing Arachidonic acid (AA) from the lipid membrane, which will be further metabolized to other pro-resolving/ anti-inflammatory lipid mediators including PGD2. I show that inducing oxidative stress in young mice as well as in human peripheral blood macrophages, results in the upregulation of PLA2G2D (probably as a counter mechanism against oxidative stress). Also increase in the expression levels of this gene during the course of SARS-CoV infection results in the upregulation of PGD2, which is completely abrogated in Pla2g2d-/- mice. I also show Pla2g2d/- middle-aged mice have low levels of PGD2 and that they are capable of mounting a strong immune response and survive the otherwise lethal SARS-CoV infection. PGD2 is also a major PG in the brain and its role has been investigated in many non-infectious setting such as stroke and Alzheimer' disease. The PGD2 binding to one of its receptors DP1 has been shown to have primarily a neuro-protective role. In chapter III, I show that PGD2/DP1 signaling has beneficial effects in the brain of mice infected with a neurotropic strain of murine hepatitis virus (MHV) (rj2.2). In agreement with the neuro-protective role of PGD2, at least 60% of DP1-/- mice succumb to a sublethal dose of rj2.2. rj2.2 infection in these mice is characterized by a delay in the induction of IFN I response and lower activation status of microglia and macrophages in the brain. I also show that abrogation of DP1 signaling results in global defects in the immune system response to infection. Notably, a genome wide expression analysis using microarray, shows that a gene, Pydc3 with putative inflammasome inhibiting function is upregulated in WT mice compared to DP1-/- mice in the CD11b population of cells which primarily comprises microglia and macrophages. In line with the predicted function of Pydc3, DP1-/- mice have higher frequency and number of IL-1β+ producing microglia in the brain. Studies are underway to determine the exact role of DP1 signaling in Pydc3 expression as well as the role of this gene in inflammasome function. Overall these studies emphasize the immuno-modulatory roles of PGs in the context of a viral infection. Thus, altering the levels of these lipid mediators at appropriate times during the course of infection might prove useful as an effective therapeutic strategy to decide the fate of an infection.

妊娠婦女接受產前檢查是促進新生兒健康的重要投入，以目前的醫療水準來看，許多新生兒疾病是可以早期發現早期治療的，對於醫療支出龐大的臺灣而言，如何達成醫療資源配置效率，同時提升國民健康水準，實為刻不容緩的任務。若能有效預防早產發生，不僅能提升國人健康素質，更能節省社會醫療資源，此為本文探討產前檢查是否能降低懷孕風險之目的。2003年SARS疫情爆發改變國人醫療利用行為引發本文探討產前檢查與生產健康關聯性的動機，本研究以SARS疫情爆發為外在衝擊，探討這段期間臺灣懷孕婦女產前檢查情形的變化，並進一步分析影響懷孕結果的因素，期望研究結果能提供政府規劃婦幼預防保健政策參考之用，同時降低婦女的懷孕風險，有效提升新生兒健康素質。本研究發現產檢次數、分娩年齡、胎次及懷孕婦女就業狀態均為影響生產健康的關鍵因素，適齡生產的婦女能避免發生早產或流產，前胎生產經驗有助於降低不良懷孕結果發生率，而自行投保的職業婦女明顯較依附投保婦女有較良好的懷孕健康。

published_or_final_version / Geography / Doctoral / Doctor of Philosophy