<div>The papain-like protease (PLP) domain of nonstructural protein 3 (nsp3) of the coronavirus (CoV) genome promotes viral replication by processing the CoV polyprotein (protease) and also antagonize innate immune responses by deubiquitinating (DUB) and deISGylating (deISG) host substrates. Selectively removing the DUB/deISG activities of PLP while keeping the protease activity intact is a potential strategy for designing a live attenuated virus. However, it is unclear in the literature the precise mechanism by which PLPs support CoV evasion of the innate immune system. Deciphering the substrate specificity of PLPs for host ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) can therefore help in the design of PLP mutants that selectively lack one activity for evaluating the DUB and deISG mechanism in CoV pathogenesis and replication. </div><div> In this dissertation, we investigate the structure and function of the single PLP (PLpro) from beta-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which are dangerous viral pathogens that emerged from a zoonotic source to cause infectious disease in the human population. Additionally, we translate the knowledge gained to the equivalent PLP2 from alpha-CoV porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV), which cause fatal disease in suckling piglets on industrial pork farms and household cats, respectively. The primary objective of this work is to rationally design PLP mutants across beta- and alpha-CoVs to help attenuate CoV infection, as no antiviral or vaccine exist for human CoVs and the efficacy of PEDV vaccines are an ongoing research topic. </div><div><br></div><div>In Chapter 1, different human, animal, and the bat origin CoV strains are introduced. The CoV life-cycle and virion structure are outlined, along with the replicase complex for viral replication. The multidomain nsp3 from alpha- and beta-CoV genomes are also described with a focus on the PLP domain and its proposed cleavage sites of the viral polyprotein. The discovery of the first viral protease DUB and the multiple activities of PLPs are defined, which includes a proposed model of how DUB versus deISG activities may act in the innate immune response. This leads into the therapeutic potential of PLP for an antiviral or live attenuated vaccine, which is followed by the introduction of live attenuated vaccines and the reverse genetics system. Next, proof of concept studies on PLP2 mutants are described and the introduction is concluded by stating the ultimate goal for the design of PLP mutants.</div><div><br></div><div>In Chapter 2, we hypothesize that the flanking ubiquitin-like (Ubl2) domain of MERS-CoV PLpro is not required for its enzymatic function. We characterize the specific activity, kinetics, substrate specificity, and inhibition of the PLpro enzyme with and without the Ubl2 domain and reveal that the Ubl2 domain does not significantly alter PLpro function. We determine the structure of the core PLpro, smallest catalytic unit to 1.9 Å resolution and observed no structural changes compared to the wild-type. Additionally, we demonstrate that a purported MERS-CoV PLpro inhibitor is nonselective in non-reducing conditions and should not be pursed for therapeutic use. We show that the core PLpro enzyme i.e. without the Ubl2 domain is a stable and robust construct for crystallization and is also thermally stable based on thermal melting studies with utility for structure-based drug design. </div><div><br></div><div>In Chapter 3, we shed light on the specificity of SARS-CoV PLpro towards Ub versus ISG15 by characterizing the specific activity and kinetic parameters of SARS-CoV PLpro mutants. In addition, the structure of SARS-CoV PLpro in complex with the C-terminal domain of ISG15 is determined and compared with the Ub-bound structure. Based on the structure and kinetic results, the altered specificities of SARS-CoV PLpro mutants Arg167Glu, Met209Ala, and Gln233Glu are compared with the wild-type. Arg167Glu mutant exhibits DUB hyperactivity and is expected to adopt a more favorable interaction with the Arg42 of Ub. At the same time, ARG167GLU contains a shorter side-chain that hinders interaction with the unique Trp123 of ISG15 for deISG activity compared to the wild-type. These results aid in the development of SARS-CoV PLpro mutants that have directed shifts in substrate specificity for Ub versus ISG15. </div><div><br></div><div>In Chapter 4, the process and antiviral activity of ISGylation is reviewed and how viruses can modulate host-derived versus virus-derived machineries to counteract ISGylation for viral infection. MERS-CoV PLpro is cross-reactive for Ub, but less is known about its specificity towards ISG15. In this study, we determine the structure of MERS-CoV PLpro bound with ISG15 to 2.3 Å resolution and reveal a small hydrophobic pocket of ISG15 that consists of P130 and W123, which differs from Ub hydrophobic patch. We design and determine the kinetic parameters for 13 PLpro mutants and reveal that MERS-CoV PLpro only has a single ubiquitin recognition (SUb1) site. Kinetic studies show that removing the charge of the R1649 greatly enhances DUB/protease activity while mutating in an Arg near R42 of Ub or ISG15 hydrophobic region is detrimental to both DUB/deISG activities. Kinetic experiments and probe-reactivity assays showed that Val1691Arg, Val1691Lys, and His1652Arg mutants are drastically reduced DUB/deISG activities compared to the wild-type. Overall, MERS-CoV PLpro mutants with alter kinetic profiles will be useful for discovery tools and DUB/deISG deficient mutants are great candidates for removing host cell antagonism activity by PLpro for live attenuated vaccines.</div><div><br></div><div>In Chapter 5, the goal is to translate the knowledge gained in Chapters 2-4 on beta-CoVs PLpro and evaluate the substrate specificity of alpha-CoVs FIPV and PEDV PLP2 for mutagenesis experiments. First, we design and purify the core PLP2 enzymes for kinetics. PLP2s are efficient DUBs that prefer Ub to ISG15 in vitro, and this preference is conserved in beta-CoV MHV PLP2 as well as alpha-CoV NL63 PLP2. We determine the structure of alpha-CoV PEDV PLP2 to 1.95 Å resolution and reveal the unique Zn-finger coordinating Cys3-His arrangement of the alpha-CoV genus that differs from past beta-CoV PLP crystal structures. To determine residues of the SUb1 site, we generate a homology model of FIPV PLP2 and overlay our PLP2 structures with MERS-CoV PLpro bound with Ub. In addition, we create electrostatic surface maps across coronaviral PLP subfamilies to evaluate the charge distribution of the SUb1 for the rational design of several FIPV and PEDV PLP2 mutants. We evaluate the turnover of PLP mutants for FRET-based substrates and reveal that His101ArgFIPV and Asn101ArgPEDV are drastically reduced in Ub-AMC activity while their peptide activities are within 2-fold of the wild-type. These mutants show delayed reactivity for Ub probes and no longer cleave Ub-chains displaying isopeptide bonds compared to the wild-type. Results from this study reveal a hot spot in both alpha- and beta-CoVs that can be used to selectively remove DUB activity of PLPs for generating a DUB deficient PLP enzyme. </div><div><br></div><div>In this dissertation, we investigate the substrate specificity of PLPs across alpha- and beta-CoVs and develop a fingerprint for Ub and also shed light on ISG15 recognition. Specifically, hot spots were identified in the SUb1 site of different PLPs, which recognize R42 and hydrophobic Ile44 of Ub. Position 97-98 of PLPs can be used to remove DUB activity by substituting an Arg, but usually effect protease function. Substituting an Arg at position 101 and 136 of coronaviral PLPs serve as the best strategy to remove DUB function while not hindering active site functionality. The DUB/deISG deficient mutants described will be useful for inhibiting the ability of PLPs to function in the innate immune response. Ultimately, this work provides a guide for identifying attenuating mutants in existing CoVs for live attenuated vaccines and also a blueprint for engineering PLPs from new emerging CoVs. </div>

Tao Dacheng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 70-75). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Content-based Image Retrieval --- p.1 / Chapter 1.2 --- SVM based RF in CBIR --- p.3 / Chapter 1.3 --- DA based RF in CBIR --- p.4 / Chapter 1.4 --- Existing CBIR Engines --- p.5 / Chapter 1.5 --- Practical Applications of CBIR --- p.10 / Chapter 1.6 --- Organization of this thesis --- p.11 / Chapter Chapter 2 --- Statistical Learning Theory and Support Vector Machine --- p.12 / Chapter 2.1 --- The Recognition Problem --- p.12 / Chapter 2.2 --- Regularization --- p.14 / Chapter 2.3 --- The VC Dimension --- p.14 / Chapter 2.4 --- Structure Risk Minimization --- p.15 / Chapter 2.5 --- Support Vector Machine --- p.15 / Chapter 2.6 --- Kernel Space --- p.17 / Chapter Chapter 3 --- Discriminant Analysis --- p.18 / Chapter 3.1 --- PCA --- p.18 / Chapter 3.2 --- KPCA --- p.18 / Chapter 3.3 --- LDA --- p.20 / Chapter 3.4 --- BDA --- p.20 / Chapter 3.5 --- KBDA --- p.21 / Chapter Chapter 4 --- Random Sampling Based SVM --- p.24 / Chapter 4.1 --- Asymmetric Bagging SVM --- p.25 / Chapter 4.2 --- Random Subspace Method SVM --- p.26 / Chapter 4.3 --- Asymmetric Bagging RSM SVM --- p.26 / Chapter 4.4 --- Aggregation Model --- p.30 / Chapter 4.5 --- Dissimilarity Measure --- p.31 / Chapter 4.6 --- Computational Complexity Analysis --- p.31 / Chapter 4.7 --- QueryGo Image Retrieval System --- p.32 / Chapter 4.8 --- Toy Experiments --- p.35 / Chapter 4.9 --- Statistical Experimental Results --- p.36 / Chapter Chapter 5 --- SSS Problems in KBDA RF --- p.42 / Chapter 5.1 --- DKBDA --- p.43 / Chapter 5.1.1 --- DLDA --- p.43 / Chapter 5.1.2 --- DKBDA --- p.43 / Chapter 5.2 --- NKBDA --- p.48 / Chapter 5.2.1 --- NLDA --- p.48 / Chapter 5.2.2 --- NKBDA --- p.48 / Chapter 5.3 --- FKBDA --- p.49 / Chapter 5.3.1 --- FLDA --- p.49 / Chapter 5.3.2 --- FKBDA --- p.49 / Chapter 5.4 --- Experimental Results --- p.50 / Chapter Chapter 6 --- NDA based RF for CBIR --- p.52 / Chapter 6.1 --- NDA --- p.52 / Chapter 6.2 --- SSS Problem in NDA --- p.53 / Chapter 6.2.1 --- Regularization method --- p.53 / Chapter 6.2.2 --- Null-space method --- p.54 / Chapter 6.2.3 --- Full-space method --- p.54 / Chapter 6.3 --- Experimental results --- p.55 / Chapter 6.3.1 --- K nearest neighbor evaluation for NDA --- p.55 / Chapter 6.3.2 --- SSS problem --- p.56 / Chapter 6.3.3 --- Evaluation experiments --- p.57 / Chapter Chapter 7 --- Medical Image Classification --- p.59 / Chapter 7.1 --- Introduction --- p.59 / Chapter 7.2 --- Region-based Co-occurrence Matrix Texture Feature --- p.60 / Chapter 7.3 --- Multi-level Feature Selection --- p.62 / Chapter 7.4 --- Experimental Results --- p.63 / Chapter 7.4.1 --- Data Set --- p.64 / Chapter 7.4.2 --- Classification Using Traditional Features --- p.65 / Chapter 7.4.3 --- Classification Using the New Features --- p.66 / Chapter Chapter 8 --- Conclusion --- p.68 / Bibliography --- p.70

I established the phage antibody library platform for the identification of specific antibodies. In the first part of my study, I tried to identify antibody against SARS-CoV. Two fragments on the spike protein, which is responsible for inducing viral entry, was chosen as target for the selection of antibody. An antibody was identified which can selectively recognize the SARS-CoV infected cells, but not non-infected cells. Although this antibody was found to retain no neutralizing ability, this specific antibody may have potential to develop for diagnostic purpose. / I utilized the phage system-based cloning method as an attractive approach to screen and identify virus-specific antibodies that can be encoded by the human genome. Once a useful phage clone is identified, unlimited amounts of human monoclonal virus-specific antibodies can be manufactured, and potentially applied clinically for prophylactic and therapeutic uses. The study focuses on two of these new infections, both of which cause severe respiratory disease: SARS and avian influenza. / Identification of specific antibodies, either for diagnostic or therapeutic use, was successfully demonstrated in the two infectious disease models. The phage antibody platform offers a fast and cost-effective method to identify phage antibodies, which can easily be converted to human viral specific monoclonal antibodies for clinical use. / In the 21st century, a number of novel infectious diseases emerged suddenly and spread rapidly, endangering the lives and well-being of people around the world. Severe acute respiratory syndrome (SARS) is a life threatening form of atypical pneumonia that ravaged Hong Kong, Taiwan, China, Canada and many cities in 2003. In the same year, novel avian influenza viruses infected human beings on two continents. Both of these diseases originated in animals and crossed over into the human population. These emerging diseases pose significant public health threats while providing a chilling reminder that another influenza pandemic could occur at any time. Thus, the development of effective therapeutics to control the disease is of paramount importance. Although several vaccines against SARS and avian influenza are available nowadays, the poor clinical performance and frequent mutation of viral strains may limit the practical use and value of the vaccines. Moreover, there are no promising antiviral drugs available for the treatment. Therefore, I aimed to develop an immunotherapy as an alternative treatment option against these diseases. / In the second part of my study, the extracellular domain of matrix protein of avian influenza virus was chosen as target for the selection of antibody. I successfully identified an antibody which can neutralize the avian influenza virus infection. This promising result indicated this antibody has potential to develop for therapeutic use and these antibodies can be easily manufactured in unlimited amounts for clinical application. / Leung, Ka Man. / Adviser: Kwok Pui Fung. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0212. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 112-123). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

The 2003 Severe Acute Respiratory Syndrome (SARS) outbreak in Canada highlights a broad range in ethical challenges, particularly in priority setting. Presently, a leading theory in ethical priority setting is Daniels’ and Sabin’s Accountability for Reasonableness (A4R), which enhances fair and legitimate procedural decision making in typical healthcare settings. A4R attempts to mitigate conflicting interests and facilitate fairness in deliberations over priority setting issues. Whether this framework may be applied to public health emergencies has yet to be examined. This qualitative study describes the outbreak through the lens of A4R and explores the applicability of A4R in atypical or emergent circumstances. Findings from 25 structured key informant interviews of public health officials suggest refinements to the framework may be required for emergency events. The presence of such a framework may minimize collateral damage during and after a response. The lessons may guide future preparedness efforts such as pandemic planning.

The 2003 Severe Acute Respiratory Syndrome (SARS) outbreak in Canada highlights a broad range in ethical challenges, particularly in priority setting. Presently, a leading theory in ethical priority setting is Daniels’ and Sabin’s Accountability for Reasonableness (A4R), which enhances fair and legitimate procedural decision making in typical healthcare settings. A4R attempts to mitigate conflicting interests and facilitate fairness in deliberations over priority setting issues. Whether this framework may be applied to public health emergencies has yet to be examined. This qualitative study describes the outbreak through the lens of A4R and explores the applicability of A4R in atypical or emergent circumstances. Findings from 25 structured key informant interviews of public health officials suggest refinements to the framework may be required for emergency events. The presence of such a framework may minimize collateral damage during and after a response. The lessons may guide future preparedness efforts such as pandemic planning.

published_or_final_version / Public Administration / Master / Master of Public Administration

The tax world as we knew it was turned upside down on 13 September 2007 when the Supreme Court of Appeal (“SCA”) announced its decision to deem the right to use an interest-free loan as an amount that accrued to the taxpayers in the case Commissioner for South African Revenue Service v Brummeria Renaissance (Pty) Ltd and others 69 SATC 205. The findings of SCA brought about a “great deal of consternation in the business world” (Loubser, 2007:20). Due to the controversy as a result of this case, SARS drafted an Interpretation Note that illustrates the reasoning and tax treatment of an interest-free loan. On 30 June 2010, Interpretation Note No 58 was finally issued by SARS, providing guidance with regard to “an amount” that “accrues” to a taxpayer for the purposes of the gross income definition. This Interpretation Note will have a significant impact on a number of taxpayers. The purpose of this study is to understand the nature of an interest-free loan and identify its tax implications. The methodology followed in this study will be that of qualitative research. This will be conducted through analyzing the nature of a loan, specifically an interest-free loan, the gross income definition, including the value and timing of such amount, and whether a deduction may be claimed in respect of an interest-free loan. Notwithstanding the above, the study also includes an investigation of other taxes inter alia capital gains tax, donations tax, value-added tax, secondary tax on companies and newly proposed dividends tax. / Thesis (M.Com. (Tax))--North-West University, Potchefstroom Campus, 2011.

The tax world as we knew it was turned upside down on 13 September 2007 when the Supreme Court of Appeal (“SCA”) announced its decision to deem the right to use an interest-free loan as an amount that accrued to the taxpayers in the case Commissioner for South African Revenue Service v Brummeria Renaissance (Pty) Ltd and others 69 SATC 205. The findings of SCA brought about a “great deal of consternation in the business world” (Loubser, 2007:20). Due to the controversy as a result of this case, SARS drafted an Interpretation Note that illustrates the reasoning and tax treatment of an interest-free loan. On 30 June 2010, Interpretation Note No 58 was finally issued by SARS, providing guidance with regard to “an amount” that “accrues” to a taxpayer for the purposes of the gross income definition. This Interpretation Note will have a significant impact on a number of taxpayers. The purpose of this study is to understand the nature of an interest-free loan and identify its tax implications. The methodology followed in this study will be that of qualitative research. This will be conducted through analyzing the nature of a loan, specifically an interest-free loan, the gross income definition, including the value and timing of such amount, and whether a deduction may be claimed in respect of an interest-free loan. Notwithstanding the above, the study also includes an investigation of other taxes inter alia capital gains tax, donations tax, value-added tax, secondary tax on companies and newly proposed dividends tax. / Thesis (M.Com. (Tax))--North-West University, Potchefstroom Campus, 2011.

The purpose of the study The purpose of this research project was to develop and test an educational program for preparing Taiwanese nursing students for clinical practice. Study background The SARS outbreak revealed that health care professionals were ill-prepared for coping with the disease epidemic in terms of the rapid transmission of the infection, the high mortality and morbidity rate among health care workers, and the significant impacts on the public and health care personnel. Frontline nurses were the group at highest risk of becoming infected, as they are the health care personally that provide direct health care to infected patients. However, to date the ability of Taiwanese frontline nurses to respond to such a disease epidemic has not been examined. Study design This research project incorporated a three phase design, presented in the form of two separate studies. A small qualitative exploratory study was undertaken to validate the assumptions emerging from international literature regarding the preparedness nurses in managing an infection outbreak. The information gained was used to construct an infection control education program (Study I). A quasi-experimental design, using pre- and post-tests and experimental and control groups was then used to test the effectiveness of the education intervention (Study II). Participants A purposive sampling technique was used in the qualitative exploratory study, whereby six Taiwanese nurses who had provided direct nursing care to patients with SARS were interviewed. A convenience sampling approach was utilised in the quantitative study, which aimed to test the effectiveness of educational intervention. This, second study, had 175 participants in total, 80 in the experimental group and 95 in the control group. All participants were enrolled in the first semester of their fourth year in a five-year nursing program in two selected junior nursing colleges. The education intervention The purpose-designed standard and additional precautions (SnAP) program was the intervention. The experimental group received a SnAP program which consisted of 16 hours of classes over 16 weeks. The control group received a conventional education program. Data collection and instrument Data were collected at three time points during the study (baseline, four months, six month) using validated instrument. The reliability and validity of the instrument was established in a pilot study with a Taiwanese population prior to the present study. Data analysis t-tests and chi-square analyses were performed to assess any differences across demographic variables and baseline outcome variables between the experimental and control groups. Two-way repeated measures ANOVAs were used to examine the scores of the intervention and control groups across three time points. Results The data revealed that, at six months following the education program, there was a statistically significant improvement in the knowledge (F [2,180] =13.53, p=0.001) and confidence (F [2,94] =4.88, p= 0.01) of infection precautions in the intervention group compared to the control group. Also, the means of knowledge and confidence in intervention group showed a consistently increased across three time points; whereas, the mean of confidence relating infection control management in the control group resulted a drop at time 3. Although the application skills relating to infection control procedures did not show a statistically significant change during this period (F [2, 174] = 2.54, p=0.081), there were minor improvements in these scores at the six-month follow-up assessment. Conclusion The SnAP program had a positive impact on Taiwanese nursing students' readiness for clinical placement and potential outbreak of disease epidemics. Participation increased their knowledge about infection control precautions, their ability to properly use these specific precautions, and their confidence in solving infection-related issues in clinical practice.

See front file / Health Studies / DLITT ET PHIL (HEALTH ST)