Etacrynsäure – ein langjährig eingesetztes Diuretikum – und verschiedene ihrer Derivate sind als nichtpeptidische Inhibitoren von Cystein-Proteasen bekannt. Einige Amid-Derivate besitzen bezüglich des SARS-CoV sowohl enzymhemmende (SARS-CoV-Mpro) als auch antivirale Wirkung. Das elektrophile Strukturfragment des Moleküls, das als Michael-Akzeptor eine Reaktion mit dem active-site Cystein-Rest der Protease eingehen kann, ist die a,b-ungesättigte Carbonyleinheit in der Acyl-Seitenkette. Ausgehend von der Etacrynsäure als Leitstruktur wurden durch Strukturvariationen neue Etacrynsäure-Derivate, bevorzugt Amid-Derivate, dargestellt. Zusätzlich zu diesen Verbindungen wurden außerdem weitere Etacrynsäure-Derivate synthetisiert, die durch Biotin- und Deuterium-Markierung als Modellsubstanzen zum In-vitro-Nachweis des Wirkstoffes in biochemischen und spektroskopischen Assays dienen. Als deuterierte Spezies wurde mit der d9-Etacrynsäure ein hochdeuteriertes Wirkstoff-Analogon dargestellt. Der Grad der Deuterium-Markierung konnte durch Kupplung von d13-n-Hexylamin an die d9-Etacrynsäure noch auf ein d22-Amid-Derivat erweitert werden. Insgesamt konnten neben dem direkten d9-Analogon des Wirkstoffes drei weitere Amid-Derivate erhalten werden. Zur Testung der erhaltenen Substanzen an der SARS-CoV-Mpro war die Synthese eines FRET-Substrats erforderlich. Dafür wurde mittels automatisierter SPPS ein Dekapeptid mit dem FRET-Donor-Akzeptor-Paar Anthranilsäure – Nitrotyrosin hergestellt. Das Substrat besitzt die Sequenz H2N-Abz-Ser-Val-Thr-Leu-Gln-Ser-Gly-Tyr(NO2)-Arg(Mts)-COOH. Der Km-Wert für die SARS-CoV-Mpro wurde unter Berücksichtigung des inner filter effect zu 190 ± 23 µM bestimmt. Zur Evaluation der enzymhemmenden Aktivität wurden die synthetisierten Inhibitoren an den drei Enzymen SARS-CoV-Mpro, SARS-CoV-PLpro und Cathepsin L getestet. Als potenteste Inhibitoren wurden das Bromanisol MS07 (Ki = 52.7 µM, SARS-CoV-Mpro) sowie das Norethyl-Derivat der Etacrynsäure MS21 (Ki = 22.8 µM, Cathepsin L) identifiziert. Alle getesteten Verbindungen zeigten an der SARS-CoV-PLpro keine Aktivität. Im Rahmen des SFB 630 wurden mit den synthetisierten Verbindungen weitere Testungen an Bakterien, den Protozoen Leishmania major und Typanosoma brucei brucei sowie an Makrophagen durchgeführt. Dabei zeigten die Verbindungen MS38, MS40 und MS41 eine besondere Wirksamkeit. Es konnten zwar im Wachstumstest sowie im Biofilmtest Aktivitäten gegen den Bakterienstamm Staphylococcus epidermidis sowie die Hemmung der Biofilmbildung von S. epidermidis festgestellt werden. Die zur Hemmung notwendigen Mindestkonzentrationen lagen oberhalb der Konzentrationen, die auch cytotoxisch auf Makrophagen wirkt. Ähnliches gilt für die Aktivität an L. major. Bezüglich T. b. brucei zeigen diese drei Verbindungen jedoch eine erhöhte Aktivität mit MHK-Werten von < 1 µM. Das bedeutet, dass für eine Hemmung eine Konzentration erforderlich ist, die um Faktor 4 bis 11 unterhalb der cytotoxischen Konzentration liegt. Dieser Wert konnte nur noch durch das Biotin-markierte Derivat MS57 (IC50 T. b. brucei: 0.69 µM; IC50 Makrophagen: 33.8 µM) übertroffen werden. Weitere Untersuchungen mit der Biotin-markierten Verbindung MS57 zeigten zudem eine Hemmung von Falcipain-2 und -3 sowie von Plasmodium falciparum mit IC50-Werten von 3.0 µM (FP-2) 11.9 µM (FP-3) und 9.0 µM (P.f.). Weiterhin konnte mit Hilfe der Massenspektrometrie eine Bindung von MS57 an FP-2 nachgewiesen werden. Diese findet aber offensichtlich nicht im aktiven Zentrum des Enzyms statt. Die deuterierten Derivate der Etacrynsäure kamen als Modellsubstanzen zur Etablierung eines CARS-Mikroskopie-Assays zur spektroskopischen Wirkstoffdetektion zum Einsatz. Dabei war es bisher möglich, das CARS-Signal der d9-Etacrynsäure in DMSO-Lösung bis zu einer Konzentration von 500 µM zu erfassen. Quantenchemische Berechnungen zur Simulation des Reaktionsmechanismus des active-site-Cystein-Restes der SARS-CoV-Mpro mit Michael-Systemen als elektrophilem warhead zeigten, dass es sich dabei um einen zweistufigen Prozess handelt, bei dem nach erfolgter Deprotonierung des Thiols der Angriff an der Doppelbindung erfolgt. Zudem ergaben die Berechnungen, dass sowohl Halogenarylsubstituenten am Carbonylkohlenstoff sowie exo-konfigurierte Systeme sich energetisch günstig auswirken. Diese Trends konnten durch kinetische NMR-Experimente durch Umsetzung einer Reihe von Modellsubstanzen mit p-Methoxythiophenolat bestätigt werden. Die Ergebnisse zeigen, dass für die betrachteten Systeme die Umsetzung innerhalb von wenigen Minuten, spätestens aber innerhalb von 90 Minuten zu 99 % erfolgt ist. / Etacrynic acid, which has been used as a diuretic drug for many years, and several derivatives thereof are well-known as non-peptidic inhibitors of cysteine proteases. Some amide derivatives possess both, enzyme inhibiting (SARS-CoV-Mpro) and antiviral properties (SARS-CoV, TGEV, MHV). The a,b-unsaturated carbonyl unit located in the acyl side chain serves as an electrophilic scaffold which is supposed to react as a Michaeltype acceptor with the active site cysteine residue of the protease. Additionally, etacrynic acid derivatives labelled either with biotin or deuterium were synthesised as model compounds for in-vitro detection in biochemical and spectroscopic assays. As a d-labelled compound a highly deuterated analogue of the known drug etacrynic acid could be obtained. The labelling was enhanced by peptide coupling of d13-hexane-1-amine with d9-etacrynic acid yielding a d22-amide derivative. In total, the deuterated etacrynic acid and three amide derivatives were synthesised. In order to evaluate inhibitory properties in fluorometric enzyme assays a FRET-pair-labelled substrate (H2N-Abz-Ser-Val-Thr-Leu-Gln-Ser-Gly-Tyr(NO)2-Arg(Mts)-COOH) was synthesised by SPPS. The Km value was determined to 190 ± 23 µM (SARS-CoVMpro). The synthesised compounds were tested against SARS-CoV-Mpro, SARS-CoV-PLpro, and human cathepsin L. Within this tested series the bromo anisole MS07 (Ki = 52.7 µM, SARS-CoV-Mpro) and the norethyl derivative of etacrynic acid (MS21, Ki = 22.8 µM, cathepsin L) turned out to be the most active compounds. No inhibition of the SARS-CoVPLpro was detected for any of the tested substances. Within the framework of the SFB 630 the antibacterial properties of the synthesised compounds as well as their activities towards the protozoa Leishmania major and Trypanosoma brucei brucei were determined. Compounds MS38, MS40 and MS41 inhibit biofilm formation of Staphylococcus epidermidis, growth of the bacteria as well as growth of Leishmania major in low micromolar concentrations. However, the compounds also display cytotoxic effects against macrophages. In contrast, growth of T. b. brucei is inhibited with IC50 values < 1 µM leading to selectivity indices of 5 to 11 with the biotin-labelled derivative MS57 displaying the highest selectivity (IC50 T. b. brucei: 0.69 µM; IC50 macrophages: 33.8 µM). Further investigations with the labelled compound MS57 showed the compound to inhibit falcipain-2 and -3 as well as Plasmodium falciparum. The IC50 values are 3.0 µM (FP-2), 11.9 µM (FP-3) and 9.0 µM (P. f.). Binding of MS57 to FP-2 could be proved by mass spectroscopic experiments. Results indicate an allosteric binding mode of MS57 to falcipain-2. The deuterated etacrynic acid derivatives served as model compounds for the development of a spectroscopic detection assay using CARS microscopy. Currently, the detection of the CARS signal of d9-etacrynic acid in a DMSO solution is possible for a 500 µM concentration. Quantum chemical calculations simulating the reaction of the active site cysteine residue of SARS-CoV-Mpro with Michael-type acceptors revealed a two step mechanism. Deprotonation of the thiol is followed by the addition of the thiolate to the double bond of the a,b-unsaturated moiety. Calculations of the energy profiles of the second reaction step showed, that the reaction rate can be enhanced by halogenated aryl residues and by exoconfiguration of the Michael-system. These trends could be confirmed by means of kinetic NMR experiments monitoring the reactions of a set of model compounds with p-methoxy thiophenolate.

Abstract Since the outbreak of sever acute respiratory syndrome (SARS). The potentially serious impact of SARS on people¡¦s accessibility to medical services should not be overlooking. Many wondered whether the fear of SARS among patient¡¦s alike changed people¡¦s seeking behaviors. Kaohsiung Area remains one of the most severely affected areas of Taiwan. We aimed to identify three problems: 1. Whether the impact of SARS influenced hospital¡¦s ambulatory patient¡¦s seeking behaviors (including overuse, underused, health and preventive behavior, fixed medical care place, and use alternative medicine)? 2. Whether the impact of SARS influenced patient¡¦s selection of hospitals level or clinics? 3. Whether the demographic and health status characteristics affected patient¡¦s seeking behaviors during or after the SARS? Questionnaires were distributed to all willing hospital¡¦s ambulatory patients of Kaohsiung Areas during 2003 Feberry to May. The sampling methodology was stratified, and cluster random sampling. The survey included questions regarding concern about 3-SARS-periods (included pre-SARS, during-SARS, after-SARS) patient¡¦s seeking behaviors (13 items of each period) and selection of hospitals level or clinics (1 item), personal demographic and health status characteristics (12 items). Of the 1834 questionnaires distributed, 1101 (60.03%) were returned. The Cronbach [alpha] for internal consistency reliability was 0.861 for the Questionnaire. We used the repeated measured CATMOD procedure in SAS (Version 8.0, SAS Institute Inc., Gary, NC, 1999-2000) to test the data of 3-SARS-periods patients seeking behaviors. We compared pre-SARS with during-SARS, and compared pre-SARS with after-SARS patient¡¦s seeking behaviors. Comparisons were considered significant at P< or =0.01 and P values were 2-tailed. Combined data for the two comparisons showed that significantly decreased the overuse such as repeated use and go to the medical centers for command cold, decreased the underused such as delay seeking care, increased health and preventive behavior, but fixed health care place deceased, the percentage of use alternative medicine¡¦s didn¡¦t significantly changed. The two comparisons of selection of hospitals level and clinics showed that significantly decreased the percentage of selection of medical center level. Male, single, educational level under high school, family income below 50,000 per month, the chronic disease patient's prescription user, the major illness/injury card user, and high use¡]use above E health care card¡^group patients were statistically significant more percentage of overuse, and less percentage of health behavior, fixed medical care place.

This study objects are focus on the tourist hotels in the north region of Taiwan. The research method adopts information collection from their websites and conducts in-depth observation and research from the content of the case-study hotel websites, in order to acquaint with their primary business contents. The related references and papers are also referred to, for analyzing and structuring the initial data. Finally, the framework of 4P (product phase, price phase, promotion phase, place phase) serves as the analytical structure to collect the main business and management contents of the tourist hotels and their applications in E-commerce. On the other hand, the interviews with the E-commerce executives in some case-study hotels also are conducted in order to make up the insufficient of website observations. Through the in-depth website observation and case-study hotel supervisor interviews, we find how the case-study hotels use E-commerce technology to provide with their services and products online in the post-SARS age and categorize their e-commerce strategies. The purpose of this research is to provide hotels with effective methods to respond to the customer requirements, avoiding vicious price competition in the same industry, and reduce the risks in hotel management through the E-commerce development trends in the tourist hotel industry. Hopefully, the research of this study can help the hotels to create better management efficacy, and to promote the prosperity in the domestic tourist hotel industry. Keywords: E-Commerce¡BSARS¡BTourist Hotels

Abstract This thesis focuses on the SARS epidemic situation in 2003, conducting the comparison research. The compared objects are Singapore and Taiwan government¡¦s strategies. When Singapore and Taiwan have been through repeatedly the same crisis, which policies and measures have both countries separately made? Why the Singapore government's strategies were more successful? But Taiwan's strategies were actually thought awaits improvements. This article first introduces SARS epidemic situation development in 2003, after understanding event background, gradually will discuss the focal point to gather in Singapore and Taiwan. Then, the writer will make the analysis to the Singapore government as well as the Taiwan government SARS epidemic situation crisis management. The crisis management can be separated to three phases: crisis prevent phase, crisis handle phase and crisis restore phase, and the writer will discuss each phase. Finally, proposes regarding the government whole related SARS epidemic situation crisis management view and the suggestion.

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Using a multiple stage age of infection model, we derive an expression for the basic reproduction number, Ro. We apply this method to find Ro in analogous treatment models. We find, in the model without treatment, Ro depends only on the mean infective period, and not on the infective distribution. In treatment models, Ro depends on the mean infective and mean treatment period, as well as the distribution of the infective period, but not on the distribution of the treatment period. With an explicit formula for Ro and the final size relation, we provide a practical alternative to evaluating the effect of treatment and other control measures. We compare our models to previous models of SARS and TB.

Using a multiple stage age of infection model, we derive an expression for the basic reproduction number, Ro. We apply this method to find Ro in analogous treatment models. We find, in the model without treatment, Ro depends only on the mean infective period, and not on the infective distribution. In treatment models, Ro depends on the mean infective and mean treatment period, as well as the distribution of the infective period, but not on the distribution of the treatment period. With an explicit formula for Ro and the final size relation, we provide a practical alternative to evaluating the effect of treatment and other control measures. We compare our models to previous models of SARS and TB.

Zugl.: Innsbruck, Univ., Diss., 2005

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 194-218) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leave 126-134). Also available in print.