<p>This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an <em>N</em>-acyl-(4<em>R</em>)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated.</p> / Report code: LIU-TEK-LIC-2006:46.

<p>Since the discovery in 1989 of hepatitis C virus (HCV) as the infectious agent responsible for the vast majority of post-transfusion non-A non-B hepatitis, blood transfusions are no longer a source for HCV transmission in Sweden. Anti-HCV testing was implemented for all blood donations in 1992. Since then intravenous drug use (IDU) has become the major route of transmission in the western world. Six genotypes and more than 80 subtypes of HCV have now been identified world-wide. These genotypes and subtypes are determined by genetic divergences between the HCV strains. Subtypes 1a, 1b, 2b, 2c, and 3a have global spread, while the other subtypes have a more limited geographical distribution. Little was known on the prevalence of HCV among blood donors and on which genotypes and subtypes of HCV were circulating in Sweden before the testing of all blood donations was implemented. The prevalence of anti-HCV was therefore investigated in sera sent to the Swedish Institute for Infectious Disease Control (SMI) from 412 patients; 241 were sampled between 1970 and 1991 before the universal screening in 1992, while 171 were sampled between 1992 and 2002. The samples derived from 193 (47%) blood donors, (104 sampled before, and 89 after 1992), and from seven other groups of patients. Two groups had suspected known routes of infection, intravenous drug use (IDU) 33 patients and hemodialysis, 16 patients, while it was unknown for the other patients. Anti-HCV was detected in 120 (29%) samples. The highest frequency was found among IDUs, (91%). Before general screening was implemented, 2.8% of the blood donors were positive for hepatitis C, whereas 28% of those sampled after 1992 were anti-HCV positive. Those latter samples were sent to SMI due to anti-HCV reactivity in a primary test at the blood centre. HCV RNA could be detected by PCR in 56 (47%) of the anti-HCV positive samples, the subtype could be determined by sequencing in 45 (80%) of those. The subtypes found were 1a in 31 %, 1b in 18%, 2b in 22%, and 3a in 27%. One sample was of subtype 2c. There was a tendency of increase of genotype 2 and a decrease in subtype 1a with time. 1a was found in 38% of the samples collected before 1992, while it was only found in 19% of the samples from 1992 or later. On the other hand genotype 2 was found in 17% sera sampled before 1992 and in 37% of the samples collected 1992 or later. It is not known if this genotype has recently been introduced into Sweden. Further analysis on larger series of samples is needed to confirm these preliminary results.</p> / AcknowledgmentsI would like to express my gratitude to several people who have been supportive in different ways throughout this project.First of all, I want to thank my supervisor Helene Norder, for giving me the possibility to do my diploma thesis at the Department of Virology, Swedish Institute for Infectious Disease control (SMI) and for helping me during this study and for the many insightful conversations during the design and development stages of the application, and also for the many helpful comments and suggestions on the text of the thesis.I want to express my appreciation to my laboratory supervisor Regina Wallin, Camilla Jern and Josefine Ederth for helping me during the procedure for this study. Then, I want to thank my examiner Magnus Johansson from the Södertörns university collegefor his advice on writing this paper. Finally, I would like to thank my family and specially my mother Bahar Hamid for always supporting me during my whole life.Last, but not least, I would like to thank my friends Annika Andersson and Yourdons Yemane for being encouraging, understanding and always supportive.

Hepatitis C is a blood-borne disease affecting 130-170 million people worldwide. The causative agent, hepatitis C virus (HCV), infects the liver and is the major reason for chronic liver disease worldwide. The HCV NS3 protease, a key enzyme in the virus replication cycle, has been confirmed to be an important target for drug development. With the recent release of two HCV NS3 protease inhibitors onto the market and an arsenal of inhibitors in clinical trials, there are now hopes of finally combating the disease. However, the success of treatment relies heavily on the ability to overcome the emergence of drug-resistant forms of the protease. The main focus of this thesis was on designing and synthesizing novel inhibitors of the NS3 protease with a unique resistance profile. Efforts were also made to decrease the peptide character of the compounds, with the long-term goal of making them into more drug-like compounds. Special attention was devoted to developing inhibitors based on a phenylglycine in the P2 position, instead of the highly optimized and commonly used P2 proline. Around ninety acyclic and macrocyclic inhibitors have been synthesized and biochemically evaluated. P2 pyrimidinyloxy phenylglycine was successfully combined with an aromatic P1 moiety and alkenylic P1´ elongations, yielding a distinct class of HCV NS3 protease inhibitors. Macrocyclization was performed in several directions of the inhibitors via ring-closing metathesis. Only the macrocyclization between the P3-P1´ residues was successful in terms of inhibitory potency, which suggests that the elongated P1-P1´ residue is oriented towards the P3 side chain. The metathesis reaction was found to be significantly more dependent on the substrate than on the reaction conditions. It was also found that the P3 truncated inhibitors were able to retain good inhibitory potency, which initiated the synthesis and evaluation of a series of P2-P1´ inhibitors. The potential of the P3-P1´cyclized inhibitor and the smaller, acyclic P2-P1´ as new potential drug leads remains to be determined through pharmacokinetic profiling. Gratifyingly, all the inhibitors evaluated on A156T and D168V substituted enzyme variants were able to retain inhibitory potency towards these as compared to wild-type inhibition.

This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an N-acyl-(4R)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated. / Report code: LIU-TEK-LIC-2006:46.

This thesis was undertaken to assess the characteristics of individuals who smoke crack cocaine and to examine the health-related risks and healthcare needs of this population. A literature review of 147 published articles was conducted to synthesize evidence regarding behaviours associated with crack use and to assess the risks of disease transmission through crack smoking behaviours. Qualitative interviews were subsequently conducted with thirty Ottawa residents who smoke crack to learn about their experiences with healthcare and harm reduction services. Results identified barriers related to accessing primary healthcare and drug treatment programming among people who smoke crack and gaps within existing harm reduction services. Individuals who smoke crack represent a marginalized population who are often missed through traditional health promotion and harm reduction programming. There is a need for increased coverage of current programming and a reduction of factors which currently hinder the delivery and effectiveness of crack-specific harm reduction programs.

This thesis was undertaken to assess the characteristics of individuals who smoke crack cocaine and to examine the health-related risks and healthcare needs of this population. A literature review of 147 published articles was conducted to synthesize evidence regarding behaviours associated with crack use and to assess the risks of disease transmission through crack smoking behaviours. Qualitative interviews were subsequently conducted with thirty Ottawa residents who smoke crack to learn about their experiences with healthcare and harm reduction services. Results identified barriers related to accessing primary healthcare and drug treatment programming among people who smoke crack and gaps within existing harm reduction services. Individuals who smoke crack represent a marginalized population who are often missed through traditional health promotion and harm reduction programming. There is a need for increased coverage of current programming and a reduction of factors which currently hinder the delivery and effectiveness of crack-specific harm reduction programs.

Deux types de problématiques différentes peuvent motiver l'approche structurale d'une macromolécule. Soit la fonction de cette macromolécule est inconnue et l'on détermine sa structure afin de mieux connaître sa fonction ; cette démarche était l'un des défis du développement de la génomique structurale. Soit la fonction est connue et la structure va permettre de comprendre comment cette molécule remplit son rôle biologique.<br />Durant ma thèse, j'ai eu l'occasion de pouvoir aborder deux projets structuraux très différents, qui correspondent à ces deux approches de biologie structurale. C'est pourquoi mon manuscrit décrit ces deux projets dont la démarche scientifique est différente et complémentaire pour ma formation en biologie structurale. <br />Dans un premier temps, je décrirai les résultats obtenus sur le projet PAB0955, débuté en stage de DEA et poursuivi pendant les deux premières années de ma thèse. Ce projet avait pour objectif de déterminer la fonction de la protéine d'après sa structure. La protéine PAB0955 est de fonction biologique inconnue, elle hydrolyse le GTP et elle n'a pas de proche homologue dont la structure est connue. La démarche scientifique de ce projet a été de déterminer la structure de cette protéine, puis d'analyser sa structure en la comparant de manière systématique à l'ensemble des protéines ATPases et GTPases. Notre étude a été conduite dans l'objectif extraire un maximum d'information fonctionnelle à partir des données structurales.<br />Dans une deuxième partie, je décrirai notre étude sur des protéines du système immunitaire humain débuté en troisième année de thèse. Nous nous sommes intéressés à des molécules impliquées dans la reconnaissance d'antigène : le récepteur du lymphocyte T (TCR) et la molécule du complexe majeur d'histocompatibilité (CMH). L'objectif de ce projet est de comprendre comment un complexe antigène-CMH sélectionne un répertoire de lymphocyte T spécifique. L'objectif étant de déterminer le lien entre les caractéristiques structurales d'un complexe antigène-CMH et la diversité et la fréquence des lymphocytes T activés par ce complexe. En résumé corréler les observations structurales avec les données immunologiques dont nous disposons. En déterminant les bases structurales du caractère antigénique d'un peptide présenté par une molécule CMH nous pourrons, à plus long terme, produire des peptides efficaces dans le cadre d'une vaccination

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

This thesis was undertaken to assess the characteristics of individuals who smoke crack cocaine and to examine the health-related risks and healthcare needs of this population. A literature review of 147 published articles was conducted to synthesize evidence regarding behaviours associated with crack use and to assess the risks of disease transmission through crack smoking behaviours. Qualitative interviews were subsequently conducted with thirty Ottawa residents who smoke crack to learn about their experiences with healthcare and harm reduction services. Results identified barriers related to accessing primary healthcare and drug treatment programming among people who smoke crack and gaps within existing harm reduction services. Individuals who smoke crack represent a marginalized population who are often missed through traditional health promotion and harm reduction programming. There is a need for increased coverage of current programming and a reduction of factors which currently hinder the delivery and effectiveness of crack-specific harm reduction programs.

In this dissertation we present mathematical models that help answer health policy questions relating to HIV and Hepatitis C (HCV), and analyze bias in Markov models of disease progression. We begin by developing a Markov decision process model that examines the timing of testing and treatment for diseases with asymptomatic periods such as HCV. We explicitly consider secondary infections, false positives and negatives, and behavioral modification from information from test results. We derive sufficient conditions for testing and/or treating in a dynamic environment, i.e., when unscheduled patients arrive. We also develop a detailed simulation model for general testing and/or treating for HCV. A key finding is that the current policy recommendations on testing for HCV may be too restrictive, and that it is cost-effective to test the overall population if done at the appropriate times. The Markov models used in the study of HCV motivated the next topic where we examine bias in Markov models of diseases. We examine models in which the progression of the disease varies with severity and find sufficient conditions for bias to exist in models that do not allow for transition probabilities to change with disease severity. We apply the results to HCV and find that the bias is significant depending on the method used to aggregate the disease data. We close with a discussion on a specific question in HIV policy where we develop a Bernoulli process transmission model in which, for a given individual, each risky person-to-person contact is treated as an independent Bernoulli trial. Using the model and data from the Urban Men's Health Study, we estimate the affect that interventions at venues, namely bathhouses, in which high-risk behavior takes place would have on HIV transmission.