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acase@tulane.edu

Cholera is an epidemic infectious diarrhoeal disease that for centuries has proven its frightening ability to cause rapid and widespread loss of human life. All symptoms associated with cholera are a result of rapid dehydration due to infection by pathogenic strains of the bacterium Vibrio cholerae. The damaging effects associated with cholera are mainly attributed to the toxin, which is secreted by the bacterium and infects cells lining the gastrointestinal tract. A sialidase, also secreted by the bacterium, is believed to facilitate toxin uptake by the gastrointestinal epithelium. V. cholerae sialidase is therefore a potential target for therapeutic intervention. A survey of the literature reveals that sialidases from different species share common features with respect to their structure, substrate specificity and catalytic mechanism. The unsaturated sialic acid, Neu5Ac2en, inhibits most exosialidases with a dissociation constant of inhibitor of -10-4 to-10-6 M and has frequently been used as a template in the design of more potent sialidase inhibitors. In the case of V. cholerae sialidase, there have been no inhibitors reported to date that are significantly more potent than Neu5Ac2en itself The present research aimed to develop a range of mimics of Neu5Ac2en, which contain various substituents to replace the C-6 glycerol side chain, as potential inhibitors of V cholerae sialidase. The x-ray crystal structure of V cholerae sialidase was used to explore potential interactions between active site residues and C-6 modified Neu5Ac2en mimetics of known inhibitory potency. Opportunities for interactions within the glycerol side chain pocket in the active site of V cholerae sialidase are discussed. A novel synthetic strategy was developed for the synthesis of a series of glucuronidebased Neu5Ac2en mimetics starting from readily available GIcNAc. This approach was employed for the preparation of Neu5Ac2en mimetics that contained an ether or thioether substituent as replacement of the glycerol side chain of Neu5Ac2en. Progress was also made towards the synthesis of a series of C-6 acylamino Neu5Ac2en mimetics. Analysis by 1H NMR spectroscopy showed that the acylamino derivatives adopted a half-chair conformation that was similar to the conformation of Neu5Ac2en but different to the conformation adopted by the ether and thioether derivatives prepared. The inhibitory activity of the C-6 ether and thioether Neu5Ac2en mimetics prepared was evaluated in vitro using an enzyme assay. It was found that most of the derivatives inhibited V. cholerae sialidase with a K1 of approximately 1O-4 M. The derivatives containing a hydrophobic side chain were found to be slightly more potent compared to derivatives with more hydrophilic side chains. A more detailed study of binding interactions between the C-6 thioether Neu5Ac2en mimetics and V cholerae sialdiase was carried out using STD 1H NMR spectroscopy and computational molecular modelling.

Thesis (M.A.)--McGill University, 1997. / Includes bibliographical references.

Thesis (M.Sc.(Epidemiology))--University of Pretoria, 2003. / Also available online.

Utilizing medical manuals, medical records, newspapers, and letters, the history of the management of epidemics from 1803-1902 will be explored. This thesis weaves together and explores the political history of the nineteenth century by analyzing the contribution of doctors and reformers in the management of diseases. This thesis explores the intersection between the construction of a public health system and the implementation of these practices by political actors and physicians. The history of the management of disease is analyzed from the introduction of the mass vaccination campaign, in Spain, in 1803. This thesis first analyzes the development of a public health system focused on prevention. It then challenges the system created by examining how effective these measures were against the multiple waves of cholera to hit Spain. It then addresses the important role reformers had in the late nineteenth century. It was through their efforts that doctors and reformers became explicitly linked to new ideas of citizenship and responsibility. This paper emphasizes both continuity in the importance of health care, but also the transformations in the discourse of public health responsibility. Ultimately, it centers liberalism and an emerging middle class within the discussion of a health policy.

Cholera, an acute diarrheal diseases caused by the intestinal infection of the pathogenic bacterium Vibrio cholerae, continues to be a global killer in the world today. PrtV, a secreted zinc metalloprotease, is a potent cytotoxic virulence factor of V. cholerae. The 102 kDa full length multi-domain PrtV protein undergoes several N and C terminal modifications before being secreted as a 81 kDa pro-protein. The activation of the pro-protein is calcium dependent. The removal of calcium triggers auto-proteolysis to give a stable active protease with the catalytic zinc binding domain. The aim of the thesis was to study the structure and function of the PrtV protein. The results from paper I, identified the end product of the maturation of PrtV as the stable 37 kDa M6 active domain, and not a 55 kDa complex as reported earlier. Results also showed the this 37 kDa active M6 domain alone was sufficient for catalytic activity. A revised model for the maturation of PrtV was proposed. Individual domains were isolated from the PrtV protein by domain phasing methods. This included the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25 kDa fragment (residues 589-839). The isolated domains were recombinantly over expressed as fusion proteins to increase expression and solubility. The PKD1 domain was purified to homogeneity and crystallized. The structure of the PKD1 domain reported in paper II, was solved by X-ray crystallography at an atomic resolution of 1.1 Å. From the structure, a previously unknown calcium binding site was identified at the N-terminal of the PKD1 domain. The structure also revealed two conformations for the PKD1 domain depending on free or bound calcium. From the structure, a function of the PKD1 domain as a protector of the cleavage site in the linker region between the M6 domain and the PKD1 domain in the presence of calcium was elucidated. A new model for the activation of PrtV was given. In paper III, the structure of the N-terminal domain solved by NMR spectroscopy was reported. The structure revealed two well defined helices but a third predicted helix was found to be unstructured.

In the fall of 1892, fear of cholera was pervasive in North America. Ten years into the fifth international cholera epidemic -- that lasted from 1881 to 1896 -- cholera had been raging in the Middle East, India, and Europe, but the disease had yet to cross the Atlantic Ocean. The maritime traffic of immigrants from Europe was continuous, and each migrant ship potentially carried the disease. Doctors, government officials, and politicians were not asking 'will cholera come?', but rather when. While no one got sick or died of cholera in the city of Toronto in 1892, the crisis and fear of imminent cholera was very real. Drawing on archival research, this dissertation maps how a cholera crisis was shaped by urgency, immediacy, and speculation on the future. My argument will show how the geography of an epidemic is not limited to the presence of a disease. If crises are times of profound activity, how does this event need to be substantiated in order to produce change? This dissertation follows how cholera was integral to producing an object called proliferating life that held together: migrating populations, growing cities, and degeneration; marshland as the source of disease; the medical theory of zymosis that explained how disease outbreaks got out of control; and Malthusian 'laws' of population. Health experts used correlation and synecdoche to visualize these relations. However, these experts needed a stable institutional base to articulate both their fears and their recommendations, which included: professionalized expanding health boards, as social infrastructures; reclaiming Toronto’s marshland of Ashbridge's Bay; and a health ideology built upon the fear of future epidemics, immigration, and a growing economic rationale for health. By the early 20th century, state health became instrumental to a "national vitality", a practice of government intervention that I frame as bureaucratic bio-economy.

In the fall of 1892, fear of cholera was pervasive in North America. Ten years into the fifth international cholera epidemic -- that lasted from 1881 to 1896 -- cholera had been raging in the Middle East, India, and Europe, but the disease had yet to cross the Atlantic Ocean. The maritime traffic of immigrants from Europe was continuous, and each migrant ship potentially carried the disease. Doctors, government officials, and politicians were not asking 'will cholera come?', but rather when. While no one got sick or died of cholera in the city of Toronto in 1892, the crisis and fear of imminent cholera was very real. Drawing on archival research, this dissertation maps how a cholera crisis was shaped by urgency, immediacy, and speculation on the future. My argument will show how the geography of an epidemic is not limited to the presence of a disease. If crises are times of profound activity, how does this event need to be substantiated in order to produce change? This dissertation follows how cholera was integral to producing an object called proliferating life that held together: migrating populations, growing cities, and degeneration; marshland as the source of disease; the medical theory of zymosis that explained how disease outbreaks got out of control; and Malthusian 'laws' of population. Health experts used correlation and synecdoche to visualize these relations. However, these experts needed a stable institutional base to articulate both their fears and their recommendations, which included: professionalized expanding health boards, as social infrastructures; reclaiming Toronto’s marshland of Ashbridge's Bay; and a health ideology built upon the fear of future epidemics, immigration, and a growing economic rationale for health. By the early 20th century, state health became instrumental to a "national vitality", a practice of government intervention that I frame as bureaucratic bio-economy.

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