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Developing tools to study carbohydrate processing enzymes in AmpC β-lactamase antibiotic resistance and OGA-based neurodegenerative research

Date created: 
Gram-negative bacteria
β-lactam antibiotics resistance
Disaccharide substrate
Membrane permease
Transporter enzyme ampG
Live-cell assay
Caged fluorescence
Coupled assay
High-throughput scale

Carbohydrates are one of the four main classes of biological macromolecules in nature, alongside lipids, proteins, and nucleic acids. They serve in a wide range of cellular functions fundamental to the existence of biological organisms. These functions include but are not limited to cellular metabolism, energy production and storage, structural support, signaling, and recognition. The ubiquitous presence of carbohydrates in organisms has led to the study of their role in various maladies, such as neurodegenerative and infectious diseases. Deciphering the role of carbohydrates in these diseases allows for the possibility of developing treatments for associated diseases. In this manner, it is necessary to develop tools to exploit and test our understanding of these mechanisms. Existing methodologies may need to be adapted for application to larger scale experimental designs, such as those used in chronic dosing studies using preclinical animal models or high-throughput automated screening assays. This thesis describes improvements to previously published methods in the synthesis of one such chemical tool, Thiamet-G, a small molecule inhibitor used to study the carbohydrate processing enzyme O-GlcNAcase, which has been linked to neurodegenerative diseases including Alzheimer’s and Parkinson’s Disease. This thesis also seeks to apply the concepts developed during creation of a live cell assay towards creation of a new experimental approach suitable for large scale high through-put screening of compound libraries. Such an application would allow for the efficient pursuit of inhibitors of the bacterial protein AmpG - a transporter that is essential for inducible AmpC β-lactamase-driven antibiotic resistance.

Document type: 
This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
David Vocadlo
Science: Department of Chemistry
Thesis type: 
(Thesis) M.Sc.