PublisherThe University of Arizona.
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AbstractDiabetes is an epidemic in developed nations. Glucokinase (GK) is vital for glocose sensing, and is directly implicated in particular forms of diabetes. Studying pancreatic cells with altered GK activity would facilitate studies, but current methods for altering proteomes are lacking. Porous phospholipid nanoshells (PPNs) have traditionally been used as platforms for biologically derived nanosensors, though their biocompatibility and protease resistance well suits them as enzyme delivery agents. GK kinetics were investigated with an enzyme coupled reaction to determine the effect of encapsulation. It was determined that encapsulation increased the Hill coefficient by 5.8% and the S(0.5) by 1.8%. This small deviation may not be significant in physiological conditions. To observe a recovered function in cell lines upon reintroducing GK, constitutively expressed GK must first be knocked down with siRNA. As initial work toward an siRNA knockdown, immunoblotting conditions were optimized resulting in a detection limit below 10 ng of GK. Immunoblotting verified suspected constitutive expression of GK in INS-1 cell lines. While further investigation is necessary to demonstrate the utility of GK-containing PPNs for cell delivery, this thesis outlines the generation and characterization of this novel enzyme delivery platform.
Degree ProgramHonors College
Biochemistry and Molecular Biophysics