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Title page for ETD etd-03232017-120814
|Type of Document
||Brady, Sonia Karoline
||Probing the mechanics of cellulose and nucleus associated machinery using optical tweezers
|Matthew J. Lang
|John T. Wilson
- optical trapping
- molecular motor
- single molecule
- bacterial cellulose synthesis
|Date of Defense
A fundamental understanding of biology is necessary for the engineering of biological systems and the successful employment of process and product development across a variety of industries. In an effort to truly understand the underlying mechanisms of some of these systems, the use of techniques that allow for detailed characterization of system components in isolated environments, often benefiting from the detection of hidden populations and their unique pathways, is required. Single molecule techniques are well suited for such studies. Here we use single molecule optical trapping based assays to investigate three biological systems: cellulose degradation by cellobiohydrolase 1 from Trichoderma reesei (TrCel7A), cellulose synthesis by bacterial cellulose synthase A and B (BcsAB), and the mechanotransduction of forces at the nuclear membrane, with nanometer and picoNewton precision in real time.
Cellulose, a major component of cell walls, is the most abundant polymer on earth and, as a glucose polymer, a valuable source of sugar for fermentation into biofuels. Unfortunately, the stability of cellulose makes much of its sugar backbone inaccessible without the help of cellulases. Through direct measurement of individual TrCel7A molecules, we find a system that processively hydrolyzes cellulose in discrete single cellobiose (glucose dimer) steps with little regard for opposing forces.
While biofuels provide motivation for cellulose degradation research, biofilms proved the motivation for cellulose synthesis. Cellulose is also a component of biofilms, stable antibiotic resistant bacterial environments that are responsible for hundreds of thousands of health care derived infections each year. BcsAB reveals a highly biochemically controlled complex whose activity is quenched with assisting forces of approximately 6-7 pN.
Last, we turn our attention to the mechanotransduction of signals through the cell to the nucleus where mechanical based genetic reprogramming can result. Exactly how those signals are delivered from the cytoskeleton to the nucleus is unknown. By focusing on single interactions between cytoskeletal components of hMSCs and nesprin proteins on the nuclear membrane, we find that cell growth conditions can dictate the forces required for a conserved nuclear response.
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