2013 Distinguished Master's Thesis Award Winner
John Anderson, Master of Science in Biological Sciences, earned EIU's top research honor for his master's thesis titled, Desiccation Tolerance in Insect Cells.
Michael Menze, Ph.D., assistant professor of biological sciences, served as faculty mentor.
Thesis Abstract
Our society is in need of cost effective cell-preservation methods to facilitate advances in fields which utilize recombinant gene expression (e.g. to produce therapeutics such as insulin) and engineer tissues and organs in vitro. Current methods of cell preservation require freezing at ultra-low temperatures (-135°C), and toxic compounds such as dimethyl sulfoxide. We explored alternative preservation methods using ‘lessons learned from nature’ to stabilize cells in the dry state at room temperature. Several organisms in nature can tolerate severe water loss including larva of the chironomid Polypedilum vanderplanki and embryos of the brine shrimp Artemia franciscana. These animals accumulate both LEA proteins and the sugar trehalose to maintain viability upon rehydration. LEA proteins are intrinsically disordered in solution, but increase in ordered secondary structure as water is removed and stabilize macromolecules during water-limited states. Trehalose is a glass-forming sugar that can stabilize proteins and membranes through reduced molecular mobility in the glassy state. Trehalose must be present on both sides of the cell membrane to confer protection, but cell membranes are impermeable to trehalose in the absence of a specific transporter. To increase membrane permeability, we transgenically over-expressed trehalose transporters in the cell membrane of insect cells. Cells loaded with trehalose and slowly dried maintain little viability when rehydrated, but ultra-fast dried cells employing a spin-drying apparatus, which quickly removes liquid water in a matter of seconds, maintained viability after water removal and rehydration. This thesis investigated the question whether the overexpression of a trehalose transporter localized in the cytoplasmic membrane of insect cells will facilitate intracellular loading of trehalose and demonstrates increase viability after fast drying of trehalose loaded cells. Furthermore, cellular tolerance to osmotic stress was increased by LEA proteins. These novel findings may help to engineer desiccation tolerant cells and tissues in the future.
