Publication Date


Date of Final Oral Examination (Defense)


Type of Culminating Activity


Degree Title

Doctor of Philosophy in Biomolecular Sciences



Major Advisor

Daniel Fologea, Ph.D.

Major Advisor

Juliette Tinker, Ph.D.


Denise Wingett, Ph.D.


Xinzhu Pu, Ph.D.


Pore-forming toxins secreted by various evolutionarily distant organisms are important components of their innate defense mechanisms. These toxins may kill the target cells by inserting un-regulated channels into the plasma membrane. Tampering with the otherwise well-controlled membrane permeability alters cell homeostasis by contributing to un-controlled dissipation of both chemical and electrical gradients, which is often an essential component of virulence mechanisms leading to cell death. However, the same ability to create nanoscopic conducting pathways, i.e. nanopores, has been exploited for creating powerful tools in nano-biotechnology. Single nano-channels reconstituted in artificial planar lipid membranes are extremely versatile sensors that are capable of detection, identification, and characterization of single molecules. In addition, the changes in permeability induced by pore-forming toxins reconstituted in artificial and natural lipid membrane systems are exploited for numerous biomedical, scientific, and bio-technological applications. Many of these applications, underlying principles, and limitations are briefly described in the introduction section of this dissertation.

To overcome many of the restrictions presented by currently used pore-forming toxins, we propose to use lysenin channels for both stochastic sensing and for the achievement of controlled membrane permeability. Lysenin is a pore-forming toxin extracted from the earthworm E. foetida that inserts large nanopores in natural and artificial lipid membranes containing sphingomyelin. Chapter 2 of the presented work is focused on exploring the use of single lysenin nanopores for stochastic sensing of human angiotensin II, a short hormone peptide,which is highly relevant for the pathophysiology of cardiovascular diseases. Besides a traditional analysis of the interactions between lysenin channels and peptides, we succeeded to employ high sensitivity liquid chromatography – mass spectroscopy analyses to demonstrate the passage of un-altered peptide molecules through open lysenin channels.

In Chapter 3 we exploit the unique regulatory mechanisms presented by lysenin channels to achieve controlled permeability over artificial and natural lipid membranes. We demonstrate that ATP molecules may reversibly regulate the macroscopic ionic conductance of lysenin channels inserted into planar lipid membranes. Lysenin reconstitution into spherical membranes (liposomes) enables a two-way control over membrane permeability by using multivalent metal cations capable of inducing reversible ligand-induced gating of the channels. Live cell analyses demonstrate that lysenin channels allow transport of non-permeant molecules in Jurkat leukemia and ATDC5 chondrogenic cells. In addition, extended control over membrane permeability is achieved by using chitosan molecules as irreversible blockers of the macroscopic conductance. Survival rate estimations indicate that the permeabilized cells maintain a satisfactory viability rate for further use. Therefore lysenin may be used for the controlled transport of ions and molecules in living systems.


Included in

Life Sciences Commons