Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 77-100). Also available in print.

Engineered protein systems have been created to impart new functions, or “re-program” mammalian cells for applications including cancer and HIV/AIDS therapies. The successful development of mammalian cells for re-programming will depend on having well-defined, modular systems. Migration is a particularly important cell function that will determine the efficiency and efficacy of many re-programming applications in vivo, and Rho proteins are responsible for regulation of cell migration natively. While there have been several reports of photo-activated Rho proteins, no strategy has been developed such that Rho proteins and cell migration can be controlled by a variety of extracellular stimuli that may be compatible with signaling in large organisms. Here, several methods are described for engineering Ca2+-sensitive Rho proteins so that the large, natural toolbox of Ca2+-mobilizing proteins can use the Ca2+ intermediate to activate Rho proteins in response to a variety of exogenous stimuli, including chemicals, growth factors, and light. First, an unreported calmodulin binding site was identified in RhoA. This knowledge was used to create a tandem fusion of RhoA and calmodulin that mediated Ca2+-sensitive bleb retraction in response to a variety of Ca2+-elevating chemicals. Ca2+-mobilizing modules including channelrhodopsin-2 and nicotinic acetylcholine receptor α4 were used for light- and acetylcholine-dependent bleb retraction. Second, a more robust morphology switch was created by embedding a calmodulin binding site into RhoA to enable Ca2+-responsive bleb formation. A wider range of Ca2+-mobilizing modules were also used here including LOVS1K/Orai1 and vascular endothelial growth factor 2. Combining Ca2+-mobilizing and Ca2+-responsive modules increased amoeboid-like cell migration in wound closure and transwell assays. Finally, the embedded peptide design was applied to Rac1 and Cdc42 to enable control of new morphologies and migration modes. The modular Ca2+ control over Rho proteins developed here is an important contribution to cell re-programming because it shows that control over cell migration can be rewired in a way that is flexible and tunable.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Surangani Dharmawardane. Vita. Includes bibliographical references.

published_or_final_version / Pathology / Master / Master of Philosophy

The division of one cell to form two cells, or cytokinesis, is fundamental to the development of all known multi-cellular organisms, as well as the propagation of life between generations. The intracellular mechanisms that mediate the physical deformation of the cell membrane during division have proven to be remarkably robust, with multiple processes functioning together to achieve bisection. Here, I present my doctoral work, which seeks to illuminate the dynamic molecular interplay that coordinates and drives cytokinesis in the Caenorhabditis elegans single-cell zygote. In Chapter 1, I begin with an introduction on cytokinesis and the many proteins known to regulate cell division. Chapter 2 presents a detailed review of three intracellular signaling molecules that mediate the spatial control of cytokinesis, known as Rho family small GTPases. In Chapter 3, I present work in which we inactivated specific cytokinesis protein functions at precise stages of the division process, in order to map out the first “temporal atlas” of essential cytokinetic functions. In Chapter 4, I present evidence that the GTPase CDC-42 and the cortical polarity machinery sequester cytokinesis-inhibiting proteins away from the division plane and protect the fidelity of cytokinesis. Chapter 5 lays out preliminary evidence that another GTPase, RAC-1, is a suppresser of cytokinesis and must be inactivated in the division plane specifically by a spindle-associated regulatory protein. Through this body of work, I have attempted to elucidate the underpinnings of the complex intracellular orchestra that drives cytokinesis. This work provides valuable insight, not only into how this vital process occurs, but also how the disruption of its components could lead to the development of complex diseases like cancer.

No description available.

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 262-280). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 262-280). Also available online.