Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, December 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1981. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 87-89).

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Allen G. Harmsen. Includes bibliographical references (leaves 134-149).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / On t.p. "F̳u̳" is superscript. Includes bibliographical references (leaves 131-161). Also available in electronic version.

Thesis (M.S.)--Villanova University, 2006. / Biology Dept. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

We propose a biochemically plausible mechanism for constructing digital logic signals and gates of significant complexity within living cells. These mechanisms rely largely on co-opting existing biochemical machinery and binding proteins found naturally within the cell, replacing difficult protein engineering problems with more straightforward engineering of novel combinations of gene control sequences and gene coding regions. The resulting logic technology, although slow, allows us to engineer the chemical behavior of cells for use as sensors and effectors. One promising use of such technology is the control of fabrication processes at the molecular scale. / DARPA/ONR Ultrascale Computing Program under contract N00014-96-1-1228 and by the DARPA Embedded Computing Program under contract DABT63-95-C130.

<p>The complex process of visually interpreting our environment begins with the task of detecting the light that enters our eyes. This task is performed by the rod and cone photoreceptors, which both contain a highly evolved sensory cilium called the outer segment. The outer segment is a specialized cellular compartment that contains all of the protein machinery involved in converting the initial light signal into an electrical signal that can be ultimately transmitted to the brain. Outer segments are cylindrical structures that envelop an array of individual, densely packed membrane discs. Discs are renewed throughout the lifetime of a photoreceptor, with older material being shed at the tip and new material added at the base of the outer segment. Many studies conducted over the past 35 years conclude that disc formation starts with evagination of the plasma membrane at the outer segment base, followed by membrane expansion and, in the case of rods, subsequent disc enclosure. Despite the intense interest in the topic, the molecular mechanisms governing how outer segment discs are formed and renewed are not well understood. </p><p>The focus of this dissertation centers on elucidating the molecular role of peripherin/retinal degeneration slow (rds) in outer segment disc morphogenesis, including study of peripherin/rds trafficking from its site of synthesis in the endoplasmic reticulum to its site of function in the outer segment. Peripherin/rds is expressed specifically in photoreceptor outer segments, where it fulfills a critical role in assembling and/or maintaining the structure of this organelle. Mutation or loss of peripherin/rds in humans is often associated with visual impairments, and its knockout in mice results in rudimentary ciliary stumps completely lacking disc structures. </p><p>We found that early outer segment morphogenesis steps in mice lacking peripherin/RDS proceed normally for the first week. However, in the second week of postnatal development at the onset of disc formation, mice lacking peripherin/RDS produce extracellular vesicles next to their connecting cilia rather than discs. We characterized these vesicles and determined that they are enriched in outer segment proteins, are ~230 nm in size, and are formed as outward buds of the plasma membrane. These characteristics allowed us to classify these extracellular vesicles as ciliary ectosomes. Furthermore, we determined that ectosome shedding is arrested upon expression of the peripherin/rds C-terminal cytoplasmic sequence, which allows for the accumulation of excessive membranous material. Thus, we conclude that peripherin/rds transforms the functional dynamics of photoreceptor primary cilium from shedding massive amounts of ectosomes to retaining these membranes in the outer segment to eventually become photoreceptor discs. This novel function of peripherin is performed by its C-terminal cytoplasmic sequence and represents the first step in disc morphogenesis. </p><p>Finally, the morphogenesis study of peripherin/rds is complemented by a study of its trafficking. Understanding how peripherin is delivered from the site of its synthesis is critical for its function at the outer segment. We show that the peripherin/rds targeting sequence is confined within ten amino acid residues, which do not overlap with the putative fusogenic domain, and that only a single amino acid within this region is irreplaceable, a highly conserved valine at position 332.</p><p>Collectively, these studies shed considerable light on the molecular role played by peripherin. Peripherin is a photoreceptor specific protein that transforms the primary sensory cilium into a specialized sensory cilium capable of building the discs required for efficient photon capture. While work in this direction provides a significant advance in our understanding of peripherin’s role in disc morphogenesis, questions such as whether peripherin participates in disc enclosure, remain to be solved.</p> / Dissertation

<p> Regulation of barrier function is critical for patients who suffer from inflammatory diseases such as acute respiratory distress syndrome (ARDS) and sepsis. A major regulator of endothelial barrier function is vascular endothelial cadherin (VE-cad). Cellular levels of VE-cad are known to be regulated by p120 catenin. Loss of p120 leads to decreased barrier function as a result of the endocytosis of VE-cad. However, recent work from our lab shows that expression of an endocytic defective VE-cad mutant was not able to rescue barrier function, as measured using transendothelial electrical resistance (TEER). In contrast, expression of a non-phosphorylatable VE-cad mutant was able to restore barrier function independent of p120 binding. These results suggest that endocytosis is not the only mechanism regulating VE-cad localization to the cell-cell junctions, but rather the phosphorylation state of the protein may play a more critical role to stabilizing VE-cad at the junction. In order to investigate junctional stability of VE-cad, we created a recombinant form of VE-cad by cloning mEos2 into a plasmid containing the VE-cad gene. This fluorophore is photoconvertible, thus allowing for tracking protein movement at the cell-cell junction. The VE-cad proteins, labeled with mEos2 at the C-terminus, were introduced via adenoviral infection into human umbilical vein endothelial cells (HUVEC). Initially, mEos2 fluoresces green, in order to induce photoconversion, a 405nm laser is directed in a specific region of interest (ROI) at the junction. A conformational change in the mEos2 protein will cause irreversible red fluorescence. Tracking the change in fluorescence intensity in the ROI will provide insight into the localization of VE-cad at endothelial cell junctions. We now have a model that can be used to test junctional localization and stability of endocytic defective and non-phosphorylatable mutants of VE-cad.</p><p>