Graduation date: 2001

A novel protocol for the study of protein-nucleic acid interactions is presented and demonstrated to be feasible. The protocol combines photochemical crosslinking techniques and mass spectrometric methods into a new strategy for identifying protein domains or amino acid residues that are in close contact with nucleic acid in protein-nucleic acid complexes. Identifying nucleic acid binding domains in proteins provides a starting point for understanding structure-function relationships in protein-nucleic acid complexes. The protocol can be divided into three parts: 1) Cross linking of the protein-nucleic acid complex by irradiation with ultraviolet light and subsequently verifying the crosslinking by mass spectrometry; 2) Mass spectrometric peptide mapping of crosslinked protein-nucleic acid complexes to identify crosslinked peptide-nucleic acid hybrids; 3) Tandem mass spectrometric sequencing of peptide-nucleic acid hybrids to localize the crosslinked amino acid residue(s). The experimental data described in this dissertation documents our efforts to establish and implement this analytical protocol. Using several different protein-nucleic acid systems and different crosslinking techniques, we have demonstrated the feasibility of a mass spectrometric based approach to structurally characterize UV-crosslinked protein-nucleic acid complexes. Matrix-assisted laser desorption/ionization mass spectrometry was for the first time demonstrated to be highly effective for detection and molecular weight determination of intact, UV-crosslinked protein-nucleic acid complexes and for molecular weight determination of synthetic and UV-crosslinked peptide-nucleic acid hybrids. Electrospray ionization mass spectrometry and tandem mass spectrometry was demonstrated to be effective for analysis of synthetic peptide-nucleic acid hybrids and, in conjunction with HPLC, for peptide mapping of a protein. The first application of MALDI mass spectrometry to the characterization of crosslinked peptide-nucleic acid hybrids isolated from a photochemically crosslinked protein-nucleic acid complex demonstrate that the new protocol can be used to identify nucleic acid binding domains in proteins. / Graduation date: 1995

Graduation date: 2003

To understand the chemistry of life processes in detail is largely a challenge of resolving them in their native, cellular environment. Cell culture, first developed a century ago, has proven to be an essential tool for reductionist studies of cellular biochemistry and development. However, for the technology of cell culture to move forward and address increasingly complex problems, in vitro environments must be refined to better reflect the cellular environment in vivo. This dissertation work has focused on the development of methods to define cellular microenvironments using the high resolution, 3D capabilities of multiphoton lithography. Here, site-specific photochemistry using multiphoton excitation is applied to the photocrosslinking of proteins, providing the means to organize bioactive species into well-defined 3D microenvironments. Further, conditions have been identified that enable microfabrication to be performed in the presence of cells -- allowing cell outgrowth and motility to be directed in real time. In addition to the intrinsic chemical functionality of microfabricated protein structures, 3D protein matrices are shown to respond mechanically to changes in the chemical environment, enabling new avenues for micro-scale actuation to be explored. Complex 2D and 3D protein photocrosslinking is further facilitated by integrating transparency and automated reflectance photomasks into the fabrication system. These advances could be transformative in efforts to fabricate precise cellular scaffolding that replicates the morphological (and potentially biochemical) features of in vivo tissue microenvironments. Finally, these methods are applied to the study of microorganism behavior with single-cell resolution. Microarchitectures are designed that allow the position and motion of motile bacterial to generate directional microfluidic flow -- providing a foundation to develop micro-scale devices powered by cells. / text

Bibliography: leaves 163-172. / vi, 172 leaves ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Organic Chemistry, 1993

No description available.

Call number: LD2668 .T4 1985 J54 / Master of Science

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING / Includes bibliographical references. / by Elizabeth Hsiu-Yun Chen. / M.S.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Elliot Chaikof; Committee Member: Marc Levenston; Committee Member: Robert Nerem; Committee Member: Vincent Conticello; Committee Member: Yadong Wang. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis: M.S., Massachusetts Institute of Technology, Department of Mechanical Engineering, 1980 / Includes bibliographical references. / by Dennis P. Orgill. / M.S. / M.S. Massachusetts Institute of Technology, Department of Mechanical Engineering