Implantation of tissue engineered pancreatic constructs can provide for a physiologic regulation of blood glucose levels. A major concern in designing such constructs is ensuring sufficient oxygenation of the cells, as oxygen is usually the limiting nutrient affecting cell viability and function. Furthermore, in vivo factors influencing construct oxygenation often lead to implant failure, and are detected primarily on end physiologic effects. The ability of perfluorocarbons (PFCs) to dissolve large amounts of oxygen and their high fluorine content makes these compounds a potentially valuable oxygen delivery tool and good 19F Nuclear Magnetic Resonance (NMR) markers for dissolved oxygen concentration (DO). Experimental studies and simulations showed that although the addition of 10 vol% PFC increased construct oxygenation, this improvement was minimal and had limited benefits on the growth and function of encapsulated bTC-tet cells under normoxic and hypoxic conditions. A dual PFC method that utilizes 19F NMR spectroscopy was developed to noninvasively monitor DO within a tissue construct and in its surroundings. In vitro studies using an NMR-compatible bioreactor demonstrated the feasibility of this method to monitor the DO within alginate beads containing metabolically active bTC-tet cells, relative to the DO in the culture medium, under perfusion and static conditions. In vivo, the method was capable of acquiring real-time DO measurements in murine models. Measured DO can be correlated with the physiological state of the implant examined post-explantation and was compatible with the therapeutic function of the implant.

<p>This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy. </p><p>The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days. </p><p>The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus.</p><p><b>Keywords:</b> protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein</p>

<p>This thesis concerns different areas of biomolecular nuclearmagnetic resonance spectroscopy (NMR). In the first part of thethesis a new formalism for simulations of NMR pulse sequencesis introduced. The formalism is derived both from classicalmechanics and quantum mechanics and is presented forhomonuclear and heteronuclear spin systems. The formalism hasalso been adapted to systems in chemical exchange. Simulationsof pulse sequences should be more straightforward using the newformalism.</p><p>In the second part of the thesis the NMR solution structuresof two ribosomal proteins are described. The ribosome isresponsible for protein production in all living cells and tounderstand the mechanism of the ribosome it is important toknow the three dimensional structure. In this thesis thestructures of S16 and S19, two of the proteins in the smallribosomal subunit, are presented. S16 is a mixed α /βprotein with a five-stranded parallel-antiparallel β-sheetand two α -helices. S19 is s mixed α/β proteinwith a three-stranded parallel-antiparallel β -sheet, oneα -helix and a short 3₁₀-helix.</p><p>In the third part of the thesis a program for semiautomaticassignment of NMR-spectra is presented. Assigning resonances inthe NMR spectrum is a labor-intensive process, which can takelong time. In semiautomatic assignment a computer program aidsthe user in finding assignments but leaves all decisions to theuser, thus speeding up the process. The program described inthis thesis is a new version of ANSIG, called Ansig forWindows. The program runs on PCs under Windows and has severaltools for semiautomatic assignment.</p><p><b>Keywords:</b>nuclear magnetic resonance, structuredetermination, ribosomal proteins, NMR simulations, NMR theory,NMR assignment software, semiautomatic assignment</p>

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 118-123). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 128-134). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 118-123). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 128-134). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 202-216).

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xv, 190 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 153-180).

Thesis (Ph. D.)--University of California, Riverside, 2008. / Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed March 10, 2010). Includes bibliographical references. Also issued in print.