Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2004. / Includes bibliographical references (leaves 231-240). / As part of the non-specific immune response to infection, activated macrophages synthesize nitric oxide (NO) at relatively high rates, thereby creating local concentrations of NO that are toxic to the invading microorganisms. However, high rates of NO synthesis can also damage host tissues, especially if prolonged (as in chronic inflammations). Although NO can act directly, much of its cytotoxicity and genotoxicity may be due to peroxynitrite (ONOO-), formed by the rapid reaction of NO with superoxide (02-)- Superoxide is a byproduct of mitochondrial respiration and of certain cytosolic reactions, and it is present in virtually all cells. Thus, intracellular ONOO- formation is inevitable when cells are exposed to NO, which diffuses readily across cell membranes. Superoxide is generated also by an NADPH oxidase on the external membrane surface of macrophages, thereby delivering it to the extracellular fluid at significant rates. The research in this thesis aimed to determine the concentration profiles of NO and related species in cell cultures and tissues. Toward that end, the diffusion and reactions of NO, 02, and 02- were studied by mathematical modeling and by experimental work with macrophage cultures. The results obtained will be useful in designing NO toxicity experiments and in determining potentially toxic conditions in body tissues. A mathematical model was developed to predict the intracellular concentrations of NO, 02-, and ONOO- in suspension cell cultures exposed to NO and/or peroxynitrite. Steady-state concentrations were computed as a function of radial position within an idealized spherical cell, with a distinction being made between cytosolic and mitochondrial values. / (cont.) Potential sources of peroxynitrite include intracellular generation in mitochondria and cytosol and (depending on the type of experiment) diffusion of extracellular peroxynitrite into the cell. The relative importance of extracellular and intracellular sources was estimated for a wide variety of conditions. The calculated mitochondrial concentrations were generally 5-10 times higher than the cytosolic values. For the baseline conditions, including an NO concentration of 1 [mu]M and no peroxynitrite in the medium, the cytosolic peroxynitrite concentration was estimated as [approx.] 2 nM. The extracellular peroxynitrite concentration required to double the cytosolic level was [approx.] 25 nM, and an extracellular concentration of -100 nM was needed to effect a five-fold increase. Another approach for studying NO and ONOO- toxicity involves the co-culture of activated macrophages (producers of NO and 02-) with "target cells" (which make 02- but not NO). Because peroxynitrite concentrations in such experiments are too small and localized to measure, reaction-diffusion models were developed for situations ranging from isolated cells to many cells randomly distributed on a plate. The average concentration inside randomly distributed target cells increased with increasing macrophage number density, as entry of extracellular peroxynitrite grew in importance relative to intracellular formation. For high cell densities, large peroxynitrite membrane permeabilities, and low rates of intracellular synthesis, the surrounding macrophages were calculated to double or triple the peroxynitrite concentration in an average target cell ... / by Nitesh Nalwaya. / Ph.D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/29376 |
| Date | January 2004 |
| Creators | Nalwaya, Nitesh, 1979- |
| Contributors | William M. Deen., Massachusetts Institute of Technology. Dept. of Chemical Engineering., Massachusetts Institute of Technology. Dept. of Chemical Engineering. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 240 leaves, 7398492 bytes, 7398061 bytes, application/pdf, application/pdf, application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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