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

Thesis. 1976. M.S.--Massachusetts Institute of Technology. Dept. of Mechanical Engineering. / Microfiche copy available in Archives and Engineering. / Bibliography: leaves 95-97. / by David R. Lampe. / M.S.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1980. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. / Bibliography: leaves 121-122. / by Michael P. Shepley. / M.S.

Sphingolipids are a complex family of molecules that participate in many aspects of cell structure and function, including an essential cellular process known as autophagy. Autophagy is a degradation and recycling pathway whereby intracellular components are sequestered into double-membrane vesicles, known as autophagosomes, for subsequent fusion with lysosomes and degradation. Autophagy takes part in cell survival, host immune defense against pathogens, and other biological processes, but is also sometimes lethal. Ceramide, sphingosine 1-phosphate, and more recently dihydroceramide have been shown to induce autophagy, which opens an interesting new field of cell regulation by sphingolipids. This dissertation describes two new cases in which sphingolipids participate in the induction of autophagy: a) RAW264.7 cells treated with Kdo2-Lipid A, a lipopolysaccharide sub-structure with endotoxin activity equal to LPS; and b) MCF7 cells treated with fenretinde, a chemotherapeutic agent which has shown success in clinical trials. It also analyzes the structural properties of fenretinide that contribute to its ability to modulate sphingolipid metabolism through inhibition of dihydroceramide desaturase, thereby elevating dihydroceramide and induction of autophagy. Autophagy was monitored by following the redistribution of GFP-LC3 into discrete punctate vesicles in response to the agents and by Western blotting; in parallel, the sphingolipid composition of the cells was monitored by liquid chromatography, electrospray ionization tandem mass spectrometry. These analyses revealed that Kdo2-Lipid A and fenretinide induce profound changes in sphingolipid metabolism in RAW264.7 and MCF7 cells, respectively, and that one of the purposes for increased de novo biosynthesis is to enable the production of autophagosomes, as the autophagic response was inhibited by myriocin. These studies have uncovered a direct link between sphingolipid metabolism and autophagy, which could pave the way for new therapeutic interventions for the treatment of pathogenic infection and be clinically useful in enhancing the efficacy of current cancer treatment strategies.

At present, compared to our scientific knowledge of how natural biosystems function, we have practically no knowledge of how to engineer biosystems, i.e., how to design, build, repair, maintain, operate, and modify them in a rational and knowledgeable manner. Thus, the EcoCyborg research project was established with the basic intent of making a contribution to the science and engineering of biosystems. / This thesis covers three important arms of study within the overall framework of the EcoCyborg project. The first area is the development and implementation of a workbench for simulation based on composite models. The workbench was designed to accommodate easy integration, synchronization and communication of a number of independent modules. It was developed for use under OS/2 Warp, an operating system that supports multi-process simulation. The functionality of the workbench was tested with a composite model of an EcoCyborg. This composite model contained submodels of an ecosystem, two control systems, and several forcing functions. / The second area is the development and implementation of control systems for biosystems. The control system was intended to guide an ecosystem so that its behavior would best achieve the goals of the controllers which, in this case, were related to the populations of the ecosystem's species. The major part of the work on the control systems was focused on the Pavlovian controller. It mainly covered the investigation of the possibilities to develop and to implement a physiological-level Pavlovian controller. The thesis also presents results from a preliminary investigation on the design and implementation of a cognitive controller. From this, a relatively simple cognitive controller was developed to complement the action of the Pavlovian controller. / The third area is the experimental use of the workbench, and of the Pavlovian and cognitive controllers. For this, ecosystems having various constitutions and their forcing functions were modeled and used in the simulation. Pavlovian controllers with on-off, proportional and proportional plus integral mechanisms were used to control the populations. The cognitive controller was used to maintain minimum population of the various species in the ecosystem. / In general, the workbench that was developed was found suitable to accommodate the simulation of an EcoCyborg composite model. The tools that ware developed for constructing and implementing the Pavlovian controller were tested and found practical and effective for use in constructing Pavlovian controllers of various configurations. The results of the work on the cognitive controller provided useful information for its further development.

The muscarinic acetylcholine type 2 receptor (M2 mAChR), a member of the GPCR superfamily, is found throughout the parasympathetic nervous system where it controls pulmonary, urinary, and cardiac function, and neurotransmission. The molecular mechanisms that regulate M2 mAChR availability at the cell surface are an important component in controlling these physiological events. Since beta-arrestin proteins are known to regulate the activity of other GPCRs, we sought to identify their role in regulating M2 mAChR activity, a topic that remains contentious in the field. To achieve this goal we utilized mouse embryonic fibroblasts (MEFs) derived from beta-arrestin knockout mice lacking one or both isoforms (MEF KO1, KO2, or KO1/2 cells) in addition to exogenous expression of beta-arrestin mutants. This study demonstrates that agonist-induced internalization of M2 mAChR is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles suggesting it behaves as a class B receptor. Next, we sought to identify beta-arrestin s function in regulating the post-endocytic trafficking (down-regulation) of the M2 mAChR. MEF KO1/2 cells were unable to down-regulate M2 mAChRs whereas MEF KO1 or KO2 cells retained the ability to do so. In MEFwt cells, both M2 mAChR and beta-arrestin exhibited basal ubiquitination that increased following agonist stimulation. Receptor degradation appeared to be regulated by the ubiquitination status of beta-arrestin 2, since expression of a chimeric â-arrestin 2 form fused to ubiquitin increased both constitutive and agonist-promoted down-regulation, whereas expression of a beta-arrestin 2 mutant lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, significantly blocked degradation while internalization and stable association remained intact. Upon further analysis, the beta-arrestin 2K18R, K107R, K108R, K207R, K296R mutant blocked delivery of M2 mAChR to the late endosome/lysosome, presumably where degradation occurs. Inhibition of proteasome-dependent recycling of ubiquitin blocked receptor down-regulation without affecting internalization or the ubiquitination state of the M2 mAChR while ubiquitination of beta-arrestin 2 diminished significantly. These results support a role for ubiquitinated beta-arrestin in mediating M2 mAChR sorting and degradation in the lysosome. Collectively, these studies give us new insight on the function of beta-arrestin in regulating the activity of the M2 mAChR.

Thesis (M.S.)--University of Michigan, 1986.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves [80]-101). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves [80]-101). Also available on the Internet.

Thesis (M.S.)--University of Michigan, 1986.