Knox, John David.2009-03-232009-03-2319911991Source: Dissertation Abstracts International, Volume: 53-09, Section: B, page: 4463.9780315704800http://hdl.handle.net/10393/7617http://dx.doi.org/10.20381/ruor-15428This thesis presents the results of my investigation of the process of cell-mediated cytotoxicity and in particular the role of the microtubules and intermediate filaments in MTOC/GA orientation. Immunofluorescence microscopy was used to determine if the orientation of the MTOC is accompanied by the posttranslational modification of tubulin. My results indicate that, if a more stable subset of microtubules is formed during the orientation of the MTOC towards the TC, it does not persist long enough for the posttranslational modification of tubulin to occur. Immunofluorescence microscopy also demonstrated that conjugation results in the formation of filamentous vimentin aggregates in CTLs similar to those induced by microtubule disassembling drugs. However, intermediate filament aggregates are not formed in all conjugated CTLs and there is no evidence to suggest that their formation is required for lytic activity. A hyperthermia treatment of 42$\sp\circ$C for 30 min results in a marked inhibition, followed by a transient recovery of cytolytic activity that peaks within 6 h. The close correlation observed between the loss and recovery of microtubule organization and the loss and recovery of cytolytic activity suggest that the initial inhibitory effect of hyperthermia on cytolytic activity is due to the disruption of MTOC-microtubule organization. The microtubules of taxol-treated cells remain associated with the centrosome, but the characteristic radial array is replaced by large bundles. This microtubule organization has no effect on centrosome organization, orientation of the MTOC, nor on cytolytic ability. The results show that the normal radial organization of microtubules is not required for the functional polarization of CTL, but suggest that this process is dependent upon centrosome organization. It is proposed that 50% of the cytolytic activity of the effector cells used in this study is due to the synthesis and secretion of a lytic protein subsequent to TC binding and that the remaining activity is occurring via a protein synthesis-independent apoptotic pathway. To what extent the recovery of cytolytic activity is dependent upon the recovery of each of the pathways could not be determined. The final results presented suggest that the long-term loss in cytolytic activity may be due to hyperthermia-induced effector cell death. (Abstract shortened by UMI.)216 p.Biology, Cell.Thesis