Thesis (Ph.D. )--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on October 7, 2008 ). Research advisor: Paul A. Ney, M.D. Document formatted into pages (xi, 122 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 78-110).

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "May 1997." Typescript. Vita. Includes bibliographical references (leaves 147-162). Also available on the Internet.

Utilization of iron by hemoglobin-producing cells is highly efficient. The acquisition of iron from plasma requires the binding of diferric transferrin (Tf) to its cognate receptor (Tf-R) on the erythroid cell membrane, followed by internalization of the Tf - Tf-R complexes via receptor-mediated endocytosis. Through a poorly understood mechanism, iron is targeted to mitochondria, the site of heme biosynthesis. We believe that a direct interaction between iron-containing endosomes and mitochondria is essential for iron transfer to mitochondria and its efficient incorporation into heme. / In order to illustrate the interaction between endosomes and mitochondria, we have employed flow cytometry. Flow cytometry analysis of reticulocytes (erythrocyte precursors which still synthesize hemoglobin) stained with fluorescent dyes specific to mitochondria and endosomes revealed three distinct populations: mitochondria, endosomes and a population labeled with both dyes. This double-labeled population suggests a population composed of endosomes associated with mitochondria. Using non-fluorescent diferric-Tf, we were able to remove the double population, leaving only the endosomal and the mitochondrial population. This finding has confirmed that the double population is the result of the interaction between the two organelles. / Additionally, we established a cell-free assay consisting of fluorescent mitochondria and endosomes isolated from erythroid cells. Using confocal microscopy, we demonstrated a colocalization between the two organelles. We repeated the assay using fluorescent mitochondria and endosomes isolated from HeLa spinner cells. Using the mitochondrial uncoupler CCCP, we were able to significantly reduce the colocalization between the two organelles, indicating that the interaction between the organelles is specific and that the mitochondrial potential is a requirement for organellar interaction. / Based on our results from flow cytometry and confocal microscopy, we conclude that a specific and direct interaction exists between the two organelles.

Diss. (sammanfattning) Göteborg : Göteborg University, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves: 147-162). Also available on the Internet.

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Most existing chemotherapeutics lack adequate specificity for transformed cells and therefore have high rates of collateral damage to normal tissue. Moreover, such therapies often depend on p53 to induce cell death and are ineffective on the large number of human cancers that have lost p53 function. The discovery of novel p53-independent cancer therapies is therefore of significant interest. The Chicken Anemia Virus protein Apoptin selectively induces apoptosis in transformed cells in a p53-independent manner while leaving normal primary cells unaffected. This selectivity is thought to be largely due to cell type-specific localization: in primary cells Apoptin is cytoplasmic, whereas in transformed cells the protein localizes to the nucleus. The basis for this cell type-specific localization remains to be determined. In this study, Apoptin is revealed to be a nucleo-cytoplasmic shuttling protein whose localization is mediated by an N-terminal nuclear export signal (NES) and a C-terminal nuclear localization signal (NLS). Both signals are required for cell type-specific localization, as Apoptin fragments containing either the NES or NLS fail to localize differently between transformed and primary cells. Significantly, cell type-specific localization can be rescued in trans by co-expression of the two separate fragments, which are able to interact through an Apoptin multimerization domain. Interestingly, this multimerization domain overlaps with the NES suggesting that these two activities may be functionally coupled in cytoplasmic retention in primary cell types. Factors present in transformed cells induce localization of Apoptin to the nucleus where a biochemically distinct, more soluble form of the protein exists. Using affinity-purification and mass spectroscopy it was found that, specifically in transformed cells, Apoptin is associated with APC1, a subunit of the anaphase-promoting complex/cyclosome (APC/C). The APC/C is required to establish a mitotic cell-cycle checkpoint, and its inhibition results in G2/M arrest and apoptosis. Expression of wild type Apoptin in transformed cells inhibits APC/C function and induces G2/M arrest and apoptosis, whereas Apoptin mutants that are unable to associate with APC1 have no effect. In p53 null cells, ablation of APC1 by RNA interference induces a G2/M arrest and apoptosis analogous to that observed following Apoptin expression. Furthermore, Apoptin was found to induce the formation of PML bodies and to recruit APC/C subunits to these nuclear structures suggesting a mechanism involving sequestration and subsequent inhibition of the APC/C. Thus, the results of this study clarify Apoptin cell type-specific localization behavior and explain the ability of Apoptin to induce apoptosis in transformed cells in the absence of p53. This study advances a newly emerging field of viral mechanisms of apoptosis involving G2/M arrest and APC/C modulation. The resultant p53-independent apoptosis suggests that the APC/C may be an attractive target for the development of anti-cancer drugs.