An integrative biophysical and bioanalytical approach to studying the Fe distribution in isolated mitochondria was developed. This procedure involved large-scale growths, the inclusion of a chelator in isolation buffers and an anaerobic isolation protocol. Electron microscopy confirmed that mitochondrial membranes were intact and that samples were largely devoid of contaminants. The Fe-ome-the sum of all Fe species in mitochondria--was studied using a combination of EPR, Mossbauer Spectroscopy, Electron Absorption, ICP-MS and Protein analysis. Isolated mitochondria were packed prior to analysis to improve the S/N ratio. The residual buffer content of sample pellets was determined by use of a radio-labeled buffer. There was essentially no difference in the packing efficiency of mitochondria isolated from respiring and fermenting cells. The determined packing factor, 0.80, was used to calculate concentrations of individual species in neat mitochondria. The Fe-omes of mitochondria isolated from cells grown on respiring, respirofermenting and fermenting media were determined. Neat mitochondria contained ~ 750 mM Fe, regardless of whether the cells had been grown on respiring or fermenting media. The Fe distribution of respirofermenting samples (which can undergo respiration and fermentation simultaneously) was nearly identical to that of respiring mitochondria. Fermenting samples had a very different Fe-distribution. Nearly 40 % of the iron in respiring mitochondria was present in respiratory complexes including cytochrome c, cytochrome bc1, succinate dehydrogenase, and cytochrome c oxidase. Fermenting mitochondria contain an Fe-ome dominated by non-protein centers. Approximately 80 % of the Fe was present as a combination of nonheme HS Fe2+, nonheme Fe3+ and Fe3+ nanoparticles. These centers were present in roughly equal amounts. The remaining 20 % of the Fe was present as respiratory complexes which have concentrations ~ 1/2 to 1/3 that of respiring mitochondria. A model is presented in which the nonheme HS Fe2+ species serves as a feedstock for Fe/S and heme biosynthesis. When the cell is growing on respiring media, this metabolic reservoir diminishes as respiratory complexes are constantly synthesized. Under fermentative growth, the metabolic pool increases due to the reduced demand for respiration-related prosthetic groups.

Electron paramagnetic resonance (EPR) and Mössbauer spectroscopy were used to examine the distribution of iron in mitochondria from Saccharomyces cerevisiae. These organelles were packed into EPR and Mössbauer cuvettes, affording spectra with unprecedented signal/noise ratios. EPR spectra of as-isolated intact mitochondria exhibited fourteen distinct signals, some of which were assigned according to previously reported g-values obtained using isolated proteins. Signals from adventitious manganese (II) and iron (III) were largely removed when mitochondria were isolated in buffers supplemented with the metal chelators EDTA or EGTA. Signals were simulated and intensities were quantified to afford spin concentrations and estimates of the concentration of EPR-active species in mitochondria. The effects of treating samples with chemical modifiers were examined. Packed samples were analyzed for protein and metal content, affording averaged values of 50 mg/mL [protein], 590 µM [Fe], 340 µM [Cu], and 17 µM [Mn]. 57Fe-enriched intact mitochondria isolated in the presence of metal chelators exhibited Mössbauer spectra dominated by three components. Approximately 60% of the 57Fe in the sample gave rise to a quadrupole doublet, most of which was diamagnetic. The parameters of this doublet are typical of S = 0 [4Fe-4S]2+ clusters and S = 0 ferrous heme groups. Spectra of samples reduced with dithionite, pH 8.5, suggested that at least half of this doublet arose from [4Fe-4S]2+ clusters. The second major component exhibited in the Mössbauer spectra arose from high-spin ferrous ions (10%-30%). The third major component (15%) came from iron exhibiting magnetic hyperfine interactions and is likely reflected in the Fe-containing species observed by EPR. The results presented here suggest that mitochondria contain ~ 600 µM of Fe overall, ~ 200 – 400 µM organized as [4Fe-4S]2+ clusters, with about 25 µM due to the [4Fe-4S]2+ cluster of aconitase. Approximately 60 µM – 200 µM of the Fe in mitochondria is high-spin ferrous ions, ~ 40 µM as the Rieske S = 1/2 [2Fe-2S]+ cluster of cytochrome bc1, and ~20 µM as the S = 1/2 [2Fe-2S]+ cluster of succinate dehydrogenase. The high-spin ferric hemes of the a3:CuB site of cytochrome oxidase and cytochrome c peroxidase each account for ~ 4 µM of Fe.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb 2, 2010). Includes bibliographical references.

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Kinesiology and Health Science. / Typescript. Includes bibliographical references (leaves 71-78). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38823

Species within the family Cimicidae (bed bugs) are hematophagous ectoparasites of mammals and birds. Many cimicids are of socio-economic importance. Despite the global resurgence of these pests, there is currently a paucity of information regarding the mitochondrial (mt) DNA sequences of cimicids. Therefore, I used a PCR-based primer walking strategy to amplify and sequence the near complete mitogenome of the common bed bug (Cimex lectularius), and several mitochondrial gene regions of the Eastern bat bug (Cimex adjunctus) and swallow bug (Oeciacus vicarius). I compared the mitochondrial genetic variability between C. lectularius from two populations to look for molecular markers useful for population genetic studies. Furthermore, the mt DNA sequences of these species of medical and veterinary importance were compared to those of other heteropterans to infer the evolutionary relationships of species in the family Cimicidae.

Cells adapt their metabolism to satisfy changing bioenergetic and biosynthetic needs. Investigation of metabolic reprogramming in cancer has provided insight into the metabolic control of proliferation and survival. While the predominant focus of this field has been aerobic glycolysis (the Warburg effect), increasing evidence points to a complex landscape of tumor metabolic circuitries beyond aerobic glycolysis, including varied degrees of mitochondrial contribution to tumor metabolism. To investigate alternative metabolic programs compatible with tumor growth, we turned to Diffuse Large B-cell Lymphoma (DLBCL), a highly heterogeneous disease encompassing discrete clusters or subtypes defined by tumor-intrinsic genetic distinctions. In one classification scheme, a B-cell receptor (BCR)/proliferation cluster identified BCR-dependent DLBCLs with elevated expression of BCR signaling components. A second subset, OxPhos-DLBCL, displayed increased expression of mitochondrial oxidative phosphorylation genes, and was insensitive to BCR inhibition. However, the functional attributes of OxPhos-DLBCLs and the nature of their BCR-independent survival were unknown. Upon integrative analyses of DLBCL subtypes, we uncovered quantitative proteome- and metabolome-level signatures associated with differences in nutrient and energy metabolism. Specifically, BCR-DLBCLs have greater glycolytic flux typical of the Warburg phenotype. Unlike BCR-DLBCLs, OxPhos-DLBCLs channel the majority of glucose-derived pyruvate into mitochondria, display elevated mitochondrial electron transport chain (ETC) activity, ATP production, and fatty acid oxidation (FAO). Importantly, these metabolic distinctions are associated with subtype-selective survival mechanisms. Moreover, acute inhibition of BCR signaling in BCR-DLBCLs increased their FAO capacity, thus revealing a reciprocal relationship between BCR and FAO. Further dissection of mitochondrial function in OxPhos-DLBCLs indicates that increased mitochondrial metabolism is integrated with at least two homeostatic mechanisms that help maintain ETC activity and FAO capacity. In particular, OxPhos-DLBCLs harbor robust protein-level enrichment of mitochondrial translation factors required for the synthesis of mitochondrial-DNA-encoded ETC subunits. Inhibition of the mitochondrial translation pathway is selectively toxic to OxPhos-DLBCLs. A second mitochondrial homeostatic pathway, mitochondrial network dynamics, also proved relevant to OxPhos-DLBCLs. Compared to BCR-DLBCLs, OxPhos-DLBCLs display a fragmented mitochondrial network that supports their FAO capacity. Overall, these findings demonstrate previously unappreciated metabolic heterogeneity in molecular subsets of DLBCL and uncover BCR-independent survival mechanisms linked to mitochondrial FAO, protein translation, and network architecture.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Dynamic mitochondrial transport into axons and dendrites of neuronal cells is critical for sustaining neuronal excitability, synaptic transmission, and cell survival. Failure of mitochondrial transport is the direct cause of some neurodegenerative diseases, and an aggravating factor for many others. Mitochondrial transport regulation involves many proteins; factoring prominently among them are the atypical mitochondrial GTPase Miro and the Milton/TRAK adaptor proteins, which link microtubule (MT) motors to mitochondria. Motors of the kinesin family mediate mitochondrial transport towards the plus ends of microtubules, while motors of the dynein family mediate mitochondrial transport towards the minus ends. Selective use of these motors determines the ultimate subcellular distribution of mitochondria, but the underlying control mechanisms remain poorly understood. Drosophila Miro (dMiro) is required for kinesin-driven transport of mitochondria, but its role in dynein-driven transport remains controversial. In Chapter 2 of this study, I show that dMiro is also required for the dynein-driven transport of mitochondria. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to analyze the function of dMiro's N- and C-terminal GTPase domains, respectively. We show that dMiroT25N causes lethality and impairs mitochondrial distribution and transport in a manner indistinguishable from dmiro null mutants. Our analysis suggests that both kinesin- and dynein-driven mitochondrial transport require the activity of Miro's N-terminal GTPase domain, which likely controls the transition from a stationary to a motile state irrespective of the transport direction. dMiroT460N reduced only dynein motility during retrograde axonal transport but had no effect on distribution of mitochondria in neurons, indicating that the C-terminal GTPase domain of Miro most likely has only a small modulatory role on transport. Furthermore, we show that commonly used substitutions in Miro's GTPase domains, based on the constitutively active Ras-G12V mutation, appear to cause neomorphic phenotypic effects which are probably unrelated to the normal function of the protein. In mammalian neurons, kinesin and dynein motors are linked to mitochondria via a Miro complex with the adapter proteins TRAK1 and TRAK2, respectively. Differential linkage of TRAK-motor complexes provides a mechanism for determining the direction of transport and controlling mitochondrial distributions within the cell. Drosophila has only one TRAK gene homolog, Milton, which expresses several protein isoform. Milton has been previously been shown to facilitate mitochondrial transport by binding to kinesin and dMiro, a role analogous to TRAK1. However, the question whether Milton might be able mediate dynein-based transport in a manner similar to TRAK2 has remained unknown. In Chapter 3 of this study, I show that protein isoforms A and B of Milton, generated through alternative mRNA splicing, facilitate differential motor activities analogous to mammalian TRAKs. Specifically, overexpression (OE) of Milton-A caused a mitochondrial redistribution and accumulation at axon terminals, which requires kinesin-driven MT plus end directed transport; while OE of Milton-B caused a redistribution of axonal mitochondria into the soma, which requires dynein-driven MT minus end directed transport. I further show that Milton-motor complex binding to mitochondria requires Miro exclusively, and that transport with either of the motor complexes absolutely requires the activity of Miro's N-terminal GTPase domain. Together, these results suggest that Miro controls the transition of mitochondria from a stationary to a motile phase. Thereafter the direction of transport is likely determined by an alternative binding of opposing Milton/TRAK-motor complexes to Miro, a process which appears to be regulated by a Miro-independent mechanism.

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