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A survey of natural isolates of N. intermedia revealed five variants with respect to growth phenotype. These variants showed stop-start behavior in growth tubes, sometimes never attaining the full tube length. These strains are mainly.female sterile. The stop-start phenotype was not transmitted via the male parent in any cross. In two crosses, maternal transmission was demonstrated. One strain was investigated for the presence of virus-like particles, but none were found. Cytochrome spectra show a deficiency of cytochromes a. and/or b_ relative to the amount of c. In respiration studies, the one strain tested proved to be cyanide resistant and salicyl hydroxamic acid sensitive. These characteristics are also found in extranuclear mutants of N. ovassa. However, the analogy no longer holds at the mitochondrial ribosome level. An analysis of four of the strains disclosed that three are large subunit deficient. Only small subunit deficient strains have been found among the cytoplasmic mutants of N. ovassa. Restriction enzyme analysis was also carried out on the variants. It was found that they possess additional mitochondrial DNA compared to normal N. intermedia. One Eco RI fragment of M.W. 2.4 x 10 was found to be common to all variants tested, but is not found in the normal strains. There were other DNA differences which were unique to each abnormal strain. These findings suggest that the basis for the abnormal phenotypes .is some heritable factor associated with the mitochondria. / Science, Faculty of / Botany, Department of / Graduate

Following neuronal injury, calcium signaling plays a critical role in promoting repair processes. Injury produces an initial cytosolic calcium elevation mediated by calcium entry from the cut site, plasma membrane channels, and intracellular storage compartments. Subsequently, a variety of signaling factors are involved in promoting growth cone formation and axon outgrowth and guidance, some of which include DLK-1, CaMP, CED-3, CED-4, and calreticulin. Specific proteins mediating calcium transport have also been reported to significantly affect regenerative outgrowth, particularly inositol triphosphate receptors, voltage-gated calcium channels, and ryanodine receptors. Given that mitochondria can store intracellular calcium and regulate cytosolic calcium levels, we hypothesized that the mitochondrial uniporter (MCU) may play a significant role in neuronal regeneration. We found that inhibiting calcium entry into the mitochondria via a loss of function mutation in MCU significantly enhances axonal outgrowth following laser axotomy of single neurons in C. elegans. This effect is calcium-dependent, with the MCU mutant regenerative phenotype reverting to baseline levels when mutants are chronically treated with the calcium chelator EGTA. We also find that sub-cellular calcium signals at the axon cut site are significantly reduced in MCU mutants, while basal levels of calcium and axon guidance remain unaffected. These findings suggest that mitochondrial calcium regulation plays a significant role in the regeneration of single neurons, and that inhibition of MCU activity may be a promising avenue for the treatment of clinical syndromes derived from axonal injury, such as spinal cord injury. / 2017-11-03T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Abnormal blood vessel growth from pre-existing blood vessels, termed pathological angiogenesis, is a common characteristic of vascular diseases like proliferative diabetic retinopathy (PDR) and wet age-related macular degeneration (wet AMD). Retinal detachment, hemorrhage, and loss of vision are only some of the debilitating consequences of advanced pathological angiogenesis. Current therapeutics targeting these blood vessels are ineffective in many patients. We previously identified a novel angiogenesis target, ferrochelatase (FECH), from the heme synthesis pathway, and found that FECH inhibition is antiangiogenic in cell and animal models. Heme synthesis occurs in mitochondria, where FECH inserts Fe2+ into protoporphyrin IX (PPIX) to produce heme. However, the relationship between heme metabolism and angiogenesis is poorly understood. I sought to understand the mechanism of how FECH and thus, heme is involved in endothelial cell function. First, I determined the energetic state of retinal and choroidal endothelial cells, previously uncharacterized. I found that mitochondria in endothelial cells had several functional defects after heme inhibition. FECH loss changed the shape of mitochondria and depleted expression of genes maintaining mitochondrial dynamics. FECH blockade elevated oxidative stress and depolarized mitochondrial membrane potential. Heme depletion had negative effects on cellular metabolism, affecting oxidative phosphorylation and glycolysis. Mitochondrial complex IV of the electron transport chain (cytochrome c oxidase) was decreased in cultured human retinal endothelial cells and in murine retina ex vivo after FECH inhibition. Supplementation with heme partially rescued phenotypes of FECH blockade. Additionally, I discovered that partial loss-of-function Fech mutation in mice caused PPIX accumulation with no change in normal vasculature, as assessed by fundoscopy. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis. My studies identify a novel role of a heme synthesis enzyme in blood vessel formation and provide an opportunity to exploit these findings therapeutically for patients with PDR and wet AMD.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

In S. cerevisiae, the accumulation of dysfunctional mitochondria activates a retrograde signal that is mediated through multiple cytosolic regulators. Central to activation is the cytosolic regulator Rtg2p which through its interaction with Mks1p, promotes the nuclear translocation of Rtg1p/3p. Nuclear localized Rtg1p/3p promotes transcription of target genes. Prior work has shown Rtg2p interaction with Mks1p is required for downstream signaling, however the Mks1p binding site within Rtg2p is unknown. To identify this motif, random mutations were generated in RTG2 and a red-white screening strategy was used to assess 14,001 clones. Sequence analysis identified four mutants with amino acid mutations in the carboxy-terminus of Rtg2p that gave rise to defects in CIT2 transcription and loss of Mks1p interaction. Relative to RTG2, all mutants had reduced Rtg2p protein half-lives. Together these results suggest that the carboxy-terminal domain of Rtg2p is essential for retrograde signaling as it may contain the Mks1p binding site.

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No description available.