Mitochondria are essential organelles that provide the cell with energy and are involved in many housekeeping processes. Maintaining a healthy population of mitochondria is vital for the proper functioning of cells and the presence of dysfunctional mitochondria may lead to cellular damage and cell death. Neurons are particularly susceptible to the consequences of mitochondrial damage as they have high energy needs and are post-mitotic. The clearance of damaged mitochondria by autophagy, or mitophagy, has emerged as an important quality control mechanism. The Parkinson's disease related proteins phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and Parkin have been identified as important regulators of mitophagy in mammalian cells, directly linking mitophagy to neurodegeneration. The role of these two proteins in this mitophagy is further explored in the first part of this dissertation. We propose a model whereby a cleavage product of PINK1 in the cytosol binds Parkin and prevents its translocation to mitochondria, which is regarded as the initiating step in Parkin/PINK1 mitophagy. Upon the occurrence of mitochondrial damage, however, full-length PINK1 accumulates on the mitochondrial outer membrane (MOM) and recruits Parkin, marking the damaged mitochondria for mitophagy. In the second part, we assess mitophagy in a cellular model based on disease caused by mutations in mitochondrial DNA (mtDNA). We find that the mere presence of damaged mitochondria in the cell does not activate mitophagy. Rather, this process is a complex interplay between mitochondrial membrane potential, levels of PINK1/Parkin and the activation of general macroautophagy. The final part of this dissertation describes the development and validation of a new method to study mitophagy. MitophaGFP, a red-green tandem fluorescent protein targeted to the MOM, changes color from yellow to red once mitochondria enter lysosomes, the final step of the mitophagy process. This new probe allows us to quantitatively and qualitatively assess mitophagy and fulfills a need in the mitophagy field. The work described in this dissertation contributes to elucidate the mechanisms underlying mitophagy regulation in mammalian cells. Its findings can serve as a basis to further explore the importance of mitophagy as a quality control mechanism and the role of its defect in neurodegeneration.

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Thesis (M.Sc.)--York University, 2008. Graduate Programme in Higher Education. / Typescript. Includes bibliographical references (leaves 91-96). 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:MR38784

Aging is a biological process that has many detrimental effects due to the accumulation of oxidative damage to key biomolecules due to the action of free radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each form has been shown to reduce a specific enantiomer of bound and free oxidized methionine. Effects of Msr have yet to be studied in the major developmental stages of Drosophila melanogaster despite the enzymes elevated expression during these stages. A developmental timeline was determined for MsrA mutant, MsrB mutant, and double null mutants against a wild type control. Results show that the Msr double mutant is delayed approximately 20 hours in the early/mid third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon is due to an issue gaining mass. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2013. / FAU Electronic Theses and Dissertations Collection

The mitochondrion is the powerhouse of the cell. Therefore, it is critical to the homeostasis of the cell that populations of mitochondria that are damaged or in excess are degraded. The process of targeted elimination of damaged or excess mitochondria by autophagy is called mitophagy. In this report, analysis of the mitophagy regulators PINK1/PARKIN and BNIP3L and their roles are assessed in the lens. PARKIN, an E3 ubiquitin ligase, has been shown to play a role in directing damaged mitochondria for degradation. While BNIP3L, an outer mitochondrial membrane protein, increases in expression in response to excess mitochondria and organelle degradation during cellular differentiation. We have shown that PARKIN is both induced and translocates from the cytoplasm to the mitochondria in human epithelial lens cells upon oxidative stress exposure. In addition, our findings also show that overexpression of BNIP3L causes premature clearance of mitochondria and other organelles, while loss of BNIP3L results in lack of clearance. Prior to this work, PARKIN mediated mitophagy had not been shown to act as a protective cellular response to oxidative stress in the lens. This project also resulted in the novel finding that BNIP3L-mediated mitophagy mechanisms are required for targeted organelle degradation in the lens. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Biological homeostasis relies on protective mechanisms that respond to cellular oxidation caused primarily by free radical reactions. Methionine sulfoxide reductases (Msr) are a class of enzymes that reverse oxidative damage to methionine in proteins. The focus of this study is on the relationship between Msr and dopamine levels in Drosophila. Dopaminergic neurons in Drosophila have comparable roles to those found in humans. A deficit in dopamine leads to the onset of many neurological disorders including the loss of fine motor control—a neurodegenerative condition characteristic of Parkinson’s disease (PD). We found that dopamine levels in the heads of MsrAΔ/ΔBΔ/Δ mutants are significantly reduced in comparison to MsrA ⁺/⁺ B⁺/⁺ heads. In addition, wefound protein and expression levels are markedly reduced in an Msr-deficient system. Our findings suggest an important role for the Msr system in the CNS. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

by Rebecca McGreal. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web. / The central premise of this dissertation is that the small heat shock protein (sHSP), (Sa(BB-crystallin is essential for lens and retinal pigmented epithelial (RPE) cell function and oxidative stress defense. To date, the mechanism by which it confers protection is not known. We hypothesize that these functions could occur through its ability to protect mitochondrial function in lens and RPE cells. To test this hypothesis, we examined the expression of (Sa(BB-crystallin/sHSP in lens and RPE cells, we observed its localization in the cells, we examined translocation to the mitochondria in these cells upon oxidative stress treatment, we determined its ability to form complexes with and protect cytochrome c (cyt c) against damage, and we observed its ability to preserve mitochondrial function under oxidative stress conditions in lens and RPE cells. In addition to these studies, we examined the effect of mutations of (Sa(BB-crystallin/sHSP on its cellular localization and translocation patterns under oxidative stress, its in vivo and in vitro chaperone activity, and its ability to protect cyt c against oxidation. Our data demonstrated that (Sa(BB-crystallin/sHSP is expressed at high levels in the mitochondria of lens and RPE cells and specifically translocates to the mitochondria under oxidative stress conditions. We demonstrate that (Sa(BB-crystallin/sHSP complexes with cyt c and protects it against oxidative inactivation. Finally, we demonstrate that (Sa(BB-crystallin/sHSP directly protects mitochondria against oxidative inactivation in lens and RPE cells. Since oxidative stress is a key component of lens cataract formation and age-related macular degeneration (AMD), these data provide a new paradigm for understanding the etiology of these diseases.

Conclusions. Our results showed that conventional in vitro FAO rate assay and acylcamitine profiling in cultured skin fibroblasts could be simplified and performed simultaneously by using the new combined method. Using the new derived parameter, TC, the new method can also reliably detect CUD. Together with molecular studies of ETFA, ETFB and ETFDH genes, the method could detect and confirm a diagnosis of MADD. No definitive case of FAOD was found in a cohort of sixteen SUD cases by the combined method. The new combined method has the potential to be applied as second-line laboratory test for detecting FAOD. (Abstract shortened by UMI.) / First-line laboratory investigation of patients with FAOD is based on some specialized biochemical tests in body fluids including blood/urine carnitine and acylcarnitines, urine organic acids and acylglycines. Further confirmatory tests involve functional assessments of the FAO pathway in cultured skin fibroblasts. The two conventional methods are tritium water release assay for determination of total FAO rate and acylcarnitine profiling for detection of individual FAOD. Based on the basic principles of these two methods, I hypothesized that the total FAO rate assay could be combined with the acylcarnitine profiling if an appropriate substrate and measurement technique were used. In this thesis, I described the development and validation of such assay. In view that the conventional methods cannot reliably detect CUD and MADD, I attempted to test a new derived parameter namely total even-chain acylcarnitines (TC) using the proposed new combined assay for detection of CUD. As mutation analysis of MADD in Chinese patients has not been carried out, a genomic approach was developed to confirm the diagnosis of MADD detected by the new combined assay. Also, the new assay was applied to detect FAOD in a cohort of sudden unexpected death (SUD) Chinese infants. / Mitochondrial fatty acid beta-oxidation (FAO) is a vital process for energy production in most tissues, particularly the skeletal muscle and heart. It also plays an important role to maintain sufficient energy supply through hepatic ketone body formation during fasting and stress. Most fatty acid oxidation disorders (FAOD) may lead to hypoketotic hypoglycaemia, hepatic dysfunction, encephalopathy, skeletal myopathy and cardiomyopathy. / Molecular studies of genomic DNA from five MADD patients in three unrelated Chinese families were performed. Four novel mutations in ETFDH gene were found in two families: one deletion (c.1355delG) and three missense (c.250G>A, c.409C>T and c.1400G>C) mutations. In another family, only one splice site (IVS2+1G>A) mutation in ETFA gene was identified. / The definitive diagnosis in nine CUD patients were made by a very low rates of carnitine uptake (<5% of control) as determined by the conventional radioactive carnitine uptake assay in cultured skin fibroblasts. When using the new combined FAO rate and acylcamitine profiling method, the calculated TC levels of these patients were all below 9.3 nmol/mg protein/96h and were well separated from the control group. At this cutoff value, ROC analysis gave a sensitivity of 100% and specificity of 87.5%, the AUC was 0.993, (p<0.001). Therefore, both conventional radioactive carnitine uptake assay and our new combined assay could be useful for the diagnosis of CUD. / Law, Lap Kay. / Advisers: Christopher Lam Wai Kei; Nelson Tang Leung Sang. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3420. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 177-202). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

[Truncated abstract] Mitochondria are semi-autonomous organelles found in almost all eukaryotic cells to contain more than 1000 different proteins. The majority of these proteins are encoded in the nucleus, translated in the cytosol and imported into mitochondria. The overall aim of this study was to characterise the regulation of nuclear-encoded mitochondrial proteins (NEMP). This was carried out in the plant, Arabidopsis thaliana, using the alternative oxidase (AOX) as a model. Specifically, the aims were to i) determine how regulation of NEMP interact with known regulatory pathways/mechanisms; ii) determine if the pattern of coexpression observed for NEMP are due to co-regulation, and iii) to determine whether mitochondrial retrograde regulatory pathways interact with known chloroplast regulatory pathways. AOX1c is one of five genes encoding AOX in Arabidopsis. It is expressed in a variety of organs and is not induced by stress. Thus, its regulation was characterised in order to gain insight into the regulation of NEMP under normal growth conditions. Analysis of the promoter of AOX1c revealed cis-acting regulatory elements (CAREs) common to both AOX1c from Arabidopsis and AOX2b from soybean. Additionally, Site II elements, previously shown to be involved in the regulation of the proliferating cell nuclear antigen, are present in the upstream promoter region of AtAOX1c and were shown to be strong negative regulators of AtAOX1c expression. AOX1a is a gene encoding AOX that is induced at a transcript level, by many stress treatments. BA signalling and provide evidence of at least one common factor between chloroplastic and mitochondrial retrograde regulatory pathways, i.e. ABI4. ... The above results reveal that the regulation of NEMP are integrated with the mainstream regulatory pathways that control gene expression for a variety of proteins in various locations. Although this is not unexpected, it does raise the question of how mitochondrial function impacts, or feeds back, to alter these pathways, i.e. how mitochondrial retrograde signals affects the regulation of genes encoding proteins in a variety of locations. The observed interaction of mitochondrial and plastid retrograde regulatory pathways at the level of ABI4, suggests that mitochondrial signals have the potential to act as a powerful regulators of many cellular functions. Although interaction between mitochondrial and other organelles at a cellular level has been known for some time, there is still much work left to be done to define the network of molecular interactions that exists to regulate and integrate the expression of NEMP with all other proteins in the cell. This study reveals that interactions also occur at regulatory steps that have to potential to regulate many function in organelles, even if no direct metabolic link exists. However, this study has only begun to uncover these interactions at a molecular level.

Thesis (Ph. D.)--University of Texas at Austin, 2009. / Title from PDF title page (University of Texas Digital Repository, viewed on Sept. 9, 2009). Vita. Includes bibliographical references.