Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/01/15). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on June 19, 2009) Includes bibliographical references.

Thesis (Ph.D.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Hanjoong Jo; Committee Member: Ajit Yoganathan; Committee Member: Dean P. Jones; Committee Member: Kathy K. Griendling; Committee Member: W. Robert Taylor

Thesis (Ph. D.)--Florida State University, 2005. / Advisor: W. Ross Ellington, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Jan. 26, 2006). Document formatted into pages; contains x, 82 pages. Includes bibliographical references.

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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.

The acquisition of beta cell identity and function is a multistage process that involves the sequential regulation of specific factors and signals. The maintenance of beta cell identity and function is a process of comparable importance that requires persistent and continuous regulation. Loss of beta cell identity and/or reprogramming represents an important feature of beta cell dysfunction in genetic models of diabetes, as well as in patients with type 1 and type 2 diabetes. The factors and mechanisms involved in the acquisition and maintenance of beta cell identity are still not well understood. Nevertheless, several beta cell developmental transcription factors have been found to be important in the maintenance of its functional identity during the postnatal stage. Nkx2.2 is a transcription factor that is critical for the development and differentiation of beta cells both in mice and humans. In adults, Nkx2.2 is expressed in the entire beta cell population. However, due to the perinatal lethality of the Nkx2.2 null mice, the study of its function in adult beta cells has remained elusive. For my dissertation work, I explored the function and mechanism of action of Nkx2.2 in the adult beta cell. I deleted Nkx2.2 specifically in beta cells during their maturation and in adults. Deletion of Nkx2.2 in beta cells caused rapid onset of diabetes due to the loss of insulin and the down-regulation of many beta cell functional genes. Concomitantly, Nkx2.2-deficient beta cells acquired non-beta cell endocrine features, resulting in populations of completely reprogrammed cells and bi-hormonal cells that have hybrid endocrine cell morphological characteristics. Molecular analysis in mouse and human islets revealed that Nkx2.2 is a conserved master regulatory protein that controls the acquisition and maintenance of a functional monohormonal beta cell identity by directly activating critical beta cell genes, and actively repressing genes that specify the alternative islet endocrine cell lineages. This study demonstrates the highly volatile nature of the beta cell; it is necessary to actively maintain expression of genes involved in beta cell function, but to also maintain repression of closely related endocrine gene programs. These findings have potential applications that include the optimization of iPS cell differentiation protocols that aim to differentiate functional beta cells that remain safely locked into that identity state; as well as in future therapies that attempt to restore beta cells into a functional state.

Chan, Chi Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 103-127). / Abstracts in English and Chinese. / Thesis Assessment Committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / 摘要 --- p.iv / Abstract --- p.vi / List of Abbreviations --- p.viii / List of Figures --- p.xi / List of Tables --- p.xiii / Table of Content --- p.xiv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Classification of the aldehyde dehydrogenase superfamily --- p.1 / Chapter 1.2 --- Structures and catalytic mechanism of ALDH --- p.4 / Chapter 1.3 --- Multiple functions of ALDH --- p.8 / Chapter 1.4 --- Antiquitin - background and recent discoveries --- p.12 / Chapter 1.5 --- Aim of study --- p.19 / Chapter Chapter 2 --- Mitochondrial and Cytosolic Localizations of ALDH7A1 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Cell culture --- p.26 / Chapter 2.2.2 --- Subcellular fractionation --- p.26 / Chapter 2.2.3 --- Western blot analysis --- p.27 / Chapter 2.2.4 --- Flow cytometric analysis of mitochondria in WRL68 cells --- p.28 / Chapter 2.2.5 --- Transient transfection of various EGFP constructs --- p.29 / Chapter 2.2.6 --- Immunofluorescence staining --- p.31 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Presence of ALDH7A1 in cytosol and mitochondria in WRL68 cells --- p.33 / Chapter 2.3.2 --- Mitochondrial-targeting N-terminal sequence in ALDH7A1 --- p.34 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- In silico and in vitro subcellular localization studies on ALDH7A1 --- p.40 / Chapter 2.4.2 --- Significance of mitochondrial and cytosolic localizations of ALDH7A1 --- p.45 / Chapter 2.4.3 --- Comparison of animal ALDH7A and plant ALDH7B enzymes --- p.48 / Chapter Chapter 3 --- "ALDH7A1: A Potential Regulator for Cell Growth, Cell Cycle and a Potential Biomarker for Cancer (Stem) Cells" / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.55 / Chapter 3.2.1 --- Cell synchronization --- p.55 / Chapter 3.2.2 --- Semi-quantitative determination of DNA amount in synchronized cells --- p.55 / Chapter 3.2.3 --- Total protein extraction --- p.55 / Chapter 3.2.4 --- Western blot analysis --- p.57 / Chapter 3.2.5 --- Immunofluorescence staining --- p.57 / Chapter 3.2.6 --- Expression and purification of ALDH7A1 and its mutant --- p.57 / Chapter 3.2.7 --- Kinetic analysis of ALDH7A1 and its mutant --- p.58 / Chapter 3.2.8 --- Generation of native ALDH7 A1 and mutant for transfection --- p.58 / Chapter 3.2.9 --- Generation of stable cell line transfectants --- p.59 / Chapter 3.2.10 --- 2D cell culture and ultra-low attachment cell culture --- p.59 / Chapter 3.2.11 --- Collection of total cell lysates --- p.60 / Chapter 3.2.12 --- Western blot analysis --- p.60 / Chapter 3.2.13 --- Growth analysis --- p.61 / Chapter 3.2.14 --- Aldefluor assay --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Expression level of ALDH7A1 at different phases of the cell cycle --- p.62 / Chapter 3.3.2 --- Subcellular distribution of ALDH7A1 in synchronized cells --- p.64 / Chapter 3.3.3 --- Changes in the expression level of key cell cycle regulators and the growth rate after ALDH7A1 knockdown --- p.68 / Chapter 3.3.4 --- Absence of catalytic activity in the purified ALDH7A1 mutant C302S --- p.68 / Chapter 3.3.5 --- Over-expression of ALDH7A1 variants in HEK293 cells --- p.73 / Chapter 3.3.6 --- Growth rates of cells overexpressing different ALDH7A1 variants --- p.73 / Chapter 3.3.7 --- Expression level of ALDH7A1 in various 2D cell types and stem-like cells --- p.76 / Chapter 3.3.8 --- Aldefluor assay on cells over-expressing different ALDH7A1 variants --- p.79 / Chapter 3.4 --- Discussion --- p.82 / Chapter 3.4.1 --- Nuclear localization of ALDH7A1 --- p.82 / Chapter 3.4.2 --- Potential role of ALDH7A1 in cell cycle --- p.86 / Chapter 3.4.3 --- Non-catalytic role of ALDH in cell growth and development --- p.86 / Chapter 3.4.4 --- Relationship between ultra-low attachment culture and stem-like cells --- p.89 / Chapter 3.4.5 --- Up-regulation of ALDHs in cancer and CSCs and the evaluation of applicability of Aldefluor assay in CSC isolation --- p.93 / Chapter 3.4.6 --- Comparison on ALDH7A1 expression level in primary and stem-like cells --- p.98 / Chapter Chapter 4 --- Future Prospects / References --- p.103

Fusaric acid (FA) (5-butylpicolinic acid) is a divalent ion chelating agent that has low affinity for Ca2+ and Mg2+ and a high affinity for other essential metal ions such as Fe2+ Mn2+, Zn2+ and Cu2+. Its mode of action therefore may involve its interference with various transition metal ions and thus may be analogous to picolinic acid. Fusaric acid inhibits the proliferation of numerous cell lines in vitro. In the current in vitro study the effects of FA on peripheral blood lymphocytes was studied. Lymphocytes from a healthy volunteer were treated with varying concentrations of FA (3uM, 6uM, 25uM, 50uM 100uM 200uM, 400uM, and 1000uM) to assess the toxins apoptotic inducing potential. The 'Comet Assay' (Single cell gel electrophoresis), DNA fragmentation and Annexin V flous assays were employed to assess apoptosis. These assays proved that FA induces apoptosis in human lymphocytes. Lymphocytes were also incubated with phytohaemagglutinin (PHA) (10ug/ml) and increasing doses of FA (10, 50, 100 and 200uM). After 24, 48 and 72 hours of incubation an aliquot of the cells was stained with propidium iodide and subjected to flow cytometric analysis to assess the DNA configuration. Phytohaemagglutinin stimulation led to a significant increase of the S-phase of the cell cycle after 48 and 72 hours of incubation. All the PHA induced effects were reduced by co-incubation with increasing doses of FA. Lymphocytes were inhibited in the S-phase at 100 and 200uM concentration of FA. The current study shows that the in vitro inhibitory effects of FA can be demonstrated using flow cytometric technology on a cellular level. Fusaric acid leads to an inhibition of cell cycle progression in peripheral blood lymphocytes. / Thesis (M.Med.)-University of Natal, 2003.