Various agents such as heat, ultrasound, rhodamine derivatives and carbonylcyanide m-chlorophenylhydrazone are used to induce cellular damage so as to understand certain events leading to cell death using a mammalian cell culture system - HeLa S3 cells. Clonogenic assay is insensitive as it is an end-point technique for determining the effectiveness of cell-killing. A combined approach using protein synthesis, oxygen consumption, time-sequenced morphological study, flow cytometric analysis and others is used instead. Ultrasound is used also because it can produce heat especially when focussing beams are employed. However, with the present experimental set-up system, it is the formation of microbubbles (cavitations) together with microstreaming in a fluid medium and not heat, that produce the 'shattering' damage to the HeLa S3 cells in suspensions. Heating e.g. at 42oC for a brief period can produce 'thermoprotection' to other stimuli, in this case ultrasound. Mitochondria are found to be highly sensitive to heat especially at 45oC and the compounds used in this study. Multivesicular and myelinoid-multivesicular bodies are related to mitochondria. Lysosomes do not appear to play a critical role in the early events leading to cell death. It is suggested that by damaging mitochondria leading to an irreversible state of cellular 'energy crisis' could be the earliest event in cell death.

Call number: LD2668 .T4 1986 G37 / Master of Science / Genetics Interdepartmental Program

Oxidative phosphorylation couples ATP synthesis to respiratory electron transport. This coupling occurs in mitochondria, which carry DNA. Respiratory electron transport in the presence of molecular oxygen generates mutagenic reactive oxygen species (ROS) at a frequency that is itself increased by mutation. Damage to mitochondrial DNA (mtDNA) therefore accumulates within the lifespan of individual organisms. Syngamy requires motility of one gamete, and this motility requires ATP. It has been proposed that that oxidative phosphorylation is absent in the special case of quiescent, template mitochondria, and that these remain sequestered in oocytes and female germ lines. Oocyte mtDNA is thus protected from damage. Here I present evidence that female gametes, which are immotile, repress mitochondrial DNA transcription, mitochondrial membrane potential (!!m), and ROS production. In contrast, somatic cells and male gametes are seen actively to transcribe mitochondrial genes for respiratory electron carriers, and to produce ROS. I find that this functional division of labour between sperm and egg is widely distributed within the animal kingdom, and characterised by contrasting mitochondrial size and morphology. If quiescent oocyte mitochondria alone retain the capacity for an indefinite number of accurate replications of mtDNA, then "female" can be defined as that sex which transmits genetic template mitochondria. Template mitochondria then give rise to mitochondria that perform oxidative phosphorylation in somatic cells and in male gametes of each new generation. Template mitochondria also persist within the female germ line, to populate the oocytes of daughters. Thus mitochondria are maternally inherited.

by Li Yanchun. / "August 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 193-213). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Xenotransplantation of neonatal porcine islets have been demonstrated to be a viable alternative to exogenous insulin therapy for diabetes mellitus. The use of liberase has gained much success in islet isolation but factors such as batch-to-batch variation and deterioration of a batch with storage time have hampered the quality and reproducibility of tissue dissociation. Islet culture aims to optimise islet survival and insulin release in response to glucose challenge. However, it is difficult to recover and preserve islets in vitro. Mitochondria play a key role in the secretion of insulin from pancreatic islet cells in response to glucose stimulation. Mitochondrial dysfunction results in the induction (at mRNA and protein levels) of a molecular stress protein/heat chock protein called Cpn60. Since mitochondrial impairment will have a significant effect on the ability of in vitro cultured islet cells to function properly (i.e. release insulin in response to glucose stimulation), the expression of Cpn60 was investigated as a function of exposing neonatal porcine islet cells to various growth conditions. The best choice of media to culture neonatal porcine islet cells was found to be not heated activated serum which showed the least levels of Cpn60 expression at mRNA levels suggesting that the cells had low levels of mitochondrial stress. Neonatal porcine islet cells would be best digested in cells digested with new liberase (QC 1050) while in 2% not heat inactivated porcine serum (NPS) as this gave the lowest levels of Cpn60 expression suggesting low levels of mitochondrial stress. Although expression of Cpn60 at mRNA levels seems to be modulated during the growth of the porcine islet cells in media supplemented with different serum, heat treatment of serum and liberase content, no firm conclusion can be made with regard to the effect of the different treatments on mitochondrial health status until the porcine Cpn60 protein can be unequivocally identified.

The production of reactive oxygen species by mitochondria is implicated in mitochondrial dysfunction associated with a range of diseases and ageing. In addition, reactive oxygen species produced by mitochondria are involved in redox signalling pathways that modulate a number of cell processes. Mitochondria targeted antioxidants comprised of an antioxidant moiety linked to a lipophilic triphenylphosphonium cation have recently been used to decrease oxidative damage to mitochondria and to investigate the involvement of mitochondrial reactive oxygen species in redox signalling. These lipophilic cations are selectively accumulated by mitochondria within cells due to the mitochondria membrane potential. This thesis presents the synthesis and characterization of mitochondria targeted membrane uncoupler, cyclic nitroxide and alkyl thionitrite derivatives, all of which had the potential to influence reactive oxygen species. The biological analysis of these compounds is also presented. A triphenylphosphonium derivative of the membrane uncoupler 2,4-dinitrophenol (DNP) was anticipated to act as a self regulating protonophore. The DNP moiety would influence the scale of the membrane potential while the triphenylphosphonium cation would respond to the membrane potential. These two factors would combine so that as the membrane potential was dissipated by the uncoupler, the phosphonium cation would be released from the mitochondria and the effect of the uncoupler would thereby be nullified until the membrane potential had increased again. The compound was prepared by nitration of 3-(4-hydroxyphenyl)propyl triphenylphosphonium bromide. An untargeted derivative was also prepared by nitration of 3-(4-hydroxyphenyl)-1-propanol. Unfortunately, while this compound had appropriate acidity and lipophilicity to act as a membrane uncoupler, and did enter mitochondria in response to the membrane potential, it did not act as an uncoupler. A chemically stable targeted cyclic nitroxide based on Tempol was prepared following literature procedure, although other synthetic routes were also trialled. This compound was shown to concentrate in mitochondria in response to the membrane potential, was reduced by ubiquinol of the coenzyme Q pool, acted as a superoxide dismutase mimetic, and protected membranes against lipid peroxidation. A mitochondria targeted thionitrite or nitric oxide (NO) donor was anticipated to exhibit an effect on respiration at low oxygen concentrations as the released NO interacted with aspects of the respiratory chain. The alkyl thionitrites were synthesised from appropriate thiol precursors, several of which were prepared. Two targeted alkyl thionitrites were prepared with primary or tertiary carbon arrays next to the thionitrite functionality. Another targeted thionitrite, based on S-nitroso-N-acetylpenicillamine (SNAP), was also prepared. These compounds were difficult to characterise because of issues surrounding their stability. However, modified high resolution positive ion electrospray mass spectrometry in combination with HPLC and NMR was used to identify the compounds and to gauge the purity of the samples. Initial biological investigations verified that the primary alkylthionitrite derivative accumulated in mitochondria, released NO, and had an effect on respiration at low oxygen concentrations.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2007. / Includes bibliographical references (p. 129-140).

Sperm mobility denotes the net movement of a sperm population. Previous work implicated mitochondrial function as a basis underlying phenotypic variation in this quantitative trait. Our objective was to determine if mitochondrial function was indeed critical to expression of phenotype. Phenotype was assigned to roosters within a random bred population (n=242). A representative subpopulation (n=40) was used to correlate sperm mobility with oxygen consumption (r=0.83). In contrast, sperm mobility was independent of mitochondrial helix length in a sample of males (n=7) representing the range of phenotype observed within the population. Thus, mitochondrial function rather than number appeared to be critical to expression of phenotype. This hypothesis was tested by ultrastructural analysis of sperm midpieces. Males from the lower and upper tails of the distribution were characterized with high and low proportions of sperm containing aberrant mitochondria in 47 and 4% of the cells respectively. When sperm from average males were allowed to segregate into immobile and mobile subpopulations, 40% of immobile sperm contained aberrant mitochondria. In contrast, only 9% of sperm from the same males contained aberrant mitochondria in non-segregated populations. In conclusion, the mitochoridrion is an organelle that may account for phenotypic differences in sperm mobility. / Graduation date: 2002

Graduation date: 1994

Mitochondria have evolved to play a vital role in both the life and death of a eukaryotic cell, through involvement in numerous cellular functions, such as the proficient production of energy from ATP biosynthesis and the regulation of programmed cell death. As a result, dysfunction in the biochemical processes housed within this organelle is implicated in diverse diseases, including cancer, diabetes, and neurodegenerative disorders. Advancing mitochondrial medicine by probing the subcellular biochemistry or targeting therapeutics into this organelle has motivated the development of effective mitochondrial delivery vectors. Thus, the rational design of novel mitochondrial-specific molecules, inspired by the success of cell-penetrating peptides, is described, whereby short synthetic peptides that retain the ability to traverse the plasma membrane, yet with mitochondrial-specificity were engineered. By modulating the overall physicochemical properties, through substitutions with both natural and synthetic amino acids, and monitoring the intracellular localization by confocal fluorescence microscopy, the requisite thresholds for achieving mitochondrial accumulation with a cationic peptide were elucidated. These systematic studies led to the development of a novel class of cationic yet lipophilic peptides, referred to as mitochondria-penetrating peptides (MPPs), which are readily cell permeable and preferentially localize into the mitochondria of living mammalian cells. The mechanisms of cellular uptake and mitochondrial matrix accumulation were investigated and the results from these studies suggest that MPPs utilize the negative membrane potential across these biological membranes to drive translocation. In addition, the effects of various chemical perturbations on the cellular and mitochondrial uptake, such as sequence, structure of the cation moiety, and chirality, were examined. The information obtained from these studies provided insight into the important features of these peptides and led to the design of an optimized molecule displaying pyridinium salt side chains. Moreover, MPPs were shown to be effective mitochondrial delivery vectors for diverse and bioactive small molecule cargo. In conclusion, the extensive biological and chemical characterization of MPPs revealed the importance of balancing the opposing characteristics of positive charge and lipophilicity to attain preferential sequestration into mitochondria, as well as provided evidence that these peptides will be suitable as mitochondrial delivery vectors.