Embryos from the annual killifish Austrofundulus limnaeus have a unique and unequalled ability among vertebrates to withstand extended periods of anoxia (maximum lethal time to 50% mortality of 65 days at 25°C). In addition, tolerance of anoxia is gained and subsequently lost during the normal development of this species. Thus, anoxia tolerant and anoxia sensitive individuals can be compared within the same species, making A. limnaeus an excellent model for studying the molecular changes associated with survival of oxygen deprivation in vertebrates. The aim of this project is to analyze the molecular changes associated with anoxia tolerance in the embryos of A. limnaeus. Understanding how the cells of these embryos become tolerant to anoxia will aid in identifying novel therapeutic targets to reduce cell death following periods of ischemia in heart, brain or other tissues. Three major analyses were used to investigate the molecular changes associated with anoxia tolerance in this species. The first was a cell cycle and cell cycle arrest analysis using flow cytometry along with an immunoblot analysis of both positive and negative regulators of cell cycle progression. The second was a cell death analysis utilizing caspase-3/7 activity as well as TUNEL staining. The third was an immunoblot analysis of three different post-translational modifers (ubiquitin, SUMO-1 and SUMO-2/3). The overall findings from this study indicate that the embryos of A. limnaeus do indeed experience some degree of cellular stress (i.e. increase in ubiquitinated proteins, increase in p53 expression, evidence of DNA damage from TUNEL staining and increases in caspase activity) in response to anoxic treatment, even in their most protective state of diapause II. However, despite these observations, the whole organisms are still able to recover from anoxia and do not succumb to death. The overall low levels of TUNEL-positive cells and caspase activity relative to the positive controls indicates that the damage accrued in response to anoxic treatment is minimal. It appears that embryos are able to either "sacrifice" a certain portion of cells or they are able to repair the damage required for resumed development following anoxia.

Apoptosis is a regulated "programme" by which cells are induced to die in a manner which does not result in pathological inflammatory reactions, and involves dismantling of the cell into membrane-bound fragments that are removed by phagocytosis. This process is induced in order to remodel tissues and maintain homeostasis in cell numbers. Apoptosis may be induced via many pathways, many of which are redox-regulated, and is dysregulated in cancer cells, mainly due to mutational inactivation of certain pathways. Cancer cells also have a non-linear response to redox imbalance, a potentially exploitable characteristic for the therapeutic selective induction of apoptosis in cancer cells in mixed cell populations. Model cell culture systems are required for the selective toxicity testing of anti-cancer drugs, many of which work by inducing redox stress. In the current study, hydrogen peroxide was selected as the redox stress-inducing agent, and the test cells were an immortal, non-invasive breast epithelial cell line (MCFlOA) and its rastransfected, pre-malignant derivative (MCF10AneoT). A reliable, sensitive, cost effective and least time-consuming system for detection of apoptosis in such a system was sort and two novel methods, cytochrome c release and caspase-3 activity assays, were finally selected and compared with results seen by conventional DNA laddering and morphological examination at the light and electron microscopic level. No single procedure was found to be reliable individually. For the model system used, a combination of electron microscopy and DNA laddering was sufficient for simply detecting apoptotic cell death and necrosis. The caspase activity assay distinguished between apoptosis and necrosis, and cytochrome c release proved the most sensitive indicator of cell response. However, since cytochrome c release may be reversible and may not necessarily proceed to the downstream events of apoptosis in the time frame used in the current assays, it is not certain that cytochrome c release ultimately leads to apoptosis. However, three forms of cytochrome c were observed on western blots, the nature and significance of which remains to be determined. A comparison of the results of different methods allowed a model for the sequence of specific apoptotic events to be proposed. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Mammalian neurons exhibit extreme sensitivity to oxygen deprivation and undergo rapid and irreversible degeneration when oxygen supply is curtailed. Though several neuroprotective pathways are activated during oxygen deprivation, their analyses are masked by the complex series of pathological events which are triggered simultaneously. Such events can be analyzed in the anoxia tolerant fresh water turtle, which can inherently survive the conditions of oxygen deprivation and post-anoxic reoxygenation without brain damage. It is likely in such a model that modulation of a particular molecular pathway is adaptive rather than pathological. The major objective behind this study was to analyze the intracellular signaling pathways mediating the protective effects of adenosine, a potential neuromodulator, and its effect on cell survival by influencing the key prosurvival proteins that prevent apoptosis. In vivo and in vitro studies have shown that adenosine acts as a neuroprotective metabolite and its action can be duplicated or abrogated using specific agonist and antagonists. Stimulating the adenosine receptors using selective A1 receptor agonist N6-cyclopentyladenosine (CPA) activated the presumed prosurvival ERK and P13-K/AKT cascade promoting cell survival, and suppression of the receptor using the selective antagonist DPCPX (8- cyclopentyl-1,3-dipropylxanthine) activated the prodeath JNK and P38 pathways. The complex regulation of the MAPK's/AKT signaling cascades was also analyzed using their specific inhibitors. The inhibiton of the ERK and AKT pathway increased cell death, indicating a prosurvival role, whereas inhibiton of the JNK and p38 pathway increased cell survival in this model. In vitro studies have also shown a high Bcl-2/BAX ratio during anoxia and reoxygenation, indicating a strong resistance to cell death via apoptosis. / Silencing of the anti-apoptotic Bcl-2 gene using specific siRNA upregulated levels of prodeath BAX, thus altering the Bcl-2/BAX ratio and elevating cleaved Caspase-3 levels leading to increased cell death. Another promising neuroprotective target which we analyzed was Neuroglobin, which was induced during oxygen crisis and silencing this gene indicated that its plays a major role in modulation of ROS. This study strongly emphasizes the advantages of an alternate animal model in elucidating neuroprotective mechanisms and revealing novel therapeutic targets which could eventually help clinicians to design new stroke therapies based on naturally tolerant organisms. / by Gauri Nayak. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Cytotoxic chemotherapy is extremely important in adjuvant treatment of breast cancer. Yet, tumours frequently acquire chemoresistance that correlates with increased aggressiveness and poor prognosis. Three theories exist describing how the resistance develops: genetic, epigenetic and karyotypic theory. The epigenetic theory is the least explored. Here we analyzed the role of the epigenetic phenomena in the acquisition of drug resistance. To do so, we employed genome wide screens of microRNA and gene expression, DNA methylation and complete genome hybridization. We identified three novel microRNA interactions involved in the chemoresistant phenotype. These three microRNAs displayed depressed expression in the resistant cell lines and we were able to re-establish some level of drug sensitivity through ectopic expression of these under expressed microRNAs. In addition, we described the role of DNA methylation in impacting expression of a wide range of genes, thus, contributing to the phenotype of chemoresistance. Furthermore, we revealed a distorted global DNA methylation pattern that coincides with massive instability of the resistant genome. Finally, our results present a striking similarity between gene expression, epigenetic profiles and chromosomal aberrations in two different drug resistant cell lines. Taken together, this project suggests that the acquisition of chemoresistant phenotype is epigenetic in nature and may arise with a predictable pattern. Elucidating the specifics of this pattern may in the future prove useful in developing treatment and prognostic chemoresistance biomarkers. / xiii, 116 leaves : ill. (some col.) ; 29 cm

Indiana University-Purdue University Indianapolis (IUPUI) / Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We previously reported that chemotherapy-treated lymphoma patients acquire a deficiency of Signal Transducer and Activator of Transcription 4 (STAT4), which results in defective IFNy production during clinical immunotherapy. With the goal of further improvement in cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that exhibits immunostimulatory effects on natural killer cells (NKCs). Peripheral blood mononucleated cells (PBMCs) from healthy donors and chemotherapy-treated lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-cultuted NK cells demonstrated synergistic effects in the induction of IFNG and genes involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNy production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines have higher tumoricidal activity than those stimulated with cytokines alone using in vitro tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation at target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.

Indiana University-Purdue University Indianapolis (IUPUI) / With effective antiretroviral therapy (ART), cardiovascular diseases (CVD), are emerging as a major cause of morbidity and death in the aging population with HIV infection. Although this increase in CVD could be partially explained by the toxic effects of combined anti-retroviral therapy (ART), more recently, HIV infection has emerged as an independent risk factor for CVD. However, it is unclear how HIV can contribute to CVD in patients on ART, when viral titers are low or non-detectable. Here, we provide several lines of evidence that HIV-Nef, produced in infected cells even when virus production is halted by ART, can lead to endothelial activation and dysfunction, and thus may be involved in CVD. We demonstrate that HIV-infected T cell-induced endothelial cell activation requires direct contact as well as functional HIV-Nef. Nef protein from either HIV-infected or Nef-transfected T cells rapidly transfers to endothelial cells while inducing nanotube-like conduits connecting T cells to endothelial cells. This transfer or transfection of endothelial cells results in endothelial apoptosis, ROS generation and release of monocyte attractant protein-1 (MCP-1). A Nef SH3 binding site mutant abolishes Nef-induced apoptosis and ROS formation and reduces MCP-1 production in endothelial cells, suggesting that the Nef SH3 binding site is critical for Nef effects on endothelial cells. Nef induces apoptosis of endothelial cells through both NADPH oxidase- and ROS-dependent mechanisms, while Nef-induced MCP-1 production is NF-kB dependent. Importantly, Nef can be found in CD4 positive and bystander circulating blood cells in patients receiving virally suppressive ART, and in the endothelium of chimeric SIV-infected macaques. Together, these data indicate that Nef could exert pro-atherogenic effects on the endothelium even when HIV infection is controlled and that inhibition of Nef-associated pathways may be promising new therapeutic targets for reducing the risk for cardiovascular disease in the HIV-infected population.