Thesis (Ph. D.)--University of Virginia, 2001. / Spine title: Cell death in olfactory development. Includes bibliographical references (leaves 124-151). Also available online through Digital Dissertations.

1. The present study examined whether primary cultured rat A2B5+ cerebrocortical oligodendrocyte progenitor cells (OPC) were susceptible to Zn2+- and excitotoxin-induced cell death. 2. Initial pharmacological studies demonstrated the selective ionotropic glutamate receptor agonists' kainate and (S)-5-Iodowillardiine induced OPC death after 24-hour exposure. (S)-AMPA and L-glutamate only induced cell death in the presence of 100muM cyclothiazide (a selective AMPA receptor desensitisation blocker). The selective AMPA- receptor antagonists, GYKI 52466 and Evans' Blue, attenuated 300muM kainate-induced toxicity and therefore suggested OPC excitotoxic insult was via AMPA receptor activation. 3. Metabotropic glutamate receptor (mGluR) involvement was also established as (S)-DBPG (100muM), a selective group I mGluR agonist, afforded significant (p < 0.05) protection against 300muM kainate-induced toxicity. The selective mGIuR antagonist (S)-MCPG reversed the effects of (S)-DHPG protection. 4. OPC death was partially prevented by the broad-spectrum caspase inhibitor Z-VAD-fmk (100muM) at 6-hour and 24-hour paradigms. Hoechst 33342 staining revealed the presence of pyknotic nuclei following 6-hour kainate (300muM) exposure and Western Blotting using anti-caspase-3 antibody and anti-a-fodrin antibody indicated potential activation of the apoptotic executioner caspase-3. 300muM Kainate-induced OPC death did not appear to result in the activation of reactive oxygen species (ROS). 5. Zn2+ exposure over 24-hours resulted in OPC death (pECso 4.1+0.1). 100muM Zn2+- induced OPC death was not potentiated by 300muM kainate and Evans Blue afforded no protection. Nicardipine also failed to influence OPC viability. The lack of effect of kainate and nicardipine was confirmed by 65Zn2+ uptake studies. 6. 100muM and 300muM Zn2+-induced OPC death did not appear to result in activation of ROS. Hoechst 33342 staining revealed the presence of chromatin condensation with 300muM Zn2 (6-hour exposure). 100|muM and 300muM Zn2+ (24-hour exposure) was not influenced by Z-VAD-fmk or PD 150606. 7. Zn2+-induced OPC toxicity resulted in significant ATP depletion 6-hours following 300muM Zn2+ exposure (p < 0.05) and was attenuated in the presence of 5mM pyruvate. These data therefore suggest the mechanisms of Zn2+ toxicity may involve disruption of the glycolytic cascade.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2002. / Restricted until May 2005. Bibliography: leaves162-192.

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Many genes are activated to influence the self-destruction programme of the cell. This programme entails synchronised instigation and implementation of numerous subprograms. The arrival of gene targeting aided in the determining of the functions of novel genes. Such genes may have been sequenced, but not functionally characterised. The fulfillment of this requirement through gene targeting technology has swiftly developed. The mode by which DWNN operate in organisms in which it is thought to be covalently linked to some other proteins, which have a definite role in apoptosis, is not yet unraveled. This study attempted the functional characterisation of DWNN in light to the hypothesis that it may be involved in Cytotoxic T lymphocyte killing and apoptosis.

A dissertation submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science. Gauteng, Johannesburg, 2013. / Retinoblastoma Binding Protein 6 (RBBP6) is a multi-domain protein that uses its ring finger domain to interact with p53 and pRb tumour suppressor genes. The mechanism by which RBBP6 uses to degrade p53 is still unknown. Nonetheless, it is well known that RBBP6 promotes cell proliferation in several cancers by negatively regulating p53 via its E3 ubiquitin ligase activity (Ntwasa, 2008). Degradation of p53 by RBBP6 may compromise p53-mediated apoptosis in breast cancer. This study is intended to investigate the potential applications of RNA interference (RNAi) to block RBBP6 expression as well as its subsequent effect on cell growth and apoptosis. To achieve these methodologies, the following techniques were used: RT-PCR, western blotting, xCELLigence system and flow cytometry. Our studies indicate that the knockdown of RBBP6 expression by siRNA modulates p53 gene involved in cell death pathways and apoptosis, showing statistically significant gene expression differences. RBBP6siRNA significantly reduced cell index (CI) compared to the control samples and we observed an inhibition of cellular proliferation in the interval of between 24 and 48 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. These results were further confirmed by flow cytometry which showed some apoptotic activity. About 20.7% increase in apoptosis was observed in cells co-treated with RBBP6 siRNA and camptothecin when compared to camptothecin-only whereas in siRBBP6 and Staurosporine treated there was only 8.8% increase in apoptosis. These findings suggest that silencing RBBP6 may be a novel strategy to promote staurosporine- and camptothecin-induced apoptosis in breast cancer cells. Keywords: Retinoblastoma Binding Protein 6, staurosporine, camptothecin

During open orthopaedic surgical procedures, the articular cartilage covering the exposed joint surfaces can be exposed to air for prolonged periods. This exposure facilitates cartilage drying, characterised by changes to the extracellular matrix and chondrocyte death. Due to cartilage’s limited capacity for regeneration, it has been proposed that this may lead to post-operative joint degeneration. It has been proposed that chondrocyte death occurs as a result of both drying and nutritional deficiency. It is known that death during drying correlates with the drying interval and is initiated in the superficial chondrocytes, progressing to deeper layers at higher intervals. Additionally, it is well established that periodic rewetting (e.g. using 0.9% saline solution) can reduce chondrocyte death. The work presented in this thesis aimed to characterise chondrocyte death during drying, with particular reference to the chondrotoxic/protective effect of environmental variables (airflow) and surgical interventions (irrigation solutions), mechanism of injury and cell death, and the effect of in vivo drying on joint health. An ex vivo model of cartilage drying was developed and carried out on bovine and human intact cartilage and osteochondral explants, while varying environmental factors (drying interval, airflow velocity, oxygen concentration) and interventions (irrigation solutions and protective coverings. Throughout the study, cartilage drying was assessed in terms of 1) cartilage macroscopic appearance, 2) percent chondrocyte death (PCD), 3) cartilage water content, and 4) chondrocyte morphology. Histologically and fluorescently labelled samples were imaged using light and confocal laser scanning microscopy respectively, which formed the basis of the qualitative and quantitative assessments. Experimental drying at high airflow velocities had a more severe effect on cartilage appearance, PCD, and water content than in static air. This relationship was apparent in dried intact joints and osteochondral explants and in bovine and human samples. This suggests that the effects of surgical drying (where ventilation systems and airflow are routine) may be more pronounced than previously suggested and demonstrates a correlation between PCD and water-loss. Irrigation solutions supplemented with glucose (25-100 mM) had no significant effect on the PCD or water content in dried samples. Additionally, PCD was minimal in osteochondral explants cultured in the absence of glucose, even after 24 hr. This suggests that nutritional deficiency is unlikely to contribute to PCD during drying. However, chondrocyte death (in intact bovine cartilage) was reduced when drying was carried out at an oxygen concentration more reflective of the in vivo environment (5 %), which suggests that cell death during drying may be facilitated by a hyperoxic shock. Finally, in vivo cartilage drying was carried out on murine cartilage. Compared to sham operated controls, dried cartilage demonstrated a loss of surface integrity (4 weeks post-surgery) and fibrillations (8 weeks) and an increased modified Mankin score (at 4 and 8 weeks). Microscopically, an altered cartilage thickness, and chondrocyte density and arrangement were visible. These changes are comparable with changes in osteoarthritis. The results of this study demonstrate the importance of maintained cartilage hydration in order to avoid unnecessary chondrocyte death articular cartilage degeneration.

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