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Discriminative eradication of cancer cells using quantum dots functionalised with peptide-directed delivery of a pro-apoptotic peptideSwartz, Lauren Taryn January 2013 (has links)
>Magister Scientiae - MSc / The therapeutic goal of cancer treatment is to trigger selective cell death in cancer cells. To eliminate cancerous cells effectively, the anti–cancer drugs must be targeted to the affected cells. However, anti–cancer drugs are often distributed non–specifically giving rise to systemic toxicities and other adverse effects. Cancer specific peptides are useful cancer targeting agents that can be used for the targeted delivery of anti-cancer drugs. Several cancer targeting peptides and some of their corresponding protein targets have been identified. Previous work investigated the specific binding of five of these peptides (p.C, p.H, p6.1, Frop-1 and p.L) conjugated to fluorescent nanoparticles (quantum dots) to a panel of human cell lines, which included four cancerous cell lines (Caco-2, HeLa, HT29 and HepG2) and one non-cancerous cell line (KMST-6). Flow cytometry showed that the p.L peptide preferentially bind to HT29 cells; suggesting that the expression levels of the target for the p.L peptide are higher in these cells. The objective of this study was to make use of target specific functionalised quantum dots (QDs) to deliver Second mitochondria-derived activator of caspases/ Direct AIP binding
protein with low PI (Smac/DIABLO) to HT29 cells with the aim of enhancing the effects of pro-apoptotic drugs. Smac/DIABLO is a pro-apoptotic peptide that is able to interact with inhibitor of apoptosis proteins (IAPs), thereby inducing pro-apoptotic signalling. Methodology: CdSe/ZnS core-shell QDs were synthesised using the one-pot synthesis method. These QDs were characterised using photoluminescence (PL) spectroscopy, high resolution transmission electron microscopy (HR-TEM) and energy dispersive x-ray spectroscopy (EDS). The CdSe/ZnS core-shell QDs were solubilised with L-cysteine (Cys- QDs). The Cys-QDs were bi-conjugated to the p.L peptide and Smac peptide using 1-ethyl-3-
(30-dimethylamino) carbodiimide (EDC) chemistry. Cultured HT29 cells were exposed to the 10 | P a g e QD peptide bi-conjugates and fluorescence microscopy was employed to assess targeting and internalisation. The cytotoxicity of the QD peptide bi-conjugates in combinatorial treatment with ceramide was evaluated using the WST-1 Cell Proliferation assay. A commercially available QD with similar chemistry was used to carry out a comparative study to relate the efficiency of the in-house synthesized QD.
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Analysis of Mitochondrial Signaling in the Regulation of Programmed Cell DeathHui, Kelvin Kai-Wan 31 August 2011 (has links)
The involvement of mitochondrial signaling in mammalian PCD regulation has been examined extensively via biochemical analyses and cellular studies in vitro. However there still exist considerable gaps in our knowledge regarding its contribution in specific tissues and cell types during mammalian development in vivo. In addition, given the numerous pathologic conditions associated with aberrant PCD, modulation of this signaling process represents an attractive target for therapeutic intervention. In this thesis I have therefore examined the regulation of mitochondrion-mediated PCD signaling as it pertains to several forms of developmental and injury-induced cell death.
In the first component of the thesis I have examined the differential sensitivity of Bcl2 on the survival of motor neuron populations from two distinct developmental origins (alpha and gamma motor neurons), demonstrating that gamma motor neurons are preferentially affected in Bcl2 null mice. Thus, Bcl-2 plays a critical in vivo in regulating subtype-specific motor neuron survival during development. In the second study I have demonstrated that a major portion of the neuroprotective effect exerted by the immunophilins cyclosporin A and FK-506 are mediated through calcineurin signaling; rather than MOMP-mediated events as previously held. Additional findings of this study demonstrated the first neuroprotective effects of the pyrethroid insecticide cypermethrin and calcineurin-mediated control of Bad phosphorylation. Such findings establish a link between calcineurin signaling and mitochondrion-mediated cell survival.
The above studies established critical features of mitochondrion-mediated PCD in regulating survival of several neuronal subpopulations. I therefore followed these studies with an examination of how post-mitochondrial PCD signaling is regulated following MOMP permeabilization. Specifically I examined regulation of the Smac-IAP-caspase axis, investigating how combinatorial deletion of Casp3 and Diablo alter PCD progression in mouse embryonic fibroblasts. Using a series of injury stimuli in the context of biochemical and cellular analyses I have developed a model of how endogenous Smac/DIABLO regulates executioner caspase activity. Collectively these studies elucidate key aspects of mitochondrial signaling during both developmental and injury-induced PCD in vivo.
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Analysis of Mitochondrial Signaling in the Regulation of Programmed Cell DeathHui, Kelvin Kai-Wan 31 August 2011 (has links)
The involvement of mitochondrial signaling in mammalian PCD regulation has been examined extensively via biochemical analyses and cellular studies in vitro. However there still exist considerable gaps in our knowledge regarding its contribution in specific tissues and cell types during mammalian development in vivo. In addition, given the numerous pathologic conditions associated with aberrant PCD, modulation of this signaling process represents an attractive target for therapeutic intervention. In this thesis I have therefore examined the regulation of mitochondrion-mediated PCD signaling as it pertains to several forms of developmental and injury-induced cell death.
In the first component of the thesis I have examined the differential sensitivity of Bcl2 on the survival of motor neuron populations from two distinct developmental origins (alpha and gamma motor neurons), demonstrating that gamma motor neurons are preferentially affected in Bcl2 null mice. Thus, Bcl-2 plays a critical in vivo in regulating subtype-specific motor neuron survival during development. In the second study I have demonstrated that a major portion of the neuroprotective effect exerted by the immunophilins cyclosporin A and FK-506 are mediated through calcineurin signaling; rather than MOMP-mediated events as previously held. Additional findings of this study demonstrated the first neuroprotective effects of the pyrethroid insecticide cypermethrin and calcineurin-mediated control of Bad phosphorylation. Such findings establish a link between calcineurin signaling and mitochondrion-mediated cell survival.
The above studies established critical features of mitochondrion-mediated PCD in regulating survival of several neuronal subpopulations. I therefore followed these studies with an examination of how post-mitochondrial PCD signaling is regulated following MOMP permeabilization. Specifically I examined regulation of the Smac-IAP-caspase axis, investigating how combinatorial deletion of Casp3 and Diablo alter PCD progression in mouse embryonic fibroblasts. Using a series of injury stimuli in the context of biochemical and cellular analyses I have developed a model of how endogenous Smac/DIABLO regulates executioner caspase activity. Collectively these studies elucidate key aspects of mitochondrial signaling during both developmental and injury-induced PCD in vivo.
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