Mitochondrial biochemistry and its role in apoptosis

Simmons, T.D., 2004. Mitochondrial biochemistry and its role in apoptosis. PhD, Nottingham Trent University.

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Apoptosis is a form of programmed cell death that occurs throughout the lifespan of all living creatures. It is morphologically distinct from that of necrosis where one of the main distinguishing features between these two types of death is the maintenance of the plasma cell membrane during apoptosis and the avoidance of an inflammatory response. Many human disorders occur when the regulation of apoptosis fails and tissue homeostasis is lost.

Markers of apoptosis and necrosis include externalisation of phosphatidyl serine from the inner to the outer plasma cell membrane, loss of mitochondrial transmembrane potential, release of apoptogenic factors into the cytosol such as cytochrome c, DNA fragmentation and loss of plasma membrane integrity. These indicators of apoptosis and necrosis can be measured using flow cytometry or fluorescence microscopy. These methods are usually quite subjective and can involve lengthy procedures for their measurement.

This thesis describes the use of bioluminescent technology to measure the levels of ATP and ADP within samples of cells to assess cell viability, early and late apoptosis and necrosis. The bioluminescent ATP kit ViaLight HS was able to measure a 96 well white plate in 5 minutes showing sensitivity and reliability over the ATP standard range and also down to 50 cells per well giving values of 0.999 and 0.996 respectively. The ATP/ADP detection assay ApoGlow was found to correlate well with the traditional methods of measuring both apoptosis and necrosis. Bioluminescent technology was able to distinguish between apoptosis and necrosis when ApoGlow was used in conjunction with the ToxiLight assay, which measures the loss of the plasma membrane and thus dead cells with the release of adenylate kinase (AK) from the cell.

The ApoGlow assay would lend itself particularly well for use as a screening assay during testing of potentially cytotoxic drugs. Those drugs showing a response could then be investigated in more depth with other assays to confirm apoptosis and necrosis.

Item Type: Thesis
Creators: Simmons, T.D.
Date: 2004
ISBN: 9781369312782
Divisions: Schools > School of Science and Technology
Record created by: Linda Sullivan
Date Added: 28 Aug 2020 08:45
Last Modified: 08 Jun 2023 09:00

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