No description available.

The normal physiological response to bacterial infection or wounding with threat of infection, termed inflammation, has been shown to be dysregulated in certain human diseases including (but not limited to): idiopathic pulmonary fibrosis, acute lung injury, arthritis and glomerulonephritis. The earliest arriving and most abundant cell responding to an inflammatory stimulus is the neutrophil granulocyte. It has been shown that under inflammatory conditions neutrophil granulocytes have extended longevity, enhanced responsiveness and upregulated activation parameters. In the setting of non-infective, or prolonged, ineffectuallycleared infective disease where resolution of inflammation does not occur then neutrophil granulocytes may cause tissue damage which is mediated by excessive, misdirected exocytosis of toxic granule contents or by spillage of the same products from necrotic or netotic cell carcasses that have lost membrane integrity. A key process in the resolution of inflammation is the induction of apoptosis in recruited neutrophils following a successful response to an inflammatory stimulus. Cellular signalling from apoptotic cells and from professional phagocytes that have ingested apoptotic cells has been shown to favour resolution of inflammation and restoration of tissue homeostasis. Additionally, the removal of key inflammatory cells in a highly regulated, non-phlogistic fashion robustly assists the resolution process. Cyclin-dependent kinase (CDK) inhibitor drugs are being developed as anti-cancer agents as it is hypothesized that they should interfere with the enhanced cellcycling ability (increased proliferative capacity and extended longevity) which is such a key feature of cancer cell biology. The CDKs that drive the cell cycle are CDKs 1, 2, 4 and 6 and consequently agents were designed to have enhanced specificity for these targets. CDK inhibitor drugs target the ATP-binding domain of CDKs and as a result usually have activity against more than one CDK. The CDK inhibitor drug, R-roscovitine which targets CDKs 2, 5, 7 and 9 was shown to promote neutrophil apoptosis and consequently resolution of inflammation. This thesis aims to investigate the mechanism by which apoptosis is induced in neutrophil granulocytes by CDK inhibitor drugs. The first experimental chapter of this thesis explores in detail the time-course and active concentration range of CDK inhibitor drugs in comparison to known promoters and inhibitors of neutrophil apoptosis. It then dissects the apoptotic machinery which is responsible for the effects of CDK inhibitor drugs before investigating their capacity to promote apoptosis even in the presence of survival mediators relevant to the context of inflammatory disease. Flow-cytometry, light and confocal microscopy as well as western blotting for caspases, mitochondrial dissipation assay, fluorometric caspase assay and the detection of DNA laddering demonstrate that CDK inhibitor drugs promote classical neutrophil apoptosis by the intrinsic pathway and show similar kinetics of apoptosis induction to drugs that inhibit transcription. The second experimental chapter investigates the key neutrophil survival protein and bcl-2 homologue Mcl-1. By flow cytometry, western blotting and RT-PCR it is demonstrated that Mcl-1 is down-regulated at the level of transcription and that this occurs even in the presence of inflammatory mediators that would normally promote neutrophil survival. Additionally, it is shown that pro-apoptotic bcl-2 homologues are affected to a lesser degree suggesting an imbalance of bcl-2 proteins is caused by effects at a transcriptional level mediated by CDK inhibitor drugs. The third experimental chapter identifies CDKs and their binding partner cyclins in neutrophil granulocytes and investigates the impact of CDK inhibitor drugs on CDK protein levels and cellular distribution by differential lysis and western blotting as well as by confocal microscopy. The key transcriptional enzyme RNA polymerase II is also identified and the effect of CDK inhibitor drugs on phosphorylation of this enzyme is documented. Western blotting and confocal microscopy demonstrate the presence of key CDKs 2, 5, 7, 9 and cyclin binding partners of CDKs 7 and 9. It is shown that the phosphorylation of RNA polymerase II mediated by CDKs 7 and 9 is inhibited by CDK inhibitor drugs. This suggests that a key mechanism by which neutrophil apoptosis is induced by CDK inhibitor drugs is the inhibition of transcription of key proteins and suggests that neutrophils require survival proteins for functional longevity. The fourth experimental chapter addresses the production and use of HIV-tat dominant negative CDK 7 and 9 proteins to knockdown CDKs 7 and 9 in neutrophil granulocytes in vitro to provide a molecular biology surrogate for the pharmacological data already presented. The cloning, production, purification and use of HIV-tat dominant negative CDK proteins are described. The final chapter describes the use of a more specific pharmacological inhibitor of CDKs 7 and 9, DRB, in the mouse bleomycin lung injury model. Resolution of inflammation by a compound specifically targeting CDKs 7 and 9 is described. This thesis identifies CDKs 7 and 9 as key targets of CDK inhibitor drugs in neutrophilic inflammation. It shows these drugs acting at the level of transcription to drive neutrophil apoptosis by exploiting the unique dependency of neutrophils on the short-lived survival protein Mcl-1. In so doing the presence of functional and essential transcriptional machinery is identified in neutrophils and the transcriptional profile of resting, stimulated and inhibited neutrophils is delineated. These findings suggest novel approaches to the pharmacological promotion of resolution of inflammation and indicate key new targets for rational drug design. In future, it will be important to further characterize the effects of CDK inhibitor drugs on other cell-types including epithelial cells, fibroblasts and mononuclear cells. This information should prove important to the continued investigation of CDK inhibitor drugs in resolution of inflammation and also to the ongoing experimental trial of these drugs in idiopathic pulmonary fibrosis.

Master of Science / Division of Biology / Rollie J. Clem / Caspases are a conserved family of cysteine proteases that play important roles in apoptosis and innate immunity as well as other cellular processes. Eleven caspase genes have been annotated in the mosquito Aedes aegypti. Amongst these, previous studies have demonstrated functional roles for AeDronc, CASPS7 and CASPS8 in the Ae. aegypti apoptosis pathway, while CASPS18 and CASPS19 have also been functionally characterized. A previous study from our research group showed that AeIAP1 has preferential binding for CASPS7 compared to CASPS8. In this study, it was confirmed that AeIAP1 has a higher capacity to inhibit CASPS7 than CASPS8. Furthermore, five of the remaining Ae. aegypti caspases, namely CASPS15, CASPS16, CASPS17, CASPS20 and CASPS21, were characterized. An attempt was made to classify these caspases as initiator or effector caspases, based on factors such as the length of their prodomain, sequence similarity to known Drosophila initiator and effector caspases, and their substrate specificity. The functions of these caspases in apoptosis was examined in the Ae. aegypti cell line Aag2, by using RNA interference to reduce their expression and test the effect on apoptosis. Recombinant CASPS16, 17, 20 and 21 were produced in bacteria and the abilities of these recombinant proteins to cleave different caspase substrates were examined. From the resulting data, it was concluded that CASPS17 and CASPS21 are likely to be effector caspases since they preferred a effector caspase substrate. When considering the prodomain length, CASPS17 has a short prodomain, but CASPS21 has a long prodomain, which is normally associated with initiator caspases. CASPS20 did not show preference for any specific substrate and has a short prodomain. Since it did not have a specific preference of substrate, it is likely to be an effector caspase based on prodomain length. CASPS16 showed a slightly higher preference for the initiator caspase substrate WEHD, and has a long prodomain. Based on these results, CASPS16 is likely an initiator caspase. To examine the potential roles of CASPS15, 16, 17, 20 and 21 in apoptosis, their expression in Aag2 cells was knocked down using RNA interference. Successful knockdown was verified by qRT-PCR. After silencing specific caspases, the cells were exposed to two different apoptotic stimuli, ultraviolet radiation (UV) or the RNA synthesis inhibitor actinomycin D (ActD). Following apoptotic treatment, apoptosis was measured by two methods; caspase activity was measured using an effector caspase substrate, and phosphatidyl serine exposure on the outer leaflet of the plasma membrane, which occurs in apoptotic cells, was measured by Annexin V staining and flow cytometry. In cells where CASPS15, 16, 17, 20 or 21 had been knocked down and the cells were then treated with UV or ActD, it was observed that effector caspase activity and Annexin V staining were both significantly lower than in UV- or ActD-treated cells that had received control double-stranded RNA. Together these results suggest that all of these caspases are involved in apoptosis in Aag2 cells. This study serves as a starting point for further research on Ae. aegypti caspases and their roles in specific cellular processes.

No description available.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy