RestrictedArchive–Te Puna Rangahau
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Welcome to RestrictedArchive–Te Puna Rangahau, the closed repository for research outputs from Victoria University of Wellington, New Zealand.
This collection contains papers and theses authored by University staff and students.
The content in the collection was migrated from http://restrictedarchive.vuw.ac.nz in 2021.
Access to this collection is restricted to University staff and students.
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Browsing RestrictedArchive–Te Puna Rangahau by Author "Ackerley, David"
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Item Restricted Development and Applications of Nitroreductase Activated Masked Fluorophores(Te Herenga Waka—Victoria University of Wellington, 2012) Horvat, Claire Natalia; Ackerley, David; McConnell, MelanieAn emerging anti-cancer strategy is the development of cancer prodrugs for targeted therapies. Nitroaromatic prodrugs have shown great promise in this regard due to the ability of the strongly electronegative nitro group to act as a “molecular switch”, which upon reduction can activate the cytotoxic properties of other substituent groups in the molecule. A variety of nitroaromatic prodrugs in combination with oxygen-independent nitroreductase (NTR) enzymes derived from bacteria have been evaluated in various Gene Directed Enzyme Prodrug Therapy (GDEPT) models to develop next-generation cancer therapies, but further work is required to effectively study and optimise these strategies. A key limitation in GDEPT is inefficient gene delivery. To improve gene delivery it is important to be able to effectively monitor vector distribution and gene expression. Standard invasive methods involving biopsy are both cumbersome and invasive so other reporter methods such as the use of fluorescence aim to improve on these, at least in a preclinical setting. In this study we aimed to test and screen novel masked fluorophores for their ability to report on NTR activity in a GDEPT context, as well as other biological applications including identification of novel NTRs and their potential as cellular reporters of gene expression. Initially a panel of novel masked fluorophores with a blue light emission profile were screened to identify optimal probes to report on the activity of the Escherichia coli NTR NfsB. The panel was then increased to include masked fluorophores with a range of emission spectra. With the aim of creating a multiplex genetic reporter system these were screened against an extended NTR library derived from 19 different bacterial species. Certain masked fluorophores that were structural analogues of promising nitroaromatic prodrugs were also used to screen environmental DNA libraries to recover novel NTR enzymes. A high throughput flow cytometry method was developed and used to discover several NTRs with prodrug activity. In a final application, a masked fluorophore was incorporated into a targeted ablation system where activating NTRs had been placed under the control of a stem cell specific promoter, SOX2. The masked fluorophore was used to report on cellular SOX2 expression and viability prior to the addition of a cell-ablating prodrug, metronidazole. The research within this thesis has shown that masked fluorophores have the ability to accurately report NTR activity and that they have the sensitivity to differentiate between different NTRs. This imaging capability could greatly benefit pre-clinical GDEPT research. The research undertaken in this thesis has also shown that there are several other biological applications for masked fluorophore/NTR pairings beyond their use in GDEPT.Item Restricted The incidence of non-O157 Shiga toxin producing Escherichia coli in the Canterbury region - a pilot study(Te Herenga Waka—Victoria University of Wellington, 2013) Strydom, Hugo; Ackerley, David; Dufour, MurielShiga-toxin producing E. coli (STEC) are increasingly recognized as agents of sporadic and outbreak associated gastroenteritis. Although E. coli O157 has traditionally been the most commonly identified STEC serotype, infections caused by non-O157 STEC are on the rise worldwide. Difficulties in their isolation and identification contribute to underestimation of their true virulence. Existing molecular methods can be used for rapid toxin gene detection; however a major limitation is that the bacterial strain is not recovered. This research has compared molecular and culture-based methods for the detection and isolation of STEC from clinical samples. STEC multiplex PCR, real-time PCR and Multiplex Ligation-dependent Probe Amplification were compared for their ability to detect STEC, while the following enrichment broths and selective agar were compared for enrichment and isolation of STEC: tryptic soy broth, modified tryptic soy broth supplemented with vancomycin and cefsulodin, acid enrichment broth, Rainbow® Agar O157, Statens Serum Institut enteric agar, MacConkey sorbitol agar (CT-SMAC), CHROMagar™ O157 and EHEC agar. A pilot study, consisting of screening 102 faecal samples from patients presenting with diarrhoea and/or haemolytic uremic syndrome (HUS) was conducted to establish the incidence of non-O157 STEC in the Canterbury region. The outcome of the clinical trial was the detection and isolation of eight STEC, including four non-O157 STEC. Two of the non-O157 STEC isolates failed to grow on CT-SMAC, the selective media of choice for the isolation of STEC by many clinical laboratories. Acid enrichment is an effective method for the enrichment of STEC and qPCR is a rapid and a sensitive method of detecting STEC. Evidence from this study strongly suggests that incidence of non-O157 STEC in New Zealand is likely to be under reported.