subject:(nitroreductase)

Victoria University of Wellington

1. Prosser, Gareth Adrian. Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy.

Degree: 2011, Victoria University of Wellington

► Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating… (more)

▼ Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other… Advisors/Committee Members: Ackerley, David.

Subjects/Keywords: Nitroreductase; GDEPT; Directed evolution

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Prosser, G. A. (2011). Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy. (Doctoral Dissertation). Victoria University of Wellington. Retrieved from http://hdl.handle.net/10063/1718

Chicago Manual of Style (16^th Edition):

Prosser, Gareth Adrian. “Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy.” 2011. Doctoral Dissertation, Victoria University of Wellington. Accessed February 26, 2021. http://hdl.handle.net/10063/1718.

MLA Handbook (7^th Edition):

Prosser, Gareth Adrian. “Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy.” 2011. Web. 26 Feb 2021.

Vancouver:

Prosser GA. Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy. [Internet] [Doctoral dissertation]. Victoria University of Wellington; 2011. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/10063/1718.

Council of Science Editors:

Prosser GA. Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy. [Doctoral Dissertation]. Victoria University of Wellington; 2011. Available from: http://hdl.handle.net/10063/1718

Victoria University of Wellington

2. Williams, Elsie May. Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy.

Degree: 2013, Victoria University of Wellington

URL: http://hdl.handle.net/10063/4994

► There is strong interest in developing novel targeted cancer therapies. It has been known for over a century that certain viruses and bacteria can preferentially… (more)

▼ There is strong interest in developing novel targeted cancer therapies. It has been known for over a century that certain viruses and bacteria can preferentially infect and lyse cancerous cells. Clinical utility has lagged behind the initial promise of the idea; however three therapeutic agents from the oncolytic virus field are currently in Phase IIB/Phase III clinical trials. The development path of such therapies would be substantially smoothed by an ability to nonin vasively monitor the ir location in the patient’s body post-administration. This would allay fears that viral/bacterial distribution may not be confined to the tumour and provide real time information on vector localisation and replication. This could be achieved by positron emission tomography (PET) scanning if the vector expressed a reporter protein which could activate a PET suitable imaging agent. Furthermore the potency of such therapies could be increased by if this reporter protein could also act therapeutically by converting a systemically delivered benign prodrug into a potent chemotherapeutic – thus targeting the toxicity of the prodrug specifically to cancerous cells. A promising enzyme/prodrug combination is the use of bacterial nitroreductase (NTR) enzymes to activate DNA damaging prodrugs, such as the dinitrobenzamides CB1954 and PR-104A. This thesis presents work aimed at developing the ability to noninvasively image bacterial NTR expression so that these enzymes can act as both therapeutic and reporter proteins. The primary focus of this study was to achieve this by repurposing pre-existing 2-nitroimidazole (NI) PET imaging agents, originally developed for imaging tumour hypoxia. Microplate based screening strategies were developed to enable detection of 2-NI bioreductive activation by different bacterial NTRs over-expressed heterologously in Escherichia coli, and these technologies were used to screen a 58-membered library of nitroreductase candidates. Although the most widely studied NTR for enzyme/prodrug therapy - NfsB from E. coli - was found to lack activity with 2-NI substrates, numerous NTRs from the NfsA family were able to metabolise these molecules to the cell entrapped form required for PET imaging. Following this discovery, a directed evolution study was conducted to improve the native activity of the enzyme NfsA from E. coli. In this study targeted mutagenesis of active site residues was carried out, resulting in identification of several NfsA multi-site mutants that were substantially improved in their ability to activate a range of 2-NI imaging agents. In addition to repurposing existing PET probes, this work sought to identify and engineer NTRs for efficient activation of a next - generation PET probe that is designed to be substantially less responsive to hypoxia and hence give a cleaner signal for NTR imaging (i.e. low to no background resulting from tumour hypoxia). SN 33623, a novel 5-NI analogue of the existing 2-NI PET probe EF5, was designed and synthesised by our University of Auckland collaborators. It was… Advisors/Committee Members: Ackerley, David.

Subjects/Keywords: Nitroreductase; Cancer; Noninvasive imaging

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Williams, E. M. (2013). Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy. (Doctoral Dissertation). Victoria University of Wellington. Retrieved from http://hdl.handle.net/10063/4994

Chicago Manual of Style (16^th Edition):

Williams, Elsie May. “Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy.” 2013. Doctoral Dissertation, Victoria University of Wellington. Accessed February 26, 2021. http://hdl.handle.net/10063/4994.

MLA Handbook (7^th Edition):

Williams, Elsie May. “Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy.” 2013. Web. 26 Feb 2021.

Vancouver:

Williams EM. Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy. [Internet] [Doctoral dissertation]. Victoria University of Wellington; 2013. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/10063/4994.

Council of Science Editors:

Williams EM. Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy. [Doctoral Dissertation]. Victoria University of Wellington; 2013. Available from: http://hdl.handle.net/10063/4994

Victoria University of Wellington

3. Condon, Sarah. The characterisation and application of bacterial nitroreductase enzymes.

Degree: 2013, Victoria University of Wellington

URL: http://hdl.handle.net/10063/8697

► Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as… (more)

▼ Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as a front-runner of new technologies that seek to combat the growing number of cases. One developing approach to GDEPT involves the use of bacterial nitroreductase enzymes to reduce prodrug substrates, which, upon reduction to their active form, are toxic to cancer cells through DNA crosslinking. Nitroreductases have the ability to activate a variety of nitro-quenched compounds, not only anti-cancer prodrugs, but also nil bystander antibiotics and masked fluorophores, through the reduction of strongly electron-withdrawing nitro substituents on aromatic rings. My research initially sought to exploit this capability by partnering nitroreductases with nil bystander antibiotics for targeted cell ablation, as a component of a larger gene directed enzyme prodrug therapy project. This has potential to provide important safety features for removal of viral and bacterial vectors following anti-cancer gene therapy. From this, the main focus evolved into utilising nitroreductase enzymes for targeted cell ablation for applications in developmental and regenerative biology. This exploited the ability of nitroreductases to activate nil bystander antibiotics in partnership with masked fluorophores for imaging purposes. It has previously been shown that antibiotics can be applied to a nitroreductase under control of a tissue-specific promoter in a transgenic model organism, enabling controlled ablation of that tissue at precise stages of development. However, direct imaging of the nitroreductase location and activity, by application of masked fluorophore probes prior to ablation, has not previously been explored. During the course of this work, several promising combinations of nitroreductases that exhibit opposing specificities for certain combinations of masked fluorophores and nil-bystander antibiotics were identified through screening in bacterial systems. In general, these results were found to translate effectively into eukaryotic cell lines. Pairs of nitroreductases that have opposite specificities for two different antibiotic substrates offer potential for the multiplexed ablation of either (or both) of two different labelled tissues in the same transgenic model organism, according to the substrate(s) administered to that organism. Throughout this screening process, a nitroaromatic substrate (niclosamide) was identified that is, uniquely, initially toxic to Escherichia coli but becomes non-toxic upon reduction of the nitro substituent. Using niclosamide, a novel strategy with potential for identification of new nitroreductases, as well as selection-based directed evolution to improve desired activities, was explored. Advisors/Committee Members: Ackerley, David.

Subjects/Keywords: Nitroreductase; Fluorophore; Nil bystander

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Condon, S. (2013). The characterisation and application of bacterial nitroreductase enzymes. (Masters Thesis). Victoria University of Wellington. Retrieved from http://hdl.handle.net/10063/8697

Chicago Manual of Style (16^th Edition):

Condon, Sarah. “The characterisation and application of bacterial nitroreductase enzymes.” 2013. Masters Thesis, Victoria University of Wellington. Accessed February 26, 2021. http://hdl.handle.net/10063/8697.

MLA Handbook (7^th Edition):

Condon, Sarah. “The characterisation and application of bacterial nitroreductase enzymes.” 2013. Web. 26 Feb 2021.

Vancouver:

Condon S. The characterisation and application of bacterial nitroreductase enzymes. [Internet] [Masters thesis]. Victoria University of Wellington; 2013. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/10063/8697.

Council of Science Editors:

Condon S. The characterisation and application of bacterial nitroreductase enzymes. [Masters Thesis]. Victoria University of Wellington; 2013. Available from: http://hdl.handle.net/10063/8697

Victoria University of Wellington

4. Hall, Kelsi Rose. Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis.

Degree: 2019, Victoria University of Wellington

URL: http://hdl.handle.net/10063/8821

► Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E.… (more)

▼ Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E. coli. NfsA_Ec is a promiscuous enzyme that can reduce a wide range of nitroaromatic antibiotics and prodrugs. This research sought to use NfsA_Ec as a model to improve our understanding of directed evolution, and also to identify NfsA_Ec variants exhibiting improved activation with a range of nil-bystander prodrugs for use in a targeted cell ablation system in zebrafish. There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. We sought to contrast such approaches with simultaneous site-directed mutagenesis, employing a library of 252 million unique nfsA variants. To determine whether two superior NfsA_Ec variants recovered from this library could have been identified using a conventional stepwise approach we generated all possible intermediates of these two enzyme variants and recreated the most logical evolutionary trajectory for each enzyme variant. This revealed that a stepwise mutagenesis approach could indeed have yielded both of these variants, but also that very few evolutionary trajectories were accessible due to complex epistatic interactions between substitutions in these enzymes. Moreover, many conventional stepwise mutagenesis approaches such as iterative saturation mutagenesis would have failed to identify key substitutions in these variants. We also investigated the “black-box” effect of directed evolution, using NfsA_Ec and a panel of nitroaromatic compounds to model the off-target effects an evolved enzyme can have within an existing metabolic network. We found that selection for improved niclosamide and chloramphenicol detoxification also improved activity with some structurally distinct prodrugs, but not others. Using a dual positive-negative selection, we recovered NfsA_Ec variants that were more specialised for their primary activities, however this came at a cost in terms of overall activity levels. The simultaneous site-directed nfsA_Ec mutagenesis library also had practical applications, enabling recovery of NfsA_Ec variants for targeted cell ablation in zebrafish models. These models involve the selective ablation of nitroreductase expressing cells without harming adjacent cells, to mimic a degenerative disease. Several NfsA_Ec variants were identified which were highly active with the nil-bystander prodrugs metronidazole, tinidazole, RB6145 and misonidazole when expressed in E. coli. However, these NfsA_Ec variants had inconsistent activities in our eukaryotic cell model (HEK-293). To expand the utility of the core ablation system, we sought to identify pairs of nitroreductases with non-overlapping prodrug specificities, suitable for use in a multiplex cell… Advisors/Committee Members: Ackerley, David, Patrick, Wayne.

Subjects/Keywords: Directed evolution; Enzyme; Nitroreductase

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Hall, K. R. (2019). Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis. (Doctoral Dissertation). Victoria University of Wellington. Retrieved from http://hdl.handle.net/10063/8821

Chicago Manual of Style (16^th Edition):

Hall, Kelsi Rose. “Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis.” 2019. Doctoral Dissertation, Victoria University of Wellington. Accessed February 26, 2021. http://hdl.handle.net/10063/8821.

MLA Handbook (7^th Edition):

Hall, Kelsi Rose. “Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis.” 2019. Web. 26 Feb 2021.

Vancouver:

Hall KR. Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis. [Internet] [Doctoral dissertation]. Victoria University of Wellington; 2019. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/10063/8821.

Council of Science Editors:

Hall KR. Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis. [Doctoral Dissertation]. Victoria University of Wellington; 2019. Available from: http://hdl.handle.net/10063/8821

Victoria University of Wellington

5. Rich, Michelle Hedley. Discovery and directed evolution of nitroreductase enzymes for activation of prodrugs and PET imaging compounds.

Degree: 2017, Victoria University of Wellington

URL: http://hdl.handle.net/10063/9261

► Bacterial nitroreductase enzymes, which exhibit the capacity to reduce a wide range of nitroaromatic drugs, antibiotics and environmental pollutants, have shown promise in the activation… (more)

▼ Bacterial nitroreductase enzymes, which exhibit the capacity to reduce a wide range of nitroaromatic drugs, antibiotics and environmental pollutants, have shown promise in the activation of prodrugs such as CB1954 and PR-104A. Use of these prodrugs in gene-directed enzyme prodrug therapy (GDEPT) cancer treatment would allow for targeted chemotherapy in tumour cells following specific delivery of nitroreductases to these cancerous tissues, using specialised bacterial or viral vectors. However, one key limitation in nitroreductase-based GDEPT is the current inability to rapidly and non-invasively determine vector localisation and gene delivery prior to systemic administration of prodrug. Dual-purpose nitroreductases that exhibit the ability to activate both GDEPT prodrugs and radioisotope-labelled PET imaging probes, in a manner that renders them temporarily cell-entrapped for detection using a PET scanner, would facilitate clinical development of this treatment. Previous attempts to repurpose hypoxia-activated 2-nitroimidazole PET imaging probes for nitroreductase detection have suffered from relatively high background activation under hypoxia alone. The design of nextgeneration 5-nitroimidazole PET imaging probes, by our collaborators at the Auckland Cancer Society Research Centre (ACSRC), has resulted in much lower levels of hypoxia activation in vivo. This thesis describes attempts to generate improved nitroreductases that can activate a bespoke 5-nitroimidazole PET-capable imaging probe, S33. A 58-membered library of nitroreductase candidates, including enzymes from many different bacterial species and oxidoreductase families, was heterologously over-expressed in E. coli screening strains. Microplate-based screening strategies were then used to identify enzymes that exhibited the most activity with S33, based on the ability of high levels of activated S33 to induce DNA damage and (at very high levels) E. coli cell death. Following this, site-targeted libraries of two different promising nitroreductase NfsA homologues were screened for S33 activity, with selected variants from each library showing improvement in S33 activation over the parent nitroreductase. In parallel I performed error-prone PCR mutagenesis of a top NfsA variant and top NfsB variant, subjecting each to two rounds of random mutagenesis, and selecting improved variants using a specialised E. coli screening strain and fluorescence-activated cell sorting (FACS). Selected variants from the NfsB (but not NfsA) nitroreductase candidate library showed substantially improved capacity to activate S33 over the parent enzyme. As an alternative means for developing improved nitroreductase variants, two different nitroaromatic ‘anti-prodrugs’, the anthelmintic niclosamide and the antibiotic chloramphenicol, whose cytotoxic effects on E. coli can be mitigated by the presence of an over-expressed active nitroreductase, were used to select for improved S33-activating enzymes from a site-targeted NfsA library. Variants were discovered that exhibited improved… Advisors/Committee Members: Ackerley, David, Jordan, Bill.

Subjects/Keywords: Nitroreductase; Noninvasive imaging; Cancer

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Rich, M. H. (2017). Discovery and directed evolution of nitroreductase enzymes for activation of prodrugs and PET imaging compounds. (Doctoral Dissertation). Victoria University of Wellington. Retrieved from http://hdl.handle.net/10063/9261

Chicago Manual of Style (16^th Edition):

Rich, Michelle Hedley. “Discovery and directed evolution of nitroreductase enzymes for activation of prodrugs and PET imaging compounds.” 2017. Doctoral Dissertation, Victoria University of Wellington. Accessed February 26, 2021. http://hdl.handle.net/10063/9261.

MLA Handbook (7^th Edition):

Rich, Michelle Hedley. “Discovery and directed evolution of nitroreductase enzymes for activation of prodrugs and PET imaging compounds.” 2017. Web. 26 Feb 2021.

Vancouver:

Rich MH. Discovery and directed evolution of nitroreductase enzymes for activation of prodrugs and PET imaging compounds. [Internet] [Doctoral dissertation]. Victoria University of Wellington; 2017. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/10063/9261.

Council of Science Editors:

Rich MH. Discovery and directed evolution of nitroreductase enzymes for activation of prodrugs and PET imaging compounds. [Doctoral Dissertation]. Victoria University of Wellington; 2017. Available from: http://hdl.handle.net/10063/9261

University of Alberta

6. Fraser, Irene Brittany Morgan. Using zebrafish to develop a precise model of cone photoreceptor ablation and regeneration.

Degree: MS, Department of Biological Sciences, 2011, University of Alberta

URL: https://era.library.ualberta.ca/files/cj82k814h

► We have engineered a novel model of cone photoreceptor regeneration using transgenic zebrafish to induce cell ablation. Zebrafish were engineered to express the E. coli… (more)

Subjects/Keywords: ablation; photoreceptor; zebrafish; regeneration; nitroreductase; metronidazole; cone

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Fraser, I. B. M. (2011). Using zebrafish to develop a precise model of cone photoreceptor ablation and regeneration. (Masters Thesis). University of Alberta. Retrieved from https://era.library.ualberta.ca/files/cj82k814h

Chicago Manual of Style (16^th Edition):

Fraser, Irene Brittany Morgan. “Using zebrafish to develop a precise model of cone photoreceptor ablation and regeneration.” 2011. Masters Thesis, University of Alberta. Accessed February 26, 2021. https://era.library.ualberta.ca/files/cj82k814h.

MLA Handbook (7^th Edition):

Fraser, Irene Brittany Morgan. “Using zebrafish to develop a precise model of cone photoreceptor ablation and regeneration.” 2011. Web. 26 Feb 2021.

Vancouver:

Fraser IBM. Using zebrafish to develop a precise model of cone photoreceptor ablation and regeneration. [Internet] [Masters thesis]. University of Alberta; 2011. [cited 2021 Feb 26]. Available from: https://era.library.ualberta.ca/files/cj82k814h.

Council of Science Editors:

Fraser IBM. Using zebrafish to develop a precise model of cone photoreceptor ablation and regeneration. [Masters Thesis]. University of Alberta; 2011. Available from: https://era.library.ualberta.ca/files/cj82k814h

7. Godoy, Rafael Soares. Chemogenetic Ablation of Dopaminergic Neurons in the Brain of Larval and Adult Zebrafish (Danio Rerio): Phenotypes and Regenerative Ability .

Degree: 2015, University of Ottawa

URL: http://hdl.handle.net/10393/32541

► Dopamine exerts an important role in the regulation of motor activity in humans. During the progression of Parkinson’s disease, patients are faced with the progressive… (more)

Subjects/Keywords: Zebrafish; Dopaminergic neurons; Nitroreductase; Brain; Regeneration

11 more images…

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APA (6^th Edition):

Godoy, R. S. (2015). Chemogenetic Ablation of Dopaminergic Neurons in the Brain of Larval and Adult Zebrafish (Danio Rerio): Phenotypes and Regenerative Ability . (Thesis). University of Ottawa. Retrieved from http://hdl.handle.net/10393/32541

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Godoy, Rafael Soares. “Chemogenetic Ablation of Dopaminergic Neurons in the Brain of Larval and Adult Zebrafish (Danio Rerio): Phenotypes and Regenerative Ability .” 2015. Thesis, University of Ottawa. Accessed February 26, 2021. http://hdl.handle.net/10393/32541.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Godoy, Rafael Soares. “Chemogenetic Ablation of Dopaminergic Neurons in the Brain of Larval and Adult Zebrafish (Danio Rerio): Phenotypes and Regenerative Ability .” 2015. Web. 26 Feb 2021.

Vancouver:

Godoy RS. Chemogenetic Ablation of Dopaminergic Neurons in the Brain of Larval and Adult Zebrafish (Danio Rerio): Phenotypes and Regenerative Ability . [Internet] [Thesis]. University of Ottawa; 2015. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/10393/32541.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Godoy RS. Chemogenetic Ablation of Dopaminergic Neurons in the Brain of Larval and Adult Zebrafish (Danio Rerio): Phenotypes and Regenerative Ability . [Thesis]. University of Ottawa; 2015. Available from: http://hdl.handle.net/10393/32541

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

8. Ao, Xiang. The synthesis and study of novel fluorescence probes for Nitroreductase.

Degree: 2017, RIAN

URL: http://eprints.maynoothuniversity.ie/9907/

► This Thesis entitled ‘The synthesis and study of novel fluorescence probes for NTR’ is divided in 7 chapters. Chapter 1, provides an introduction to cancer,… (more)

Subjects/Keywords: synthesis; study; novel fluorescence probes; Nitroreductase

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Ao, X. (2017). The synthesis and study of novel fluorescence probes for Nitroreductase. (Thesis). RIAN. Retrieved from http://eprints.maynoothuniversity.ie/9907/

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Ao, Xiang. “The synthesis and study of novel fluorescence probes for Nitroreductase.” 2017. Thesis, RIAN. Accessed February 26, 2021. http://eprints.maynoothuniversity.ie/9907/.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Ao, Xiang. “The synthesis and study of novel fluorescence probes for Nitroreductase.” 2017. Web. 26 Feb 2021.

Vancouver:

Ao X. The synthesis and study of novel fluorescence probes for Nitroreductase. [Internet] [Thesis]. RIAN; 2017. [cited 2021 Feb 26]. Available from: http://eprints.maynoothuniversity.ie/9907/.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Ao X. The synthesis and study of novel fluorescence probes for Nitroreductase. [Thesis]. RIAN; 2017. Available from: http://eprints.maynoothuniversity.ie/9907/

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

9. Ao, Xiang. The synthesis and study of novel fluorescence probes for Nitroreductase.

Degree: 2017, RIAN

URL: http://mural.maynoothuniversity.ie/9907/

► This Thesis entitled ‘The synthesis and study of novel fluorescence probes for NTR’ is divided in 7 chapters. Chapter 1, provides an introduction to cancer,… (more)

Subjects/Keywords: synthesis; study; novel fluorescence probes; Nitroreductase

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Ao, X. (2017). The synthesis and study of novel fluorescence probes for Nitroreductase. (Thesis). RIAN. Retrieved from http://mural.maynoothuniversity.ie/9907/

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Ao, Xiang. “The synthesis and study of novel fluorescence probes for Nitroreductase.” 2017. Thesis, RIAN. Accessed February 26, 2021. http://mural.maynoothuniversity.ie/9907/.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Ao, Xiang. “The synthesis and study of novel fluorescence probes for Nitroreductase.” 2017. Web. 26 Feb 2021.

Vancouver:

Ao X. The synthesis and study of novel fluorescence probes for Nitroreductase. [Internet] [Thesis]. RIAN; 2017. [cited 2021 Feb 26]. Available from: http://mural.maynoothuniversity.ie/9907/.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Ao X. The synthesis and study of novel fluorescence probes for Nitroreductase. [Thesis]. RIAN; 2017. Available from: http://mural.maynoothuniversity.ie/9907/

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

10. Ball, Patrick. Identification and testing of enzymes and prodrugs for use in directed enzyme prodrug therapy strategies for the treatment of cancer.

Degree: PhD, 2019, Bangor University

URL: https://research.bangor.ac.uk/portal/en/theses/identification-and-testing-of-enzymes-and-prodrugs-for-use-in-directed-enzyme-prodrug-therapy-strategies-for-the-treatment-of-cancer(c07f9470-fc01-4ef9-8e23-426f320c3abd).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.793161

► Cancer is one of the leading causes of death worldwide and improving the efficacy of cancer chemotherapy treatments is one of the most pressing issues… (more)

▼ Cancer is one of the leading causes of death worldwide and improving the efficacy of cancer chemotherapy treatments is one of the most pressing issues of the day. Prodrugs hold great promise in that regard as they can be activated selectively, allowing for a more focused treatment strategy than with conventional chemotherapy drugs. Directed enzyme prodrug therapy (DEPT) is a form of cancer chemotherapy that is being developed to utilise prodrugs in combination with prodrug-activating enzymes which would be selectively delivered to a tumour site prior to prodrug administration to allow prodrug activation to occur only at the cancer site. Current DEPT strategies have their own inherent flaws based on the biological methods being used to deliver the prodrug-activating enzymes to the tumour site. Magnetic nanoparticle directed enzyme prodrug therapy (MNDEPT) is a novel approach that is being developed within our research group that seeks to overcome these problems by using gold-coated magnetic nanoparticles as the enzyme delivery system. In this study, the suitability of several enzymes and prodrugs for use in future MNDEPT treatments were tested. The study assessed a range of enzymes including two novel nitroreductases from Bacillus cereus, two Xenobiotic reductases from Pseudomonas putida and two genetically modified nitroreductases previously developed within our research group. The prodrug candidates tested were the heavily investigated nitroreductase prodrug, CB1954, and two forefront dinitrobenzamide mustard prodrugs, PR-104A and SN27686. Several enzyme/prodrug combinations tested were identified as being promising for use in future DEPT treatments, including 1619-his with CB1954, XenB-cys with CB1954 and YfkO-cys with PR-104A. The YfkO-cys/PR-104A combination was of particular promise as it displayed a Michaelis-Menten kinetic efficiency that is nearly three times greater than that shown by the heavily investigated NfnB/CB1954 combination, which displayed limited clinical performance because of the low turnover rate of CB1954 by NfnB. Furthermore, a new method of assessing the ratio of the hydroxylamine products formed from the enzymatic reduction of CB1954 using HPLC has been identified and is reported within this body of work. Building on this, new revelations about how the product ratio changes over time have come to light and this has proven that kinetics-driven changes to the product ratio can occur as the reaction proceeds over time and this is reported here.

Subjects/Keywords: 500; Nitroreductase; Prodrug; Cancer; Dept; HPCC

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APA (6^th Edition):

Ball, P. (2019). Identification and testing of enzymes and prodrugs for use in directed enzyme prodrug therapy strategies for the treatment of cancer. (Doctoral Dissertation). Bangor University. Retrieved from https://research.bangor.ac.uk/portal/en/theses/identification-and-testing-of-enzymes-and-prodrugs-for-use-in-directed-enzyme-prodrug-therapy-strategies-for-the-treatment-of-cancer(c07f9470-fc01-4ef9-8e23-426f320c3abd).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.793161

Chicago Manual of Style (16^th Edition):

Ball, Patrick. “Identification and testing of enzymes and prodrugs for use in directed enzyme prodrug therapy strategies for the treatment of cancer.” 2019. Doctoral Dissertation, Bangor University. Accessed February 26, 2021. https://research.bangor.ac.uk/portal/en/theses/identification-and-testing-of-enzymes-and-prodrugs-for-use-in-directed-enzyme-prodrug-therapy-strategies-for-the-treatment-of-cancer(c07f9470-fc01-4ef9-8e23-426f320c3abd).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.793161.

MLA Handbook (7^th Edition):

Ball, Patrick. “Identification and testing of enzymes and prodrugs for use in directed enzyme prodrug therapy strategies for the treatment of cancer.” 2019. Web. 26 Feb 2021.

Vancouver:

Ball P. Identification and testing of enzymes and prodrugs for use in directed enzyme prodrug therapy strategies for the treatment of cancer. [Internet] [Doctoral dissertation]. Bangor University; 2019. [cited 2021 Feb 26]. Available from: https://research.bangor.ac.uk/portal/en/theses/identification-and-testing-of-enzymes-and-prodrugs-for-use-in-directed-enzyme-prodrug-therapy-strategies-for-the-treatment-of-cancer(c07f9470-fc01-4ef9-8e23-426f320c3abd).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.793161.

Council of Science Editors:

Ball P. Identification and testing of enzymes and prodrugs for use in directed enzyme prodrug therapy strategies for the treatment of cancer. [Doctoral Dissertation]. Bangor University; 2019. Available from: https://research.bangor.ac.uk/portal/en/theses/identification-and-testing-of-enzymes-and-prodrugs-for-use-in-directed-enzyme-prodrug-therapy-strategies-for-the-treatment-of-cancer(c07f9470-fc01-4ef9-8e23-426f320c3abd).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.793161

University of Oklahoma

11. Wang, Bing. X-RAY CRYSTAL STRUCTURES AND CHARACTERIZATION OF THE PRODUCTS FROM THE INTERACTIONS OF MYOGLOBIN WITH NITROGEN OXIDES AND ARYLHYDRAZINES, AND NITROREDUCTASE INTERACTIONS WITH ORGANIC NITRO COMPOUNDS.

Degree: PhD, 2016, University of Oklahoma

URL: http://hdl.handle.net/11244/34616

► This thesis describes research into the roles that metalloproteins and non-metalloproteins play in the biological inorganic/organic chemistry of common nitrogen oxides. There are three main… (more)

▼ This thesis describes research into the roles that metalloproteins and non-metalloproteins play in the biological inorganic/organic chemistry of common nitrogen oxides. There are three main chapters on this work: the first details the reactions of wild-type and mutant myoglobins (Mbs) in their interactions with nitrite and nitric oxide (NO), the second deals with these Mbs and their formation of bioorganometallic derivatives when reacted with arylhydrazines, and the third deals with an FMN-dependent nitroreductase enzyme and its reactions with the clinically relevant metronidazole drug. Mutations to the distal pocket in the active site of Mb were made; specifically, ferric-aqua derivatives of the mutants were expressed, purified, crystallized, and their crystal structures solved to 1.78-1.85 Å resolution. The proteins crystallized in either the P21 or P6 space groups. The crystals were soaked with nitrite to form their O-bonded MbIII(ONO) complexes whose structures were also solved to 1.57-1.85 Å resolution. In the case of the H64A distal pocket mutant missing the H-bonding amino-acid residue in the 64th position, a water bridge was observed to form linking the protein exterior with the bound nitrite ligand, thus replacing the expected wt H64 H-bonding feature. Further, we noted that the distal pocket Val68 residue adapted its conformation to accommodate the nitrite ligands in some of these complexes. Notably, the O-binding modes observed in the four wt and mutant structures held up exceedingly well even with the variation in H-bonding capacities. These wt and mutant Mb(ONO) compounds can be reduced with sodium dithionite to their respective nitrosyl Mb(NO) products. Verification of the formation of the nitrosyl Fe-NO derivative, and not the closely related nitroxyl Fe-HNO, was provided by FT-infrared spectroscopy. Arylhydrazines and derivatives are prevalent in nature and in pharmaceutical drugs. They interact with various heme proteins resulting in deactivation of the proteins. Eleven X-ray crystal structures of the products from the reactions of wt and mutant (H64A, H64Q, V68A/I107Y) Mbs with arylhydrazines (ArNHNH2; Ar = Ph, m-tol, and p-chlorophenyl) were obtained to 1.70-1.98 Å resolution. Direct Fe-carbon bonds were observed in all these derivatives, establishing that the hydrazine -NHNH2 moieties had been released from the reagents during their reactions with the Fe centers of the Mbs. Importantly, the C-atoms coordinating to the Fe centers were the same as those that bonded to the hydrazine functional groups, implying that the carbon-based radical intermediates were formed in close proximity to the Fe centers allowing for facile and efficient reactions to give the bioorganometallic Mb-aryl products. Significant distal pocket amino acid movements were observed in some cases with the larger p-chlorophenyl ligand aryl; for example, in the H64Q-chlorophenyl derivative, the Gln64 residue swings to a position outside the pocket towards the solvent region. We report the first expression, purification,… Advisors/Committee Members: Richter-Addo, George B. (advisor), West, Ann H. (committee member), Nicholas, Kenneth M. (committee member), Li, Jun (committee member), Ruyle, Jessica (committee member).

Subjects/Keywords: crystallography; myoglobin; nitrite; nitric oxide; nitroreductase; metronidazole

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Wang, B. (2016). X-RAY CRYSTAL STRUCTURES AND CHARACTERIZATION OF THE PRODUCTS FROM THE INTERACTIONS OF MYOGLOBIN WITH NITROGEN OXIDES AND ARYLHYDRAZINES, AND NITROREDUCTASE INTERACTIONS WITH ORGANIC NITRO COMPOUNDS. (Doctoral Dissertation). University of Oklahoma. Retrieved from http://hdl.handle.net/11244/34616

Chicago Manual of Style (16^th Edition):

Wang, Bing. “X-RAY CRYSTAL STRUCTURES AND CHARACTERIZATION OF THE PRODUCTS FROM THE INTERACTIONS OF MYOGLOBIN WITH NITROGEN OXIDES AND ARYLHYDRAZINES, AND NITROREDUCTASE INTERACTIONS WITH ORGANIC NITRO COMPOUNDS.” 2016. Doctoral Dissertation, University of Oklahoma. Accessed February 26, 2021. http://hdl.handle.net/11244/34616.

MLA Handbook (7^th Edition):

Wang, Bing. “X-RAY CRYSTAL STRUCTURES AND CHARACTERIZATION OF THE PRODUCTS FROM THE INTERACTIONS OF MYOGLOBIN WITH NITROGEN OXIDES AND ARYLHYDRAZINES, AND NITROREDUCTASE INTERACTIONS WITH ORGANIC NITRO COMPOUNDS.” 2016. Web. 26 Feb 2021.

Vancouver:

Wang B. X-RAY CRYSTAL STRUCTURES AND CHARACTERIZATION OF THE PRODUCTS FROM THE INTERACTIONS OF MYOGLOBIN WITH NITROGEN OXIDES AND ARYLHYDRAZINES, AND NITROREDUCTASE INTERACTIONS WITH ORGANIC NITRO COMPOUNDS. [Internet] [Doctoral dissertation]. University of Oklahoma; 2016. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/11244/34616.

Council of Science Editors:

Wang B. X-RAY CRYSTAL STRUCTURES AND CHARACTERIZATION OF THE PRODUCTS FROM THE INTERACTIONS OF MYOGLOBIN WITH NITROGEN OXIDES AND ARYLHYDRAZINES, AND NITROREDUCTASE INTERACTIONS WITH ORGANIC NITRO COMPOUNDS. [Doctoral Dissertation]. University of Oklahoma; 2016. Available from: http://hdl.handle.net/11244/34616

12. Little, Rory Fox. Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation.

Degree: 2015, Victoria University of Wellington

URL: http://hdl.handle.net/10063/4659

► Nitroreductase enzymes are a superfamily of bacterial flavoproteins that can catalyze the reduction of aromatic nitro groups. The reduction of an aromatic nitro group, a… (more)

▼ Nitroreductase enzymes are a superfamily of bacterial flavoproteins that can catalyze the reduction of aromatic nitro groups. The reduction of an aromatic nitro group, a highly electronegative functionality, causes a large electronic shift that can profoundly affect the activity of other substituents on the aromatic ring. For example, upon nitroreduction, initially non-toxic compounds known as prodrugs can be converted into a cytotoxic form. The ability of nitroreductases to alter the activity of compounds has lead to their development as tools for multiple biotechnological applications. Of particular note is the use of nitroreductase enzymes in combination with a nitroaromatic prodrug to study the role of specific cell populations in zebrafish (Danio rerio). Zebrafish are used as model organisms to study processes such as embryonic development and tissue regeneration. By expressing a nitroreductase enzyme in a specific tissue of a zebrafish, it is possible to selectively ablate that tissue upon administration of a prodrug. The subsequent phenotypic change induced by the ablation can provide information on the physiological role of the ablated tissue, or of the regenerative processes that can be recruited to repair the damage. The goal of this thesis was to engineer or discover new nitroreductase enzymes that could expand the capabilities of cell ablation studies in zebrafish. In particular, this work sought to develop a system that would enable the dual, or multiplexed, ablation of two tissues independently within the same organism. Control over the ablation of two distinct tissues could be useful for studying tissue interactions during developmental or regenerative processes. For this to be achievable, two different nitroreductase enzymes, each possessing distinct and non-overlapping prodrug selectivities would be required. Previous studies in the Ackerley lab had identified NfsA from Escherichia coli (NfsA_Ec) and NfsA from Pseudomonas putida (NfsA_Pp) as nitroreductase enzymes that were slightly more selective for the prodrug tinidazole compared than metronidazole. In contrast the NfsB nitroreductase from Vibrio vulnificus (NfsB_Vv) was substantially more selective for metronidazole than tinidazole. To further improve the tinidazole selectivity of the NfsA enzymes, directed evolution was employed as a tool to further enhance the substrate selectivity of each enzyme. The primary outcome of this work was the evolution of an NfsA_Ec mutant that was 12 fold more selective for tinidazole over metronidazole than wild type NfsA_Ec. In addition to engineering new enzymes for cell ablation experiments, this work also sought to discover new nitroreductase enzymes from unculturable bacteria, a previously unplumbed source. The genes and gene products of unculturable bacteria can be identified and studied by expressing fragments of their DNA in a readily culturable host such as E. coli. A variety of different screening methodologies were tested for identifying nitroreductase enzymes from eDNA inserts. The compound… Advisors/Committee Members: Ackerley, David.

Subjects/Keywords: Nitroreductase; Cell ablation; Enzymes

…1.2 – Nitroreductase gene-directed enzyme prodrug therapy. 4 Figure 1.3 – The GDEPT… …Nitroreductase Multiplex Ablation System. Figure 3.2 – Structure of the 5-nitroimidazole nil-bystander… …evolution screen for enhancing the tinidazole selectivity of a mutant nitroreductase library… …SOS-R4 nitroreductase overexpressing strains. 54 Figure 3.7 – Viability of E. coli SOS-R4… …coli fffffffffffffffff SOS-R4 nitroreductase overexpressing strains. Figure 3.11 – Replica…

13 more images…

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Little, R. F. (2015). Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation. (Masters Thesis). Victoria University of Wellington. Retrieved from http://hdl.handle.net/10063/4659

Chicago Manual of Style (16^th Edition):

Little, Rory Fox. “Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation.” 2015. Masters Thesis, Victoria University of Wellington. Accessed February 26, 2021. http://hdl.handle.net/10063/4659.

MLA Handbook (7^th Edition):

Little, Rory Fox. “Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation.” 2015. Web. 26 Feb 2021.

Vancouver:

Little RF. Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation. [Internet] [Masters thesis]. Victoria University of Wellington; 2015. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/10063/4659.

Council of Science Editors:

Little RF. Directed Evolution and Discovery of Nitroreductase Enzymes for Targeted Cell Ablation. [Masters Thesis]. Victoria University of Wellington; 2015. Available from: http://hdl.handle.net/10063/4659

University of Oklahoma

13. Powell, Samantha M. THE STRUCTURAL CHARACTERIZATION OF THE INTERACTIONS OF MYOGLOBIN, HEMOGLOBIN, C. DIFFICILE NITROREDUCTASE AND CYTOCHROME P450 WITH N-CONTAINING COMPOUNDS.

Degree: PhD, 2019, University of Oklahoma

URL: http://hdl.handle.net/11244/319369

► The overall goal of my work was to probe the structural biology of drug metabolite protein interactions and to correlate this with the negative side… (more)

▼ The overall goal of my work was to probe the structural biology of drug metabolite protein interactions and to correlate this with the negative side effects associated with many prescription drugs. More specifically, the focus was on the interactions of myoglobin (Mb), hemoglobin (Hb), a Clostridioides difficile nitroreductase (NR), and cytochrome P450 BM3-HD with N-containing compounds. Using a combination of spectroscopy and X-ray crystallography, I was able to accomplish this goal. In Chapter 2, the focus was on the formation of heme-nitroso adducts of Mb and Hb generated through oxidative and reductive pathways. While I initially predicted that heme-nitroso adducts would result from the reaction of Mb and Hb with nitro- and hydroxylamine-containing compounds, what actually resulted was the determination of a variety of products in addition to the predicted N-bound nitroso adducts. X-ray crystal structures of these products revealed that some of the compounds evaluated can indeed damage Hb through the formation of hemichromes that can be correlated with early stages of heme loss, which, downstream, may cause anemia. Additionally, we observed, for the first time, that two of the compounds evaluated are capable of S-nitrosating Hb at the betaCys93 residue, an important contributor to the transportation of NO throughout the body. And finally, one of the X-ray crystal structures displays a ligand trapped in a Xe pocket, further demonstrating how large ligands can travel through the pockets within both Mb and Hb to reach the heme active site. The work presented in this chapter thus demonstrates a wide range of products that can result from the reaction of Mb and Hb with nitro- and hydroxylamine-containing compounds. In Chapter 3, the focus was on C. difficile NRs responsible for metabolizing the antibiotic Mtz. Using UV-vis spectroscopy, I confirmed that a putative NR from C. difficile does in fact function as a Type I NR. Additionally, I solved its X-ray crystal structure, one of the first two reported from the hypervirulent C. difficile strain. The X-ray crystal structure also revealed a putative binding site for Mtz. The work in this chapter provides one of the first expression, purification, characterization and crystallization of a NR from the hypervirulent C. difficile strain. The focus of Chapter 4 was on the interaction of cytochrome P450 BM3-HD with RNOs, imidazoles and arylhydrazines. Using spectroscopy, I showed that the interaction of RNOs with P450 BM3-HD result in N-bound products and the extent of product formation was dependent on alkyl group size. Imidazoles were found to bind through their N-atoms in a type II fashion. And finally, arylhydrazines were observed to initially bind in a type II manner, but with exposure to air, they formed sigma-bonded aryl-iron complexes. The work in this chapter elucidates the identity of the products in the reaction of P450 BM3-HD with RNOs, imidazoles and arylhydrazines. Together, the work presented in Chapters 2-4, shows how N-containing compounds and… Advisors/Committee Members: Richter-Addo, George B. (advisor), West, Ann H. (committee member), Bourne, Christina (committee member), Duerfeldt, Adam (committee member), Dunn, Anne K. (committee member).

Subjects/Keywords: structural biology; nitroreductase; cytochrome P450; nitrogen compounds; heme proteins

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Powell, S. M. (2019). THE STRUCTURAL CHARACTERIZATION OF THE INTERACTIONS OF MYOGLOBIN, HEMOGLOBIN, C. DIFFICILE NITROREDUCTASE AND CYTOCHROME P450 WITH N-CONTAINING COMPOUNDS. (Doctoral Dissertation). University of Oklahoma. Retrieved from http://hdl.handle.net/11244/319369

Chicago Manual of Style (16^th Edition):

Powell, Samantha M. “THE STRUCTURAL CHARACTERIZATION OF THE INTERACTIONS OF MYOGLOBIN, HEMOGLOBIN, C. DIFFICILE NITROREDUCTASE AND CYTOCHROME P450 WITH N-CONTAINING COMPOUNDS.” 2019. Doctoral Dissertation, University of Oklahoma. Accessed February 26, 2021. http://hdl.handle.net/11244/319369.

MLA Handbook (7^th Edition):

Powell, Samantha M. “THE STRUCTURAL CHARACTERIZATION OF THE INTERACTIONS OF MYOGLOBIN, HEMOGLOBIN, C. DIFFICILE NITROREDUCTASE AND CYTOCHROME P450 WITH N-CONTAINING COMPOUNDS.” 2019. Web. 26 Feb 2021.

Vancouver:

Powell SM. THE STRUCTURAL CHARACTERIZATION OF THE INTERACTIONS OF MYOGLOBIN, HEMOGLOBIN, C. DIFFICILE NITROREDUCTASE AND CYTOCHROME P450 WITH N-CONTAINING COMPOUNDS. [Internet] [Doctoral dissertation]. University of Oklahoma; 2019. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/11244/319369.

Council of Science Editors:

Powell SM. THE STRUCTURAL CHARACTERIZATION OF THE INTERACTIONS OF MYOGLOBIN, HEMOGLOBIN, C. DIFFICILE NITROREDUCTASE AND CYTOCHROME P450 WITH N-CONTAINING COMPOUNDS. [Doctoral Dissertation]. University of Oklahoma; 2019. Available from: http://hdl.handle.net/11244/319369

University of Michigan

14. Schroeder, McKenna. Immobilized Enzymes: Activity, Orientation, and Stability.

Degree: PhD, Chemical Biology, 2016, University of Michigan

URL: http://hdl.handle.net/2027.42/135760

► Enzyme immobilization is an important tool for many industrial and medical fields applications as well as biosensors. Much work has been done developing types of… (more)

▼ Enzyme immobilization is an important tool for many industrial and medical fields applications as well as biosensors. Much work has been done developing types of immobilization, yet the field lacks a comprehensive understanding of the relationship between immobilized enzyme orientation and activity/stability. This work was done to build a more complete picture of the interplay between activity, stability, orientation, and surface characteristics for immobilized enzymes. In Chapter 2, we started by making uniform, chemically defined self-assembling monolayer (SAM) surfaces functionalized with maleimide to bind NfsB through single cysteine residues. Two orientation NfsB constructs were used in these experiments, H360C and V424C. Orientation was not found to change the specific activity. In Chapter 3, SAM surfaces functionalized with EG3-MAL, EG3-OH and EG3-ME terminated linkers were made to explore the effects of surface characteristics on immobilized NfsB. We found that for mixed surfaces a mole ratio of 1:10 EG3-MAL:EG3-OH and EG3-MAL:EG3-ME resulted in a significantly higher specific activity compared to a 1:1 or 1:20 mole ratio. In these experiments, T ½ was used as a measure of thermal stability. T ½ was increase , but was unaffected by orientation. Mixed surfaces with both EG3-ME and EG3-OH with EG3-MAL held at a constant concentration were created. These mixed surfaces were used to explore the relationship between surface hydrophobicity and the activity/stability of the immobilized enzyme. T ½ was unaffected by surface hydrophobicity while specific activity nearly doubled from 100% EG3-ME surface to 100% EG3-OH surface. A variant of NfsB was created placing the cysteine on an α-helix. The α-helix anchor site didn’t change the specific activity or T ½ of immobilized NfsB. The experiments described in Chapter 4 were performed to investigate the effect of surface crowding on specific activity and stability of immobilized NfsB. Multiple surface coverage conditions from a sparse monolayer to a densely packed monolayer were used for activity and T ½ measurements. Two NfsB variants V424C and S63C showed no change in specific activity or T ½ arising from changes in surface density. NfsB-H360C showed a decrease in specific activity and T ½ at lower surface density. Advisors/Committee Members: Marsh, E Neil G (committee member), Biteen, Julie Suzanne (committee member), Chen, Zhan (committee member), Palfey, Bruce Allan (committee member).

Subjects/Keywords: Immobilized enzymes; Nitroreductase; Self-assembling monolayers; Enzyme kinetics; Enzyme stability; Enzyme orientation; Biological Chemistry; Science

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Schroeder, M. (2016). Immobilized Enzymes: Activity, Orientation, and Stability. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/135760

Chicago Manual of Style (16^th Edition):

Schroeder, McKenna. “Immobilized Enzymes: Activity, Orientation, and Stability.” 2016. Doctoral Dissertation, University of Michigan. Accessed February 26, 2021. http://hdl.handle.net/2027.42/135760.

MLA Handbook (7^th Edition):

Schroeder, McKenna. “Immobilized Enzymes: Activity, Orientation, and Stability.” 2016. Web. 26 Feb 2021.

Vancouver:

Schroeder M. Immobilized Enzymes: Activity, Orientation, and Stability. [Internet] [Doctoral dissertation]. University of Michigan; 2016. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/2027.42/135760.

Council of Science Editors:

Schroeder M. Immobilized Enzymes: Activity, Orientation, and Stability. [Doctoral Dissertation]. University of Michigan; 2016. Available from: http://hdl.handle.net/2027.42/135760

University of Toronto

15. Singh, Kamalpreet. Small Molecule Probes for Photodynamic Therapy (PDT).

Degree: 2019, University of Toronto

URL: http://hdl.handle.net/1807/99832

►

Photodynamic therapy is a clinically approved cancer treatment that utilizes singlet oxygen generated via a combination of photosensitizer (PS), light and molecular oxygen to kill… (more)

Subjects/Keywords: Activatable Probes; Coumarin; Molecular Beacons; Nitroreductase; O6-Methylguanine DNA Methyltransferase; Photodynamic Therapy; 0485

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Singh, K. (2019). Small Molecule Probes for Photodynamic Therapy (PDT). (Masters Thesis). University of Toronto. Retrieved from http://hdl.handle.net/1807/99832

Chicago Manual of Style (16^th Edition):

Singh, Kamalpreet. “Small Molecule Probes for Photodynamic Therapy (PDT).” 2019. Masters Thesis, University of Toronto. Accessed February 26, 2021. http://hdl.handle.net/1807/99832.

MLA Handbook (7^th Edition):

Singh, Kamalpreet. “Small Molecule Probes for Photodynamic Therapy (PDT).” 2019. Web. 26 Feb 2021.

Vancouver:

Singh K. Small Molecule Probes for Photodynamic Therapy (PDT). [Internet] [Masters thesis]. University of Toronto; 2019. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/1807/99832.

Council of Science Editors:

Singh K. Small Molecule Probes for Photodynamic Therapy (PDT). [Masters Thesis]. University of Toronto; 2019. Available from: http://hdl.handle.net/1807/99832

16. Chalansonnet, Valérie. Caractérisation d’activités oxydo-réductases, leur expression et régulation : applications pour le diagnostic in vitro : Characterization of oxidase-reductase activities, their expression and regulation system : outcomes for in vitro diagnosis.

Degree: Docteur es, Microbiologie, 2016, Lyon

URL: http://www.theses.fr/2016LYSE1085

►

La réduction des liaisons azo (N=N) et des fonctions nitro (NO2) chez les bactéries est liée aux azoréductases et nitroréductases, enzymes catalysant ces réactions. Leur… (more)

▼

La réduction des liaisons azo (N=N) et des fonctions nitro (NO2) chez les bactéries est liée aux azoréductases et nitroréductases, enzymes catalysant ces réactions. Leur répartition homogène permet de les utiliser pour détecter les bactéries. Cette étude vise à confirmer leur intérêt comme marqueurs métaboliques, à optimiser les réactions enzymatiques pour la détection bactérienne et à approfondir la compréhension du lien entre l’activité nitroréductase et la résistance aux nitrofuranes.L’activité enzymatique est dépendante de la quantité d’enzyme, l’hypothèse d’une augmentation de la quantité de protéines pour accroitre l’activité et optimiser la détection des microorganismes a été testée. Le suivi de la transcription de gènes d’intérêt a permis d’observer une induction de leur transcription par des composés activateurs des mécanismes de régulation et certains substrats. L’ajout d’un inducteur transcriptionnel permet donc d’optimiser l’activité envers certains substrats et par conséquent la détection des bactéries. Ces études ont aussi contribué à identifier des liens entre structure moléculaire des substrats et capacité d’induction.La création et la caractérisation de différents mutants ayant des activités nitro- ou azoréductases altérées a permis de cribler de nouveaux substrats synthétiques en vue de leur utilisation dans des applications de diagnostic. Enfin, l’obtention et l’analyse génomique de souches obtenues par mutations aléatoire et ayant une résistance accrue à la nitrofurantoïne a mis en évidence un nouveau mécanisme de résistance, mécanisme qui apparaît également dans des isolats cliniques

Azo bond (N=N) and nitro reduction in bacteria is linked to the catalytic activities of azoreductase and nitoreductase enzymes, respectively. Their ubiquitous distribution enables their use for bacterial detection. This study aims to confirm their potential as metabolic markers, to optimize the enzymatic activities for improved detection and to generally enhance our understanding of azoreductase and nitroreductase activity . Enzymatic activity relies in part on protein quantity, increasing protein amount was tested as a solution for increased activity and improved bacterial detection. For that, the transcription of selected genes was followed in the presence of regulation system activators and substrates. Some of these compounds promoted an overexpression of selected enzymes and led to a better activity toward some substrates. Consequently, increasing the enzyme amount, through over transcription, can enable a better detection of bacteria. This study also contributed to the determination of structural elements required for induction by the substrate.Mutants with lowered azo or nitro reductase activity were constructed and used to screen new synthetic substrates which could be of use for in vitro diagnostic tests. Finally, strains with a high resistance to nitrofurantoin were obtained by random mutagenesis. Their genome analysis provided evidence for a new resistance mechanism, which can also be detected in…

Advisors/Committee Members: Gilbert, Christophe (thesis director), Orenga, Sylvain (thesis director).

Subjects/Keywords: Azoréductase; Nitroréductase; Microorganismes; Régulation; Nitrofuranes; Résistance; Escherichia coli; Azoreductase; Nitroreductase; Microorganisms; Regulation; Nitrofurans; Resistance; Escherichia coli; 579

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APA (6^th Edition):

Chalansonnet, V. (2016). Caractérisation d’activités oxydo-réductases, leur expression et régulation : applications pour le diagnostic in vitro : Characterization of oxidase-reductase activities, their expression and regulation system : outcomes for in vitro diagnosis. (Doctoral Dissertation). Lyon. Retrieved from http://www.theses.fr/2016LYSE1085

Chicago Manual of Style (16^th Edition):

Chalansonnet, Valérie. “Caractérisation d’activités oxydo-réductases, leur expression et régulation : applications pour le diagnostic in vitro : Characterization of oxidase-reductase activities, their expression and regulation system : outcomes for in vitro diagnosis.” 2016. Doctoral Dissertation, Lyon. Accessed February 26, 2021. http://www.theses.fr/2016LYSE1085.

MLA Handbook (7^th Edition):

Chalansonnet, Valérie. “Caractérisation d’activités oxydo-réductases, leur expression et régulation : applications pour le diagnostic in vitro : Characterization of oxidase-reductase activities, their expression and regulation system : outcomes for in vitro diagnosis.” 2016. Web. 26 Feb 2021.

Vancouver:

Chalansonnet V. Caractérisation d’activités oxydo-réductases, leur expression et régulation : applications pour le diagnostic in vitro : Characterization of oxidase-reductase activities, their expression and regulation system : outcomes for in vitro diagnosis. [Internet] [Doctoral dissertation]. Lyon; 2016. [cited 2021 Feb 26]. Available from: http://www.theses.fr/2016LYSE1085.

Council of Science Editors:

Chalansonnet V. Caractérisation d’activités oxydo-réductases, leur expression et régulation : applications pour le diagnostic in vitro : Characterization of oxidase-reductase activities, their expression and regulation system : outcomes for in vitro diagnosis. [Doctoral Dissertation]. Lyon; 2016. Available from: http://www.theses.fr/2016LYSE1085

University of Dundee

17. Sokolova, Antoaneta Y. Nitroaromatic pro-drug activation and resistance in the African trypanosome.

Degree: PhD, 2011, University of Dundee

URL: https://discovery.dundee.ac.uk/en/studentTheses/52c1537e-4a37-446c-b62c-86df5b95b2ea ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578815

► Sleeping sickness, caused by Trypanosoma brucei, is a deadly disease that affects some of the poorest countries in sub-Saharan Africa. Although the disease prevalence is… (more)

▼ Sleeping sickness, caused by Trypanosoma brucei, is a deadly disease that affects some of the poorest countries in sub-Saharan Africa. Although the disease prevalence is declining, strengthening of the current control efforts, including introduction of more adequate chemotherapeutic options, is needed to prevent the re-emergence of yet another epidemic. Nitroaromatic compounds, such as nifurtimox (in combination with eflornithine) and fexinidazole (in clinical trials), have been recently introduced for the treatment of the second stage of sleeping sickness. These compounds are believed to act as pro-drugs that require intracellular enzymatic activation for antimicrobial activity. Here, the role of the bacterial-like nitroreductase TbNTR as a nitrodrug activating enzyme is examined through overexpression and knock-out studies in T. brucei. Multiple attempts to purify soluble recombinant TbNTR from E. coli were unsuccessful, because the recombinant protein was found to be membrane associated. In keeping with the role of TbNTR in nitrodrug activation, loss of an NTR gene copy in T. brucei was found to be one, but not the only, mechanism that may lead to nitrodrug resistance. Furthermore, in the bloodstream form of T. brucei, resistance was relatively easy to select for nifurtimox, with no concurrent loss of virulence and at clinically relevant levels. More worryingly, nifurtimox resistance led to a decreased sensitivity of these parasites to other nitroaromatic compounds, including a high level of cross-resistance to fexinidazole. Conversely, generation of fexinidazole resistance resulted in cross-resistance to nifurtimox. Should these findings translate to the field, emerging nitrodrug resistance could reverse all recent advances in the treatment of sleeping sickness, made since the introduction of eflornithine 20 years ago. Therefore, all efforts should be made to ensure nitroaromatic drugs are used only in drug combination therapies against sleeping sickness, in order to protect them from emerging resistance.

Subjects/Keywords: 615; Sleeping sickness; African trypanosomiasis; Trypanosome; Trypanosoma brucei; Trypanosomiasis; Resistance; Pro-drug; Nitroaromatic; Nitroheterocyclic; Nifurtimox; Fexinidazole; Nitroreductase; NTR; Recombinant expression; Protein

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Sokolova, A. Y. (2011). Nitroaromatic pro-drug activation and resistance in the African trypanosome. (Doctoral Dissertation). University of Dundee. Retrieved from https://discovery.dundee.ac.uk/en/studentTheses/52c1537e-4a37-446c-b62c-86df5b95b2ea ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578815

Chicago Manual of Style (16^th Edition):

Sokolova, Antoaneta Y. “Nitroaromatic pro-drug activation and resistance in the African trypanosome.” 2011. Doctoral Dissertation, University of Dundee. Accessed February 26, 2021. https://discovery.dundee.ac.uk/en/studentTheses/52c1537e-4a37-446c-b62c-86df5b95b2ea ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578815.

MLA Handbook (7^th Edition):

Sokolova, Antoaneta Y. “Nitroaromatic pro-drug activation and resistance in the African trypanosome.” 2011. Web. 26 Feb 2021.

Vancouver:

Sokolova AY. Nitroaromatic pro-drug activation and resistance in the African trypanosome. [Internet] [Doctoral dissertation]. University of Dundee; 2011. [cited 2021 Feb 26]. Available from: https://discovery.dundee.ac.uk/en/studentTheses/52c1537e-4a37-446c-b62c-86df5b95b2ea ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578815.

Council of Science Editors:

Sokolova AY. Nitroaromatic pro-drug activation and resistance in the African trypanosome. [Doctoral Dissertation]. University of Dundee; 2011. Available from: https://discovery.dundee.ac.uk/en/studentTheses/52c1537e-4a37-446c-b62c-86df5b95b2ea ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578815

University of Kentucky

18. Pitsawong, Warintra. BASES FOR BREADTH - INSIGHTS INTO HOW THE MECHANISM AND DYNAMICS OF NITROREDUCTASE CAN EXPLAIN THIS ENZYME'S BROAD SUBSTRATE REPERTOIRE.

Degree: 2014, University of Kentucky

URL: https://uknowledge.uky.edu/chemistry_etds/44

► Nitroreductase from Enterobacter cloacae (NR) is a member of a large family of homologues represented in all branches of the tree of life. However the… (more)

Subjects/Keywords: nitroreductase; flavoproteins; enzyme kinetics; pre-steady state kinetics; reduction of nitroaromatic; broad substrate repertoire; Biochemistry, Biophysics, and Structural Biology

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Pitsawong, W. (2014). BASES FOR BREADTH - INSIGHTS INTO HOW THE MECHANISM AND DYNAMICS OF NITROREDUCTASE CAN EXPLAIN THIS ENZYME'S BROAD SUBSTRATE REPERTOIRE. (Doctoral Dissertation). University of Kentucky. Retrieved from https://uknowledge.uky.edu/chemistry_etds/44

Chicago Manual of Style (16^th Edition):

Pitsawong, Warintra. “BASES FOR BREADTH - INSIGHTS INTO HOW THE MECHANISM AND DYNAMICS OF NITROREDUCTASE CAN EXPLAIN THIS ENZYME'S BROAD SUBSTRATE REPERTOIRE.” 2014. Doctoral Dissertation, University of Kentucky. Accessed February 26, 2021. https://uknowledge.uky.edu/chemistry_etds/44.

MLA Handbook (7^th Edition):

Pitsawong, Warintra. “BASES FOR BREADTH - INSIGHTS INTO HOW THE MECHANISM AND DYNAMICS OF NITROREDUCTASE CAN EXPLAIN THIS ENZYME'S BROAD SUBSTRATE REPERTOIRE.” 2014. Web. 26 Feb 2021.

Vancouver:

Pitsawong W. BASES FOR BREADTH - INSIGHTS INTO HOW THE MECHANISM AND DYNAMICS OF NITROREDUCTASE CAN EXPLAIN THIS ENZYME'S BROAD SUBSTRATE REPERTOIRE. [Internet] [Doctoral dissertation]. University of Kentucky; 2014. [cited 2021 Feb 26]. Available from: https://uknowledge.uky.edu/chemistry_etds/44.

Council of Science Editors:

Pitsawong W. BASES FOR BREADTH - INSIGHTS INTO HOW THE MECHANISM AND DYNAMICS OF NITROREDUCTASE CAN EXPLAIN THIS ENZYME'S BROAD SUBSTRATE REPERTOIRE. [Doctoral Dissertation]. University of Kentucky; 2014. Available from: https://uknowledge.uky.edu/chemistry_etds/44

19. Franco, Jefferson Honorio [UNESP]. Biotransformação de corantes dispersos do tipo azo pela ação de enzimas redutoras e oxidação fotoeletrocatalítica após pré-concentração por MIP.

Degree: 2016, Universidade Estadual Paulista

URL: http://hdl.handle.net/11449/144994

► Corantes sintéticos do tipo azo têm sido um assunto de grande preocupação ambiental devido ao potencial genotóxico e mutagênico dos produtos de biotransformação. Deste modo,… (more)

▼ Corantes sintéticos do tipo azo têm sido um assunto de grande preocupação ambiental devido ao potencial genotóxico e mutagênico dos produtos de biotransformação. Deste modo, nos últimos anos a consequência da ingestão destes corantes presentes na agua potável servida à população é discutida por diversos autores. Este estudo avalia a ação de microssomas de fígado de rato, enzimas redutoras produzidas pela bactéria Escherichia coli (E. coli) e nitroredutase imobilizada na biotransformação de três corantes dispersos que possuem grupos azo, Disperse Red 73 (DR 73), Disperse Red 78 (DR 78) e Disperse Red 167 (DR 167). A técnica de Espectrofotometria de absorção molecular na região do Uv- visível, Cromatografia Líquida de Alta Eficiência com detector de arranjo de diodos (CLAE-DAD) e Cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS) foram técnicas usadas para identificar os principais produtos gerados após os processos de degradação dos corantes. Polímeros de impressão molecular magnéticos (MMIPs) foram investigados usando reações de polimerização por precipitação para pré-concentração do corante DR 73, juntamente com a degradação por fotoeletrocatálise e subsequente análise dos produtos por LC-MS/MS. Os estudos in vitro do metabolismo de biotransformação dos corantes têxteis com microssoma de fígado de rato mostraram que as reações ocorreram preferencialmente no grupo azo e nitro dos corantes, indicando a redução destes grupos pelas enzimas do citocromo P-450. Foram obtidos dois produtos de degradação para cada corante após reação com a bactéria E. coli; o corante DR 73 originou os produtos 3-((4-aminofenil)(etil)amino)propanitrila e 4-nitroanilina, os produtos 3-((4-aminofenil)(etil)amino)propanitrila e 2-cloro-4-nitroanilina foram obtidos após reação com o corante DR78 e o DR 167 originou dimetil 3,3`-((3-acetamido-4aminofenil)azanediyl)dipropanoato e 2-cloro-4-nitroanilina, indicando a clivagem do grupo azo, possivelmente, pela enzima azoredutase, produzida pela bacteria. A enzima nitroredutase, imobilizada em partículas magnéticas modificadas com tosil, mostrou que a redução dos corantes ocorreu preferencialmente no grupo nitro, enquanto que a enzima livre no meio reacional resultou em mais de um produto de biotransformação para cada corante, atuando em mais de um sítio da molécula, comprovando a eficácia da imobilização enzimática para estudos de biotransformação e formação de produtos majoritários. A mutagenicidade dos corantes foi avaliado pelo ensaio de Salmonella/microssoma realizado nas estirpes TA 98 e TA 100, com e sem S9. De acordo com este ensaio, DR 73 foi o mais mutagênico. O MMIP para o corante DR 73 apresentou excelentes valores de religação (16 mg g−1 e 6 mg g−1, para MMIP e MNIP, respectivamente) indicando que o polímero molecularmente impresso formou cavidades específicas para retenção do corante. Através dos resultados obtidos por LCMS/MS, observou-se 100% de degradação do corante em apenas 60 min de tratamento via fotoeletrocatálise para soluções mais diluidas do mesmo,… Advisors/Committee Members: Zanoni, Maria Valnice Boldrin [UNESP], Silva, Bianca Ferreira da, Universidade Estadual Paulista (UNESP).

Subjects/Keywords: Corantes e tingimento; Toxicologia ambiental; Escherichia coli; Biotransformação (Metabolismo); Espectrometria de massa; Disperse dyes; Escherichia coli; Nitroreductase; Rat liver microssomal; Molecularly imprinted polymers; LC-MS/MS

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Franco, J. H. [. (2016). Biotransformação de corantes dispersos do tipo azo pela ação de enzimas redutoras e oxidação fotoeletrocatalítica após pré-concentração por MIP. (Thesis). Universidade Estadual Paulista. Retrieved from http://hdl.handle.net/11449/144994

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Franco, Jefferson Honorio [UNESP]. “Biotransformação de corantes dispersos do tipo azo pela ação de enzimas redutoras e oxidação fotoeletrocatalítica após pré-concentração por MIP.” 2016. Thesis, Universidade Estadual Paulista. Accessed February 26, 2021. http://hdl.handle.net/11449/144994.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Franco, Jefferson Honorio [UNESP]. “Biotransformação de corantes dispersos do tipo azo pela ação de enzimas redutoras e oxidação fotoeletrocatalítica após pré-concentração por MIP.” 2016. Web. 26 Feb 2021.

Vancouver:

Franco JH[. Biotransformação de corantes dispersos do tipo azo pela ação de enzimas redutoras e oxidação fotoeletrocatalítica após pré-concentração por MIP. [Internet] [Thesis]. Universidade Estadual Paulista; 2016. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/11449/144994.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Franco JH[. Biotransformação de corantes dispersos do tipo azo pela ação de enzimas redutoras e oxidação fotoeletrocatalítica após pré-concentração por MIP. [Thesis]. Universidade Estadual Paulista; 2016. Available from: http://hdl.handle.net/11449/144994

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Kentucky

20. Zhang, Peng. NITROREDUCTASE: EVIDENCE FOR A FLUXIONAL LOW-TEMPERATURE STATE AND ITS POSSIBLE ROLE IN ENZYME ACTIVITY.

Degree: 2007, University of Kentucky

URL: https://uknowledge.uky.edu/gradschool_diss/492

► The enzyme nitroreductase (NR) catalyzes two-electron reduction of high explosives such as trinitrotoluene (TNT), a wide variety of other toxic nitroaromatic compounds, as well as… (more)

▼ The enzyme nitroreductase (NR) catalyzes two-electron reduction of high explosives such as trinitrotoluene (TNT), a wide variety of other toxic nitroaromatic compounds, as well as riboflavin derivatives, using a tightly-bound flavin mononucleotide (FMN) cofactor. It has important environmental and clinical implications. Previous studies have focused on elucidating NRs catalytic mechanism and solving its crystal structure. In this dissertation work, we first develop and implement new strategies for labeling NR with stable isotopes, and report a completely re-designed protocol for NRs purification. Then we report the successful assignment of over half of NRs backbone resonances using 3d-NMR methods. The most significant observation is that we find a well-resolved 2d 1H-15N HSQC NMR spectrum is obtained at 37°C for NR, while the HSQC at 4°C is poorly-dispersed and comprised of sharp overlapped peaks. Thus, it would appear that NR denatures at 4°C. However, as we demonstrate, the non-covalently-bound FMN cofactor is not released at the lower temperature, based on retention of the native flavin visible-CD spectrum. Similarly, far-UV CD spectroscopy shows that the protein retains full secondary structural content at 4C. In addition, near-UV CD and Fluorine-19 NMR experiments demonstrate that under completely native conditions (neutral pH, no additives) NR maintains a high degree of tertiary structure and well-defined hydrophobic side-chain packing, ruling out the possibility of a molten-globule state. Thus, our studies report strong evidence that the dramatic low-temperature (low-T) transition near 20°C observed by NMR is not the result of protein structural changes, but rather, we propose that NR exists as an ensemble of rapidly inter-converting structures, at lower temperature, only adopting a well-defined unique structure above 20°C. Both saturation-transfer from water and solvent proton-exchange measurements support our proposed model in which the unique high-T structure is favored entropically, by release of water molecules; on the other hand, the fluxional low-T state incorporates greater water solvation at 4°C. In the latter part of this study, we present preliminary data suggesting that the flexibility implied by easy water-access to the loosely-structured state plays a role in accommodating binding of diverse substrates. Therefore, the fluxional low-T state may be functionally important. A possible direct link between the enzyme dynamics and its catalytic activity will be an area of future investigation.

Subjects/Keywords: Nitroreductase|NMR spectroscopy|backbone resonance assignment|cold-denaturation|enzyme dynamics; Chemistry

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Zhang, P. (2007). NITROREDUCTASE: EVIDENCE FOR A FLUXIONAL LOW-TEMPERATURE STATE AND ITS POSSIBLE ROLE IN ENZYME ACTIVITY. (Doctoral Dissertation). University of Kentucky. Retrieved from https://uknowledge.uky.edu/gradschool_diss/492

Chicago Manual of Style (16^th Edition):

Zhang, Peng. “NITROREDUCTASE: EVIDENCE FOR A FLUXIONAL LOW-TEMPERATURE STATE AND ITS POSSIBLE ROLE IN ENZYME ACTIVITY.” 2007. Doctoral Dissertation, University of Kentucky. Accessed February 26, 2021. https://uknowledge.uky.edu/gradschool_diss/492.

MLA Handbook (7^th Edition):

Zhang, Peng. “NITROREDUCTASE: EVIDENCE FOR A FLUXIONAL LOW-TEMPERATURE STATE AND ITS POSSIBLE ROLE IN ENZYME ACTIVITY.” 2007. Web. 26 Feb 2021.

Vancouver:

Zhang P. NITROREDUCTASE: EVIDENCE FOR A FLUXIONAL LOW-TEMPERATURE STATE AND ITS POSSIBLE ROLE IN ENZYME ACTIVITY. [Internet] [Doctoral dissertation]. University of Kentucky; 2007. [cited 2021 Feb 26]. Available from: https://uknowledge.uky.edu/gradschool_diss/492.

Council of Science Editors:

Zhang P. NITROREDUCTASE: EVIDENCE FOR A FLUXIONAL LOW-TEMPERATURE STATE AND ITS POSSIBLE ROLE IN ENZYME ACTIVITY. [Doctoral Dissertation]. University of Kentucky; 2007. Available from: https://uknowledge.uky.edu/gradschool_diss/492

21. Mercier, Claire. Caractérisation d'activités oxydo-réductases, leurs systèmes de régulation et leur distribution au sein de la population microbienne : Characterization of oxidase-reductase activities, their regulation system and their districution within the microbial population.

Degree: Docteur es, Microbiologie générale et appliquée, 2013, Université Claude Bernard – Lyon I

URL: http://www.theses.fr/2013LYO10025

►

Les activités réductases sont des activités enzymatiques largement répandues au sein des microorganismes. Parmi ces activités, il est possible de distinguer différentes familles en fonction… (more)

▼

Les activités réductases sont des activités enzymatiques largement répandues au sein des microorganismes. Parmi ces activités, il est possible de distinguer différentes familles en fonction des substrats réduits comme les azoréductases qui catalysent la réduction des ponts « azo » (N=N) et les nitroréductases qui catalysent la réduction de la fonction nitro (NO2). Ce travail vise à étudier la répartition des activités réductases telles que nitroréductase et azoréductase au sein des microorganismes à laide de substrats devenant fluorescent une fois réduits. Ce travail a pour but dévaluer la pertinence dutilisation de ces activités enzymatiques dans des tests diagnostic de détection voire didentification des microorganismes recherchés dans un cadre clinique ou industriel. Une étude approfondie de ces activités enzymatiques a été faite sur deux espèces bactériennes : Escherichia coli et Enterococcus faecalis. Létude de lactivité azoréductase sest portée sur une seule enzyme (AzoR) chez E. coli et 2 enzymes (AzoA et EF0404) chez E. Faecalis. Concernant lactivité nitroréductase, 3 enzymes (NfsA, NfsB et AzoR) ont été mises en évidence chez E. coli tandis que 5 enzymes (AzoA, EF0404, EF1181, EF0648 et EF0655) ont été identifiées comme nitroréductases chez E. faecalis. Létude in vivo menée chez E. coli a mis en évidence le rôle indispensable dAzoR dans lactivité azoréductase aérobique alors quen conditions danaérobie la protéine AzoR est impliquée, mais une voie non précisément caractérisée à ce jour, indépendante de lenzyme AzoR, prend le relais. Lactivité nitroréductase dE. coli est due majoritairement aux enzymes NfsA et NfsB

Reductase activities are widespread among microorganisms. Among these activities, it is possible to distinguish different families based on reduced substrates such as azoreductases that catalyze the reduction of “azo” bounds (N=N) and nitroreductases that catalyze the reduction of nitro group (NO2). This work aims to study the distribution of reductase activities such as nitroreductase and azoreductase in microorganisms using fluorescent substrates. This work aims to evaluate the appropriateness of using these enzyme activities in diagnostic tests for detection or identification of microorganisms sought in a clinical or industrial context. A thorough study of these enzyme activities was performed on two bacterial species: Escherichia coli and Enterococcus faecalis. The study of azoreductase activity focused on a single enzyme (AzoR) in E. coli and 2 enzymes (AzoA and EF0404) in E. faecalis. Concerning nitroreductase activity, three enzymes (NfsA, AzoR and NfsB) have been identified in E. coli while 5 enzymes (AzoA, EF0404, EF1181, EF0648 and EF0655) have been identified as nitroreductases in E. faecalis. The in vivo study in E. coli has highlighted the essential role of AzoR in aerobic azoreductase activity whereas in anaerobic conditions the protein AzoR is involved, but an independent way of the enzyme AzoR, not specifically characterized to date, takes over. The nitroreductase activity of E.…

Advisors/Committee Members: Gilbert, Christophe (thesis director), Orenga, Sylvain (thesis director).

Subjects/Keywords: Azoréductase nitroréductase Escherichia coli; Enterococcus faecalis; Methyl red; Acide 7-nitrocoumarine-3-carboxylique; NADH; NADPH; Flavine; FMN; Azoreductase nitroreductase Escherichia coli; Enterococcus faecalis; Methyl red; 7- nitrocoumarine-3-carboxylic acid; NADH; NADPH; Flavin; FMN; 579

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Mercier, C. (2013). Caractérisation d'activités oxydo-réductases, leurs systèmes de régulation et leur distribution au sein de la population microbienne : Characterization of oxidase-reductase activities, their regulation system and their districution within the microbial population. (Doctoral Dissertation). Université Claude Bernard – Lyon I. Retrieved from http://www.theses.fr/2013LYO10025

Chicago Manual of Style (16^th Edition):

Mercier, Claire. “Caractérisation d'activités oxydo-réductases, leurs systèmes de régulation et leur distribution au sein de la population microbienne : Characterization of oxidase-reductase activities, their regulation system and their districution within the microbial population.” 2013. Doctoral Dissertation, Université Claude Bernard – Lyon I. Accessed February 26, 2021. http://www.theses.fr/2013LYO10025.

MLA Handbook (7^th Edition):

Mercier, Claire. “Caractérisation d'activités oxydo-réductases, leurs systèmes de régulation et leur distribution au sein de la population microbienne : Characterization of oxidase-reductase activities, their regulation system and their districution within the microbial population.” 2013. Web. 26 Feb 2021.

Vancouver:

Mercier C. Caractérisation d'activités oxydo-réductases, leurs systèmes de régulation et leur distribution au sein de la population microbienne : Characterization of oxidase-reductase activities, their regulation system and their districution within the microbial population. [Internet] [Doctoral dissertation]. Université Claude Bernard – Lyon I; 2013. [cited 2021 Feb 26]. Available from: http://www.theses.fr/2013LYO10025.

Council of Science Editors:

Mercier C. Caractérisation d'activités oxydo-réductases, leurs systèmes de régulation et leur distribution au sein de la population microbienne : Characterization of oxidase-reductase activities, their regulation system and their districution within the microbial population. [Doctoral Dissertation]. Université Claude Bernard – Lyon I; 2013. Available from: http://www.theses.fr/2013LYO10025

22. Park, Jonathan Taejoo. Enzymatic reduction of nitro compounds to amines with nitroreductases.

Degree: PhD, Chemical and Biomolecular Engineering, 2014, Georgia Tech

URL: http://hdl.handle.net/1853/52267

► NRs are enzymes that catalyze the reduction of nitroaromatics to their corresponding nitroso, hydroxylamine, and, in limited cases, amine They have gathered interest in many… (more)

Subjects/Keywords: Biocatalysis; Enzyme; Nitroreductase; Active pharmaceutical ingredient; Nitro; Reduction; Protein; Protein engineering

…62 5 Engineering towards Nitroreductase Functionality in Ene-reductase Scaffolds viii 63… …5.3.7 Nitroreductase vs. ene-reductase functionality ...................................... 78… …Nitrobenzene NFZ Nitrofurazone NR Nitroreductase NOB Nitrosobenzene OYE Old yellow enyzme… …typhimurium 21, nitrobenzene nitroreductase (nbzA) from Pseudomonas pseudoalcaligenes 22…

14 more images…

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Park, J. T. (2014). Enzymatic reduction of nitro compounds to amines with nitroreductases. (Doctoral Dissertation). Georgia Tech. Retrieved from http://hdl.handle.net/1853/52267

Chicago Manual of Style (16^th Edition):

Park, Jonathan Taejoo. “Enzymatic reduction of nitro compounds to amines with nitroreductases.” 2014. Doctoral Dissertation, Georgia Tech. Accessed February 26, 2021. http://hdl.handle.net/1853/52267.

MLA Handbook (7^th Edition):

Park, Jonathan Taejoo. “Enzymatic reduction of nitro compounds to amines with nitroreductases.” 2014. Web. 26 Feb 2021.

Vancouver:

Park JT. Enzymatic reduction of nitro compounds to amines with nitroreductases. [Internet] [Doctoral dissertation]. Georgia Tech; 2014. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/1853/52267.

Council of Science Editors:

Park JT. Enzymatic reduction of nitro compounds to amines with nitroreductases. [Doctoral Dissertation]. Georgia Tech; 2014. Available from: http://hdl.handle.net/1853/52267

University of Maryland

23. Rodgers, Mark Andrew. A DNA REPAIR/PHASE VARIATION REPORTER SYSTEM USING A POLY-GUANINE TRACT IN A NEISSERIA GONORRHOEAE NITROREDUCTASE GENE.

Degree: Cell Biology & Molecular Genetics, 2005, University of Maryland

URL: http://hdl.handle.net/1903/3096

► Neisseria gonorrhoeae undergoes phase variation to adapt to new environments, increase pathogenesis, and evade the host immune system. This may be due to defects in… (more)

Subjects/Keywords: Biology, Genetics; Biology, Microbiology; Biology, Molecular; Neisseria; nitroreductase; DNA repair; phase variation; reporter gene; genetics

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Rodgers, M. A. (2005). A DNA REPAIR/PHASE VARIATION REPORTER SYSTEM USING A POLY-GUANINE TRACT IN A NEISSERIA GONORRHOEAE NITROREDUCTASE GENE. (Thesis). University of Maryland. Retrieved from http://hdl.handle.net/1903/3096

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Rodgers, Mark Andrew. “A DNA REPAIR/PHASE VARIATION REPORTER SYSTEM USING A POLY-GUANINE TRACT IN A NEISSERIA GONORRHOEAE NITROREDUCTASE GENE.” 2005. Thesis, University of Maryland. Accessed February 26, 2021. http://hdl.handle.net/1903/3096.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Rodgers, Mark Andrew. “A DNA REPAIR/PHASE VARIATION REPORTER SYSTEM USING A POLY-GUANINE TRACT IN A NEISSERIA GONORRHOEAE NITROREDUCTASE GENE.” 2005. Web. 26 Feb 2021.

Vancouver:

Rodgers MA. A DNA REPAIR/PHASE VARIATION REPORTER SYSTEM USING A POLY-GUANINE TRACT IN A NEISSERIA GONORRHOEAE NITROREDUCTASE GENE. [Internet] [Thesis]. University of Maryland; 2005. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/1903/3096.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Rodgers MA. A DNA REPAIR/PHASE VARIATION REPORTER SYSTEM USING A POLY-GUANINE TRACT IN A NEISSERIA GONORRHOEAE NITROREDUCTASE GENE. [Thesis]. University of Maryland; 2005. Available from: http://hdl.handle.net/1903/3096

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

24. Hoben, John Patrick. PROTEIN SUPPRESSION OF FLAVIN SEMIQUINONE AS A MECHANISTICALLY IMPORTANT CONTROL OF REACTIVITY: A STUDY COMPARING FLAVOENZYMES WHICH DIFFER IN REDOX PROPERTIES, SUBSTRATES, AND ABILITY TO BIFURCATE ELECTRONS.

Degree: 2018, University of Kentucky

URL: https://uknowledge.uky.edu/chemistry_etds/108

► A growing number of flavoprotein systems have been observed to bifurcate pairs of electrons. Flavin-based electron bifurcation (FBEB) results in products with greater reducing power… (more)

▼ A growing number of flavoprotein systems have been observed to bifurcate pairs of electrons. Flavin-based electron bifurcation (FBEB) results in products with greater reducing power than that of the reactants with less reducing power. Highly reducing electrons at low reduction midpoint potential are required for life processes of both aerobic and anaerobic metabolic processes. For electron bifurcation to function, the semiquinone (SQ) redox intermediate needs to be destabilized in the protein to suppress its ability to trap electrons. This dissertation examines SQ suppression across a number of flavin systems for the purpose of better understanding the nature of SQ suppression within FBEB and elucidates potential mechanistic roles of SQ. The major achievement of this work is advancing the understanding of SQ suppression and its utility in flavoproteins with the capacity to bifurcate pairs of electrons. Much of these achievements are highlighted in Chapter 6. To contextualize these mechanistic studies, we examined the kinetic and thermodynamic properties of non-bifurcating flavoproteins (Chapters 2 and 3) as well as bifurcating flavoproteins (Chapters 4 and 5). Proteins were selected as models for SQ suppression with the aim of elucidating the role of an intermediate SQ in bifurcation. The chemical reactions of flavins and those mediated by flavoproteins play critical roles in the bioenergetics of all lifeforms, both aerobic and anaerobic. We highlight our findings in the context of electron bifurcation, the recently discovered third form of biological energy conservation. Bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I (Nfn) and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin were studied by transient absorption spectroscopy to compare electron transfer rates and mechanisms in the picosecond range. Different mechanisms were found to dominate SQ decay in the different proteins, producing lifetimes ranging over 3 orders of magnitude. The presence of a short-lived SQ alone was found to be insufficient to infer bifurcating activity. We established a model wherein the short SQ lifetime in Nfn results from efficient electron propagation. Such mechanisms of SQ decay may be a general feature of redox active site ensembles able to carry out bifurcation. We also investigated the proposed bifurcating electron transfer flavoprotein (Etf) from Pyrobaculum aerophilum (Pae), a hyperthermophilic archaeon. Unlike other Etfs, we observed a stable and strong charge transfer band (λmax= 724 nm) for Pae’s Etf upon reduction by NADH. Using a series of reductive titrations to probe bounds for the reduction midpoint potential of the two flavins, we argue that the heterodimer alone could participate in a bifurcation mechanism.

Subjects/Keywords: Flavin; Nitroreductase; NADOX; Electron Transfer Flavoprotein; Suppressed Semiquinone; Bifurcation; Biochemistry; Biophysics; Chemistry

…1 1.2 Nitroreductase… …NITROREDUCTASE ......15 2.1 Introduction… …Nitroreductase production ........................................................55 Production of wild… …pink) for nitroreductase (NR) in the presence of 25 or 2500 µM 2-(… …x28;e.g.NAD(P)H). An oxidoreductase with many homologs, nitroreductase, is…

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Hoben, J. P. (2018). PROTEIN SUPPRESSION OF FLAVIN SEMIQUINONE AS A MECHANISTICALLY IMPORTANT CONTROL OF REACTIVITY: A STUDY COMPARING FLAVOENZYMES WHICH DIFFER IN REDOX PROPERTIES, SUBSTRATES, AND ABILITY TO BIFURCATE ELECTRONS. (Doctoral Dissertation). University of Kentucky. Retrieved from https://uknowledge.uky.edu/chemistry_etds/108

Chicago Manual of Style (16^th Edition):

Hoben, John Patrick. “PROTEIN SUPPRESSION OF FLAVIN SEMIQUINONE AS A MECHANISTICALLY IMPORTANT CONTROL OF REACTIVITY: A STUDY COMPARING FLAVOENZYMES WHICH DIFFER IN REDOX PROPERTIES, SUBSTRATES, AND ABILITY TO BIFURCATE ELECTRONS.” 2018. Doctoral Dissertation, University of Kentucky. Accessed February 26, 2021. https://uknowledge.uky.edu/chemistry_etds/108.

MLA Handbook (7^th Edition):

Hoben, John Patrick. “PROTEIN SUPPRESSION OF FLAVIN SEMIQUINONE AS A MECHANISTICALLY IMPORTANT CONTROL OF REACTIVITY: A STUDY COMPARING FLAVOENZYMES WHICH DIFFER IN REDOX PROPERTIES, SUBSTRATES, AND ABILITY TO BIFURCATE ELECTRONS.” 2018. Web. 26 Feb 2021.

Vancouver:

Hoben JP. PROTEIN SUPPRESSION OF FLAVIN SEMIQUINONE AS A MECHANISTICALLY IMPORTANT CONTROL OF REACTIVITY: A STUDY COMPARING FLAVOENZYMES WHICH DIFFER IN REDOX PROPERTIES, SUBSTRATES, AND ABILITY TO BIFURCATE ELECTRONS. [Internet] [Doctoral dissertation]. University of Kentucky; 2018. [cited 2021 Feb 26]. Available from: https://uknowledge.uky.edu/chemistry_etds/108.

Council of Science Editors:

Hoben JP. PROTEIN SUPPRESSION OF FLAVIN SEMIQUINONE AS A MECHANISTICALLY IMPORTANT CONTROL OF REACTIVITY: A STUDY COMPARING FLAVOENZYMES WHICH DIFFER IN REDOX PROPERTIES, SUBSTRATES, AND ABILITY TO BIFURCATE ELECTRONS. [Doctoral Dissertation]. University of Kentucky; 2018. Available from: https://uknowledge.uky.edu/chemistry_etds/108

25. Chan-Hyams, Jasmine. Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy.

Degree: 2020, Victoria University of Wellington

URL: http://hdl.handle.net/10063/9064

► Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses (VDEPT) and bacteria (BDEPT) to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising… (more)

▼ Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses (VDEPT) and bacteria (BDEPT) to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to a prodrug. Bacterial nitroreductases, which are able to activate a range of anti-cancer nitroaromatic prodrugs to genotoxic metabolites, are of particular interest for GDEPT. The bystander effect is crucial to the success of GDEPT. The bystander effect is a measure of how efficiently activated prodrug metabolites are transferred from gene-expressing cells to neighbouring tissues. This promotes more extensive tumour cell killing. The bystander effect has been quantified for multiple nitroaromatic prodrugs in mixed multilayer human cell cultures. Although this is a good model for VDEPT it cannot simulate the ability of these prodrug metabolites to exit the bacterial vectors relevant to BDEPT. Prior to this work there was an unmet need for an in vitro method of quantifying the bystander effect as it occurs in BDEPT, i.e. a bacterial model of cell-to-cell transfer of activated prodrug metabolites. This thesis presents a method for measuring the bacterial bystander effect in vitro in a microplate based assay that was validated by flow cytometry. In this assay two Escherichia coli strains are grown in co-culture; an activator strain expressing the nitroreductase E. coli nfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activators to the recipients. Using this method, the bacterial bystander effect of the clinically relevant prodrugs, metronidazole, CB1954, nitro-CBI-DEI, PR-104A and SN27686, was assessed. Consistent with the bystander efficiencies in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. In contrast with observations in human cell multilayers, the PR-104A and SN27686 metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, I further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products. The outcomes of this investigation highlighted the importance of evaluating enzyme-prodrug combinations in models relevant to the intended GDEPT vector, as there can evidently be profound differences in efficacy in different settings. These findings motivated an investigation into the influence of the bystander effect on certain screening strategies used for directed evolution of nitroreductases. It was observed that the bacterial bystander effect can occur during fluorescence activated cell sorting (FACS) of a nitroreductase… Advisors/Committee Members: Ackerley, David.

Subjects/Keywords: nitroreductase; enzyme engineering; PR-104A; fluorescence activated cell sorting; bystander effect; GDEPT; gene-directed enzyme-prodrug therapy; CB1954; nitro-CBI-DEI; prodrug

…1.3.2.4 Nitroreductase/nitroaromatic prodrug -------------------------------------- 11… …Nitroreductase reduction mechanism ----------------------------------------------- 11 1.4.2 Historic… …29 1.5.1.1.3 Nitroreductase activated PR-104A----------------------------------------- 29… …FACS nitroreductase library selection --------------------------------------------------- 74… …Pre-selection of nitroreductase libraries using positive selection compounds 76 2.16 HPLC…

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Chan-Hyams, J. (2020). Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy. (Doctoral Dissertation). Victoria University of Wellington. Retrieved from http://hdl.handle.net/10063/9064

Chicago Manual of Style (16^th Edition):

Chan-Hyams, Jasmine. “Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy.” 2020. Doctoral Dissertation, Victoria University of Wellington. Accessed February 26, 2021. http://hdl.handle.net/10063/9064.

MLA Handbook (7^th Edition):

Chan-Hyams, Jasmine. “Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy.” 2020. Web. 26 Feb 2021.

Vancouver:

Chan-Hyams J. Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy. [Internet] [Doctoral dissertation]. Victoria University of Wellington; 2020. [cited 2021 Feb 26]. Available from: http://hdl.handle.net/10063/9064.

Council of Science Editors:

Chan-Hyams J. Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy. [Doctoral Dissertation]. Victoria University of Wellington; 2020. Available from: http://hdl.handle.net/10063/9064

Open Access Theses and Dissertations