title: Toward catalytic antibodies capable of ergopeptine hydrolysis

authors: Dines, Mark

abstract: Animals grazing endophyte infected pasture are exposed to the toxic effects of ergopeptine compounds which are produced by the grass-endophyte interaction. Grasses containing endophytic fungi have a competitive advantage and comprise over 60% of pastureland. Catalytic antibody vaccines would permit the use of endophyte-infected grasses without the risk of animal toxicoses. The production of catalytic antibodies capable of hydrolysing ergopeptine toxins was investigated. The literature pertaining to catalytic antibodies is reviewed with particular focus on hydrolytic reactions. Model systems were used to investigate the potential of this technique.

Model phosphorus-ester and -amide compounds were synthesised and characterised. Problems were experienced forming the RPO(OCH₂CH₃)X where X is NH(C₆H₄NO₂), and R is a protected benzyl system, successful generation of model compounds where X is NHCH₂(C₆H₅) and O(C₆H₄NO₂) was achieved. The compound HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) was synthesised as a transition-state emulator for the generation of polyclonal antibodies capable of hydrolysing (C₆H₅)CH₂COO(C₆H₄NO₂) and (C₆H₅)CH₂CONH(C₆H₄NO₂). The transition-state emulator was conjugated to Bovine Serum Albumin, Thyroglobulin and Keyhole Limpet Haemocyanin. Sheep, mice and a rabbit were immunised with the resulting conjugate material.

The immune rabbit serum was found to rapidly hydrolyse ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) relative to the non-immune serum, however, purification of the immune rabbit antibodies indicated that the hydrolysis of ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) was not due to antibody catalysis. The non-purified sera from the immune sheep and mice was found to hydrolyse ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) at a similar rate to the non-immune sera and no immune sera was found to hydrolyse amide ((C₆H₅)CH₂CONH(C₆H₄NO₂)). Investigation of sera from immunised animals by ELISA indicated production of polyclonal antibodies capable of binding the HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) material conjugated to protein. This protein was different to that used as immunogen. However, the non-conjugated HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) material did not competitively inhibit polyclonal antibody binding to the protein conjugates, suggesting that the polyclonal antibodies generated required the presence of a conjugated protein environment for binding.
 
An investigation of the ergot alkaloids produced by one grass-endophyte association (Acnatherum inebrians) known to contain high levels of ergot alkaloid was conducted. Extraction methodology and reverse phase HPLC separation systems were developed for these compounds. Use of these methods enabled the purification of iso-lysergic acid amide and ergonovinine from Acnatherum inebrians. These compounds were characterised using NMR.

This thesis details the generation of a model transition-state compound and subsequent immunisation of this compound into animals. Antibodies from the immune animals were not found to catalyse the hydrolysis of amide or ester bonds. This work and the work of others suggests that a better understanding of the emulation of the amide transition-state is required before detoxification of ergopeptines by amide hydrolysising antibodies will be achieved.

text: Toward catalytic antibodies capable of ergopeptine hydrolysis Animals grazing endophyte infected pasture are exposed to the toxic effects of ergopeptine compounds which are produced by the grass-endophyte interaction. Grasses containing endophytic fungi have a competitive advantage and comprise over 60% of pastureland. Catalytic antibody vaccines would permit the use of endophyte-infected grasses without the risk of animal toxicoses. The production of catalytic antibodies capable of hydrolysing ergopeptine toxins was investigated. The literature pertaining to catalytic antibodies is reviewed with particular focus on hydrolytic reactions. Model systems were used to investigate the potential of this technique.

Model phosphorus-ester and -amide compounds were synthesised and characterised. Problems were experienced forming the RPO(OCH₂CH₃)X where X is NH(C₆H₄NO₂), and R is a protected benzyl system, successful generation of model compounds where X is NHCH₂(C₆H₅) and O(C₆H₄NO₂) was achieved. The compound HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) was synthesised as a transition-state emulator for the generation of polyclonal antibodies capable of hydrolysing (C₆H₅)CH₂COO(C₆H₄NO₂) and (C₆H₅)CH₂CONH(C₆H₄NO₂). The transition-state emulator was conjugated to Bovine Serum Albumin, Thyroglobulin and Keyhole Limpet Haemocyanin. Sheep, mice and a rabbit were immunised with the resulting conjugate material.

The immune rabbit serum was found to rapidly hydrolyse ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) relative to the non-immune serum, however, purification of the immune rabbit antibodies indicated that the hydrolysis of ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) was not due to antibody catalysis. The non-purified sera from the immune sheep and mice was found to hydrolyse ester ((C₆H₅)CH₂COO(C₆H₄NO₂)) at a similar rate to the non-immune sera and no immune sera was found to hydrolyse amide ((C₆H₅)CH₂CONH(C₆H₄NO₂)). Investigation of sera from immunised animals by ELISA indicated production of polyclonal antibodies capable of binding the HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) material conjugated to protein. This protein was different to that used as immunogen. However, the non-conjugated HOOCCH₂CH₂CONH(C₆H₄)CH₂PO(OH)O(C₆H₄NO₂) material did not competitively inhibit polyclonal antibody binding to the protein conjugates, suggesting that the polyclonal antibodies generated required the presence of a conjugated protein environment for binding.
 
An investigation of the ergot alkaloids produced by one grass-endophyte association (Acnatherum inebrians) known to contain high levels of ergot alkaloid was conducted. Extraction methodology and reverse phase HPLC separation systems were developed for these compounds. Use of these methods enabled the purification of iso-lysergic acid amide and ergonovinine from Acnatherum inebrians. These compounds were characterised using NMR.

This thesis details the generation of a model transition-state compound and subsequent immunisation of this compound into animals. Antibodies from the immune animals were not found to catalyse the hydrolysis of amide or ester bonds. This work and the work of others suggests that a better understanding of the emulation of the amide transition-state is required before detoxification of ergopeptines by amide hydrolysising antibodies will be achieved.

year: 1998

title: Privacy-preserving encoding for cloud computing

authors: Will, Mark Antony

subjects: Homomorphic Encryption

abstract: Information in the cloud is under constant attack from cyber criminals as profitability increases; user privacy is also at risk with data being mined for monetary value – the new gold. A single leak could have devastating consequences for a person or organisation, yet users have limited control over their privacy. It is becoming clear that the current model for public cloud computing is flawed, where cloud vendors and their employees can no longer be trusted to protect user data. Privacy-preserving computation in the cloud keeps data private at all times but still remains functional, thus returning control of data back to users. The cloud could then perform operations using data that it cannot comprehend. The end-user would then be able to retrieve the results from the cloud and unlock the real answers.
Homomorphic encryption is a solution for privacy-preserving processing, allowing computation over cipher text. At the time of writing, a fully homomorphic system allows arbitrary operations but requires minutes to compute an operation, whereas partially homomorphic encryption can only support a single operation, meaning it cannot be a generic solution to privacy-preserving computing. Another solution is multi-party computation, which uses a distributed approach built upon homomorphic encryption but currently suffers other limitations like reusability and lacks the ability to be truly dynamic.
The primary objective of this research is to design a solution for the cloud that offers privacy-preserving data computation but provides performance and flexibility. A novel approach for multi-party computation is developed, where the combination of encoding and distribution helps provide the balance between security, performance and utility. Privacy is maintained by each distributed entity only receiving a small portion of the actual data through encoding, where attempting to brute-force the data results in a vast number of possibilities, similar to encryption. Functions are defined with universal or custom logic and are computed quickly, as the performance overhead is no longer computational but network latency. A cloud voting application was used for analysis between existing solutions and the novel approach taken by this research, which is able to add thousands of votes per minute, giving practical privacy-preserving processing in the cloud.

text: Privacy-preserving encoding for cloud computing Information in the cloud is under constant attack from cyber criminals as profitability increases; user privacy is also at risk with data being mined for monetary value – the new gold. A single leak could have devastating consequences for a person or organisation, yet users have limited control over their privacy. It is becoming clear that the current model for public cloud computing is flawed, where cloud vendors and their employees can no longer be trusted to protect user data. Privacy-preserving computation in the cloud keeps data private at all times but still remains functional, thus returning control of data back to users. The cloud could then perform operations using data that it cannot comprehend. The end-user would then be able to retrieve the results from the cloud and unlock the real answers.
Homomorphic encryption is a solution for privacy-preserving processing, allowing computation over cipher text. At the time of writing, a fully homomorphic system allows arbitrary operations but requires minutes to compute an operation, whereas partially homomorphic encryption can only support a single operation, meaning it cannot be a generic solution to privacy-preserving computing. Another solution is multi-party computation, which uses a distributed approach built upon homomorphic encryption but currently suffers other limitations like reusability and lacks the ability to be truly dynamic.
The primary objective of this research is to design a solution for the cloud that offers privacy-preserving data computation but provides performance and flexibility. A novel approach for multi-party computation is developed, where the combination of encoding and distribution helps provide the balance between security, performance and utility. Privacy is maintained by each distributed entity only receiving a small portion of the actual data through encoding, where attempting to brute-force the data results in a vast number of possibilities, similar to encryption. Functions are defined with universal or custom logic and are computed quickly, as the performance overhead is no longer computational but network latency. A cloud voting application was used for analysis between existing solutions and the novel approach taken by this research, which is able to add thousands of votes per minute, giving practical privacy-preserving processing in the cloud.

year: 2019

title: The peopling and development of Aotearoa New Zealand: A demographic history, 1769-2020

authors: Pool, Ian

subjects: Aotearoa

abstract: This unpublished manuscript is the first demographic history of Aotearoa New Zealand, integrating 250 years of Māori and non-Māori data. It explores how development, in the broadest social, cultural and economic sense drove population changes, but was in turn driven by demographic factors. Based on extensively referenced research and analysis, the timeline starts with the earliest European settlement and finishes with the multicultural and aging society of the first decades of the twenty-first century.  It encompasses momentous changes, including: successive waves of migration, European settler demographic dominance occurring rapidly through natural increase, Māori nineteenth century cultural devastation and population decline, and twentieth-century resurgence, the Māori rural/urban exodus beginning in the 1940s, and the post-war non-Māori baby boom and baby bust. At each stage, this book demonstrates how profoundly the story of Aotearoa New Zealand is also the demographic story of its peopling.

text: The peopling and development of Aotearoa New Zealand: A demographic history, 1769-2020 This unpublished manuscript is the first demographic history of Aotearoa New Zealand, integrating 250 years of Māori and non-Māori data. It explores how development, in the broadest social, cultural and economic sense drove population changes, but was in turn driven by demographic factors. Based on extensively referenced research and analysis, the timeline starts with the earliest European settlement and finishes with the multicultural and aging society of the first decades of the twenty-first century.  It encompasses momentous changes, including: successive waves of migration, European settler demographic dominance occurring rapidly through natural increase, Māori nineteenth century cultural devastation and population decline, and twentieth-century resurgence, the Māori rural/urban exodus beginning in the 1940s, and the post-war non-Māori baby boom and baby bust. At each stage, this book demonstrates how profoundly the story of Aotearoa New Zealand is also the demographic story of its peopling.

year: 2026