dc.contributor.advisor |
Mitra, A. |
en |
dc.contributor.author |
Radjainia, Mazdak |
en |
dc.date.accessioned |
2020-07-08T04:58:54Z |
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dc.date.available |
2020-07-08T04:58:54Z |
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dc.date.issued |
2010 |
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dc.identifier.uri |
http://hdl.handle.net/2292/52131 |
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dc.description |
Full text is available to authenticated members of The University of Auckland only. |
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dc.description.abstract |
Proteins are the Worldiorses of all living organisms and carry out the majority of cellular functions. Malfunction of a particular protein or a protein complex can Iead to disease states. In principle, administration ofbioactive polypeptides that can rescue the activity of an essential but defective component of a biochemical pathway or abrogate a malignant process can Iead to cure. Unfortunately accurate targeting of such compounds into cells is difficult and a significant amount of effort is directed at gene therapy. On the other hand in some cases, in-serum active proteins such as antibodies and adipokines elicit significant potential for therapeutic development. Anthrax toxin (AT) neutralizing antibodies and the adipokine adiponectin are such candidates and these constitute the focus of structural studies reported in this thesis using transmission electron microscopy and image analysis. The aim of these studies is to shed light on the mode of action of these molecules as potential therapeutics. It has been suggested that global political instabilities and conflicts of the 21st century can escalate into biowarfare or bioterrorism involving the usage of weaponized Bacillus anthracis spores (AT) by military forces, terrorist organizations or even individuals. Prophylactic measures or post-exposure therapies are needed to counter this threat. The anthrax toxin neutralizing monoclonal antibody 1G3 has been shown to be the most protective and the only one from a panel of murine-derived neutralizing antibodies to significantly delay time to death when administered to animals infected with Bacillus anthracis spores. Results of single-particle analysis showed that 1G3 severely impacts the structure of the pore-forming protective antigen heptamer (PA63h) - the AT moiety that mediates cytotoxic lethal factor (LF) internalization - forming a dodecameric supercomplex that was easily distinguished from intact PA63h. This unconventional mode of antibody action is shared by the bivalent F(ab,)2 and to a small extent even by single-chain scFV fragments of 1G3 supporting the evidence that FC domain dependency of neutralizing antibodies probably stems from FC-receptor binding. The created supercomplex phenotype highlights the feasibility for high throughput screening for AT antidotes and vaccines precipitating analogous structural alterations in the PA63 heptamer. Adipose tissue has dual functions as energy storage and as an endocrine organ. It has not evolved to cope with modem dietary habits that Iead to obesity and associated pathologies due to adipokine dysregulation. Adiponectin regulates glucose and fatty acid metabolism, immune and inflammatory responses, cell differentiation and has been termed the "guardian angel" in the metabolic syndrome for its protective properties against atherogenic and insulin resistance. Adiponectin therapy is not available yet mainly because of the Iow abundance of the hormone and Iack of mechanistic knowledge of the exact roles the different, bioactive post-translationally modified low, middle and high molecular weight (LMW, MMW and HMW) adiponectin variants. LMW and HMW adiponectin can be visually distinguished by electron microscopy and for both three-dimensional single-particle reconstructions were generated. The LMW structure represents the smallest yet reconstruction by single-particle analysis available in literature. For the HMW, an ensemble of Conformationally varying structures was identified highlighting the immense flexibility of this system. In addition, the threedimensional structural analysis revealed that the stretch of amino acid residues comprising the hyper-variable domain (AHD) at the N-terminal non-collagenous domain is very likely involved in the assembly of LMW adiponectin trimers to insulin sensitizing HMW forms. This suggestion was validated by size exclusion chromatography experiments of chemically synthesized AHD, which showed that oligomeric products corresponding to LMW and MMW were formed. Further, the effect on the observed oligomerization process due to site-specific alterations in the amino-acid sequence of the AHD construct suggests that this N-terminal region may provide clues to understanding the assembly process and therefore possibly create an avenue of generating therapeutic adiponectin mimetic. In conclusion, the structural investigations of the two very different biological systems investigated in this thesis that share not only activity in the serum as a common denominator, but also establish the possibility of using single-particle image analysis as a tool to efficiently distinguish between different bioactive states. Such pursuits can Iead to the development or quality control of anthrax and adiponectin therapeutics. |
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dc.publisher |
ResearchSpace@Auckland |
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dc.relation.ispartof |
PhD Thesis - University of Auckland |
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dc.relation.isreferencedby |
UoA99204058614002091 |
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dc.rights |
Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. |
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dc.rights |
Restricted Item. Full text is available to authenticated members of The University of Auckland only. |
en |
dc.rights.uri |
https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm |
en |
dc.title |
Structural investigations of serum-acting proteins anthrax toxin and adiponectin |
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dc.type |
Thesis |
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thesis.degree.discipline |
Biological Sciences |
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thesis.degree.grantor |
The University of Auckland |
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thesis.degree.level |
Doctoral |
en |
thesis.degree.name |
PhD |
en |
dc.rights.holder |
Copyright: The author |
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dc.identifier.wikidata |
Q112884325 |
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