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This thesis was scanned from the print manuscript for digital preservation and is copyright the author. Researchers can access this thesis by asking their local university, institution or public library to make a request on their behalf. Monash staff and postgraduate students can use the link in the References field. Winner of the Mollie Holman Doctoral Medal for Excellence

In the last twenty years, the recovery movement in alcohol and other drugs (AOD) has emerged as a major influence on alcohol and drug policy and practice in the UK, US and Australia. In roughly the same period of time, the desistance movement has become increasingly prominent in academic criminology, and is increasingly influential in criminal justice practice, particularly in the area of\ud probation. Furthermore, the populations involved in recovery and desistance research have significant overlap, yet there has been little shared learning across these areas. The current article explores the evolution of thinking around desistance and what lessons it might offer conceptual models of recovery. It will be argued that one of the most important shared assumptions relates to\ud identity change, and the extent to which these identity changes are intrinsically social or 'relational'. The paper will advance a social identity model as a mechanism for understanding the journey to recovery or desistance and the centrality of reintegration into communities for a coherent model and public policy around addiction recovery.

α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A — on the opposite face of the molecule — more liable to adopt an ‘open’ state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin–enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the ‘open’ state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.

A gene encoding a novel component of the cellulolytic complex (cellulosome) of the anaerobic fungus Piromyces sp. strain E2 was identified. The encoded 538 amino acid protein, named Celpin, consists of a signal peptide, a positively charged domain of unknown function followed by two fungal dockerins, typical for components of the extracellular fungal cellulosome. The C-terminal end consists of a 380 amino acid serine proteinase inhibitor (or serpin) domain homolog, sharing 30 % identity and 50 % similarity to vertebrate and bacterial serpins. Detailed protein sequence analysis of the serpin domain revealed that it contained all features of a functional serpin. It possesses the conserved amino acids present in more than 70 % of known serpins and it contained the consensus of inhibiting serpins. Because of the confined space of the fungal cellulosome inside plant tissue and the auto-proteolysis of plant material in the rumen, the fungal serpin is presumably involved in protection of the cellulosome against plant proteinases. The Celpin protein of Piromyces sp. strain E2 is the first non-structural, non-hydrolytic fungal cellulosome component. Furthermore, the Celpin protein of Piromyces sp. strain E2 is the first representative of a serine proteinase inhibitor of the fungal kingdom.

Phenylpropiolyl peroxide and its p-methyl -, p-methoxy-, p-chloro -, and p-bromo-substituted analogues have been synthesised in good yield and high purity by a method involving the reaction of the corresponding carboxylic acid with 98% hydrogen peroxide and dicyclohexylcarbodiimide. The parent compound has been decomposed in a variety of solvents and the mechanism of induced decomposition in these solvents investigated. The decomposition of the peroxides in chloroform at 65.0° in the presence of equimolar (0.05M) quantities of 3,4- dichloroetyrene to inhibit the induced decomposition enabled estimation of the first-order rate constants. Application of the Arrhenius' equation to the data from decompositions carried out in chloroform at different temperatures enabled estimation of the energy of activation for the decomposition of the parent compound and the p- methyl - and p- methoxy- analogues. The p-methoxy compound had a greatly enhanced rate of decomposition and lower activation energy - suggesting a different mechanism and the possibility of a non-homolytic decomposition. The synthesis of systems designed to differentiate between bridged and classical free radical intermediates by identification of the reaction products has been investigated. The production of radicals of this nature from aldehydes and azo compounds was unsuccessful due to the difficulty experienced in synthesis of the starting materials. The synthesis of 4-t-butyl-2-bromocyclohexylformyl peroxide has been achieved from the corresponding acid with 98% hydrogen peroxide and dicyclohexylcarbodiimide. 4-t-butyl-l-cyanocyclohexene was found to undergo stereo-specific addition of hydrogen bromide to give exclusively the trans addition product, namely, trans-4-t-butyl-cis-2-bromo -l-cyanocyclohexane, under both free radical and ionic conditions. The significance of this in relation to bridged radicals is discussed. The decomposition of the bromo peroxide did not give the expected products when investigated under similar conditions to those employed for the parent compound, 4-t-butylcyclohexylformyl peroxide. This limited the amount of information that could be obtained.

The accumulation of β-amyloid (Aβ) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aβ toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aβ levels by clearing the Aβ plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aβ are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aβ toxicity is indirect was supported by the finding that Aβ is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aβ toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy is not identical to its protection against Aβ toxicity.

Severe α1‐antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1‐antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA‐encoded chemical library to undertake a high‐throughput screen to identify small molecules that bind to, and stabilise Z α1‐antitrypsin. The lead compound blocks Z α1‐antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1‐antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1‐antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that “mutation ameliorating” small molecules can block the aberrant polymerisation that underlies Z α1‐antitrypsin deficiency., A chemistry campaign has developed a small molecule that stabilises the severe Z deficiency mutant of α1‐antitrypsin. The lead compound binds to a cryptic pocket and blocks the conformational change and pathological polymerisation that underlie α1‐antitrypsin deficiency.