title: Mechanism of RNA cleavage by VapC from Pyrobaculum aerophilum

authors: Duyvestyn, Johanna Maria

subjects: VapBC

abstract: Proteins belonging to the PIN-domain family are widespread across bacteria, archaea and eukaryotes. PIN-domain structures are well characterised and contain three highly conserved acidic residues that are orientated to form the active site of the enzyme. VapC is the toxic component of a toxin-antitoxin (TA) complex, and contains a PIN-domain. The VapBC TAs are the largest TA family, and are found in expanded copy numbers in a variety of unrelated organism including the human pathogen Mycobacterium tuberculosis. It is proposed that TAs play a role in metabolic regulation, under conditions of stress. VapCs are metal dependent endoribonucleases that target specific sequences in single stranded RNA and inhibit translation by degrading mRNA transcripts. The mechanism by which VapC cleaves RNA and achieves this specificity is unknown.
 
In this thesis, VapC 2754 from Pyrobaculum aerophilum (VapCPAE2754) is analysed, and a mechanism for catalysis of RNA cleavage by PIN-domains is proposed. Mutations were made to conserved residues in the putative active site to test the hypothesized mechanism. A fluorometric kinetic assay was designed to allow differences in activity to be accurately determined, so that conclusions about the effect of the mutations could be drawn. Mutations made to the conserved acidic amino acids eliminated RNase activity, confirming that these residues are essential to catalysis. A structure for the wild-type VapCPAE2754 was determined, containing cadmium ions and an acetate bound into the active site.

Knowledge of the enzymatic mechanism of PIN domains will enable a greater understanding of this important protein family, and serve as the basis for future drug design by transition state analogue inhibiters. This is especially important in VapC enzymes due to their proposed role in the pathology of M. tuberculosis.

text: Mechanism of RNA cleavage by VapC from Pyrobaculum aerophilum Proteins belonging to the PIN-domain family are widespread across bacteria, archaea and eukaryotes. PIN-domain structures are well characterised and contain three highly conserved acidic residues that are orientated to form the active site of the enzyme. VapC is the toxic component of a toxin-antitoxin (TA) complex, and contains a PIN-domain. The VapBC TAs are the largest TA family, and are found in expanded copy numbers in a variety of unrelated organism including the human pathogen Mycobacterium tuberculosis. It is proposed that TAs play a role in metabolic regulation, under conditions of stress. VapCs are metal dependent endoribonucleases that target specific sequences in single stranded RNA and inhibit translation by degrading mRNA transcripts. The mechanism by which VapC cleaves RNA and achieves this specificity is unknown.
 
In this thesis, VapC 2754 from Pyrobaculum aerophilum (VapCPAE2754) is analysed, and a mechanism for catalysis of RNA cleavage by PIN-domains is proposed. Mutations were made to conserved residues in the putative active site to test the hypothesized mechanism. A fluorometric kinetic assay was designed to allow differences in activity to be accurately determined, so that conclusions about the effect of the mutations could be drawn. Mutations made to the conserved acidic amino acids eliminated RNase activity, confirming that these residues are essential to catalysis. A structure for the wild-type VapCPAE2754 was determined, containing cadmium ions and an acetate bound into the active site.

Knowledge of the enzymatic mechanism of PIN domains will enable a greater understanding of this important protein family, and serve as the basis for future drug design by transition state analogue inhibiters. This is especially important in VapC enzymes due to their proposed role in the pathology of M. tuberculosis.

year: 2012

title: The Howard government's foreign policy: Really realist?

authors: McCraw, David

subjects: foreign policy

abstract: Several scholars have suggested that the Howard government took a Realist approach to foreign policy, and others have claimed that it made important deviations from Realism. This article constructs a template of a Realist foreign policy and examines the Howard government's policies against it. It finds that the government scores highly on two of the indicators of Realism – emphasis on military power and lack of enthusiasm for multilateral institutions – and scores fairly well on the third, the subordination of values promotion to the advancement of interests. The Howard government, however, promoted democracy and human rights in situations in which no other Australian interest was involved, so its Realism was somewhat modified.
The foreign policy of the Howard government has been characterised by several scholars as being in the Realist tradition of all Australian Liberal–National governments. Other scholars, however, have noted an important set of departures from that tradition, sparked, they say, by the imperatives of domestic politics. This article seeks to examine more closely the question of the Realist nature of recent Australian foreign policy. It will seek to establish a template of a Realist foreign policy, assess the Howard government's policies against that template and, finally, suggest how the Howard government's foreign policy may best be characterised.

text: The Howard government's foreign policy: Really realist? Several scholars have suggested that the Howard government took a Realist approach to foreign policy, and others have claimed that it made important deviations from Realism. This article constructs a template of a Realist foreign policy and examines the Howard government's policies against it. It finds that the government scores highly on two of the indicators of Realism – emphasis on military power and lack of enthusiasm for multilateral institutions – and scores fairly well on the third, the subordination of values promotion to the advancement of interests. The Howard government, however, promoted democracy and human rights in situations in which no other Australian interest was involved, so its Realism was somewhat modified.
The foreign policy of the Howard government has been characterised by several scholars as being in the Realist tradition of all Australian Liberal–National governments. Other scholars, however, have noted an important set of departures from that tradition, sparked, they say, by the imperatives of domestic politics. This article seeks to examine more closely the question of the Realist nature of recent Australian foreign policy. It will seek to establish a template of a Realist foreign policy, assess the Howard government's policies against that template and, finally, suggest how the Howard government's foreign policy may best be characterised.

year: 2008

title: The Māori precariat: A silhouette

authors: Stubbs, Thomas

abstract: Recent financial crises and a host of punitive labour and welfare reforms have intensified socio-economic divisions across advanced nations. In Aotearoa New Zealand, the indigenous Maori peoples are subject to systemic labour market disadvantages, and are thus more likely to be affected by increased inequalities in times of economic downturn. Despite the legacies of systematic economic exclusion and discrimination in the economy, the majority of Maori demonstrate a resounding level of social resilience in the face of this hostile context. Even so, there is evidence to suggest that an unacceptable number of Maori still struggle to overcome these challenges. For instance, 13.0 per cent of Maori are in temporary work compared to 8.2 per cent of Pakeha,1 and 12.4 per cent of Maori are experiencing unemployment against 4.4 per cent. of Pakeha.2 These trends contribute to a range of socio-economic challenges, to which mainstream and Maori service providers are struggling to respond.
In this chapter, we develop a demographic silhouette to seek an understanding of socio-economically marginalised - or 'precariat' - Maori. Although we are wary of portraying Maori in an unduly negative light, we also submit that an appreciation of the extent of the issues faced by this group is necessary if our analysis is to be useful in the development of an effective response. We begin by defining the precariat. Using data obtained from Statistics New Zealand's Te Kupenga survey of Maori wellbeing, we then outline the prevalence and composition of the Maori precariat in Aotearoa New Zealand. Overall, we find that the Maori precariat comprises a substantial portion of the Maori population, particularly among females, younger age groups, and those with low or no qualifications. They are predominantly located-in regions that have high levels of seasonal employment, and in more deprived areas. We also find the value attached to Maori culture and experiences of stigma for this group are, for the most part, similar to those among the Maori non-precariat.

text: The Māori precariat: A silhouette Recent financial crises and a host of punitive labour and welfare reforms have intensified socio-economic divisions across advanced nations. In Aotearoa New Zealand, the indigenous Maori peoples are subject to systemic labour market disadvantages, and are thus more likely to be affected by increased inequalities in times of economic downturn. Despite the legacies of systematic economic exclusion and discrimination in the economy, the majority of Maori demonstrate a resounding level of social resilience in the face of this hostile context. Even so, there is evidence to suggest that an unacceptable number of Maori still struggle to overcome these challenges. For instance, 13.0 per cent of Maori are in temporary work compared to 8.2 per cent of Pakeha,1 and 12.4 per cent of Maori are experiencing unemployment against 4.4 per cent. of Pakeha.2 These trends contribute to a range of socio-economic challenges, to which mainstream and Maori service providers are struggling to respond.
In this chapter, we develop a demographic silhouette to seek an understanding of socio-economically marginalised - or 'precariat' - Maori. Although we are wary of portraying Maori in an unduly negative light, we also submit that an appreciation of the extent of the issues faced by this group is necessary if our analysis is to be useful in the development of an effective response. We begin by defining the precariat. Using data obtained from Statistics New Zealand's Te Kupenga survey of Maori wellbeing, we then outline the prevalence and composition of the Maori precariat in Aotearoa New Zealand. Overall, we find that the Maori precariat comprises a substantial portion of the Maori population, particularly among females, younger age groups, and those with low or no qualifications. They are predominantly located-in regions that have high levels of seasonal employment, and in more deprived areas. We also find the value attached to Maori culture and experiences of stigma for this group are, for the most part, similar to those among the Maori non-precariat.

year: 2017