Developing Adaptive Leaders: The Impact of the COVID-19 Pandemic onTheological Field Education in Australia and New Zealand

Trist reflects on how theological field education adpated and functioned in New Zealand and Australia

Reconfiguration, contestation, and decline: conceptualizing mature large technical systems

Large technical systems (LTS) are integral to modern lifestyles but arduous to analyze. In this paper, we advance a conceptualization of LTS using the notion of mature “phases,” drawing from insights into innovation studies, science and technology studies, political science, the sociology of infra- structure, history of technology, and governance. We begin by defining LTS as a unit of analysis and explaining its conceptual utility and novelty, situating it among other prominent sociotechnical theories. Next, we argue that after LTS have moved through the (overlapping) phases proposed by Thomas Hughes of invention, expansion, growth, momentum, and style,mature LTS undergo the additional (overlapping) phases of reconfiguration, contestation (subject to pressures such as drift and crisis), and eventually stagnation and decline. We illustrate these analytical phases with historical case studies and the conceptual literature, and close by suggesting future research to refine and develop the LTS framework, particularly related to more refined typologies, temporal dimensions, and a broadening of system users. We aim to contribute to theoretical debates about the coevolution of LTS as well as empirical discussions about system-related use, socio- technical change, and policy-making

Coupling SEC and IEF to SXRF for metallation analysis of SOD1 enzyme isolated from the human central nervous system

International audienc

Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow

Studies of the metal content of metalloproteins in tissues from the human central nervous system (CNS) can be compromised by preparative techniques which alter levels of, or interactions between, metals and the protein of interest within a complex mixture. We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and either proton or synchrotron X-ray fluorescence within electrophoresis gels to analyze the endogenous metal content of copper-zinc superoxide dismutase (SOD1) purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis and Parkinson’s disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. Validating our novel approach, we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein versus confounding metalloproteins. We analyzed tissues from nine healthy individuals and five CNS regions (occipital cortex, substantia nigra, locus coeruleus, dorsal spinal cord, and ventral spinal cord). We found that Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28, a ratio very close to the expected value of 1. Our methodological workflow can be applied to the study of endogenous native SOD1 in a pathological context and adapted to a range of metalloproteins from human tissues and other sources

Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: A Methodological Workflow

We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and high sensitivity X-ray-based metal detection within electrophoresis gels to analyze the metal content of single proteins purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. An important metalloprotein in the human central nervous system is copper-zinc superoxide dismutase (SOD1), an antioxidant enzyme linked to the aetiology of both amyotrophic lateral sclerosis and Parkinson’s disease. Abnormal SOD1 metallation is suspected to play a role in the pathogenic aggregation of SOD1 in both disorders, although data describing SOD1 metal occupancy in human tissues has not previously been reported. Validating our novel approach we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein vs confounding metalloproteins. We found Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28 in human central nervous system tissues from healthy individuals, a ratio close to the expected value of 1. Our methodological workflow can be adapted to study a range of metalloproteins from human tissues and other sources.<br /

Coupling SEC and IEF to SXRF for metallation analysis of SOD1 enzyme isolated from the human central nervous system

Copper-zinc superoxide dismutase enzyme (SOD1) is one of the 16,227 expressed proteins in the central nervous system (CNS). Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis (ALS) and Parkinson’s disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. The analysis of metals in metalloproteins under native conditions remains a difficult exercise, especially when the protein is present at physiological levels in a complex tissue. In this work, we developed a methodological workflow that allows synchrotron X-ray fluorescence (SXRF) analysis of metalloenzymes isolated from small amounts of tissue. This protocol allowed us to characterize the metallation of endogenous SOD1 in the human CNS from only 20 mg of post-mortem tissue. In a first 2D-chromatography step, a soluble protein extract is prepared from post-mortem CNS tissues and then separated according to molecular weight using Size Exclusion Chromatography (SEC), and then according to isoelectric point by native isoelectric focusing (IEF) in gel. The processed samples were analyzed by SXRF in the microprobe hutch of the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III (DESY) in Hamburg (Germany). In total, we analyzed post-mortem CNS tissue from 30 individuals (17 without neurological disease and 13 ALS cases). The Cu/Zn atomic ratio in active SOD1 from control cases was 1.12 ± 0.28 (mean±sd) and did not differ between CNS regions or individuals. To our knowledge, this is the first report of a ratio value close to the theoretical value of 1 in active SOD1 isolated from human tissue. In addition to its excellent detection limit (∼0.1 µg/g), SXRF also offers excellent repeatability (Cu/Zn ratio in SOD1 standard=0.93 +/-0.01, n=9). Furthermore, our protocol preserves both the metallation and the activity of SOD1, and reduces proteome complexity by 97.8%. 2D chromatography enriches SOD1 99-fold compared to IEF alone, and also improves the accuracy and precision of the measurements (mean Cu/Zn=1.12 vs 2.39, sd = 0.28 vs 1.48). Using this SXRF-based method we revealed that SOD1 metallation is altered in ALS. This method is applicable to other metalloproteins when only small amounts of tissue are available and excellent sensitivity and reproducibility are required