Site directed mutagenesis and purification of the cDNA for human class I aldehyde dehydrogenase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University

Aldehyde dehydrogenase (ALDH) is a key enzyme of alcohol metabolism, removing acetaldehyde which is formed as a product of the alcohol dehydrogenase reaction. If acetaldehyde is not effectively removed, acetaldehyde accumulates and produces an adverse reaction to alcohol, with nausea, flushing and increased heart rate and blood pressure. ALDH is involved in the conversion of retinal to retinoic acid (RA). RA has recently been shown to bind to receptors, which then act as nuclear transcription factors and play important roles in foetal development and maintenance of the epithelial layer in the body. Interference by ethanol and perhaps by acetaldehyde with this process is probably the cause of Foetal Alcohol Syndrome. In addition ALDH is also involved in the metabolism of catecholamine neurotransmitters, plays a role in the removal of toxic substances from the body and may have a role in protection against some chemical carcinogens. Dr. Kerrie Jones had obtained moderate levels of expression of recombinant ALDH in E. coli and constructed a number of mutants chosen on the basis of chemical modification data and sequence alignment. Mutant proteins were also expressed and assayed for enzyme activity in crude extracts. The aim of this thesis was to improve purification and yield of the expressed ALDH proteins. By the use of site-directed mutagenesis I attempted to mutate the amino acid residue Lys272 to either alanine, histidine or arginine. Future comparison of the properties of the site-directed mutants with those of the wild type enzyme will help to determine the importance of the residue (which has been replaced by mutagenesis) to catalysis

The efficacy of non-invasive brain stimulation protocols for inducing neuroplasticity

Neuroplasticity refers to the brain’s ability to change with experience. Continuous theta burst stimulation (cTBS) is a non-invasive brain stimulation technique capable of temporarily inducing neuroplasticity in the primary motor cortex (M1), as indicated by changes in the excitability of the stimulated brain region. However, cTBS-induced neuroplasticity shows large inter-individual variability, which limits its potential in research and clinical settings. The present study investigated whether down-regulating motor cortical inhibition, with cTBS applied using a lower than conventional intensity (cTBSlow), is capable of making the brain more amenable to the neuroplasticity-inducing effects of cTBS applied using the conventional intensity. Thirty-two, right-handed, healthy adults participated in two experimental sessions: 1) cTBS primed by cTBSlow; 2) cTBS primed by sham stimulation. Due to unforeseen technical issues, there were two groups: group 1 received cTBSlow with conventional bursts; group 2 received cTBSlow with reduced pulses per burst. Motor cortical excitability and inhibition were measured from an intrinsic hand muscle at baseline, between the two cTBS applications, and following cTBS. In group 1, cTBSlow reduced inhibition in M1, however, there was no systematic change in motor cortical excitability following cTBS primed by cTBSlow or primed by sham. This lack of effect may be due to unreliable neuroplasticity induction in M1 following cTBS alone. In group 2, long-lasting and less variable changes in motor cortical excitability were found following an unconventional cTBS pattern. These findings confirm the variability of cTBS-induced neuroplasticity and highlight the importance of developing novel protocols to induce less variable neuroplasticity responses

Conceptualising leadership in early childhood education in Aotearoa New Zealand

The New Zealand Teachers Council Te Pouherenga Kaiako o Aotearoa is pleased to publish this occasional paper focused on leadership in early childhood education in Aotearoa New Zealand. The project that led to this publication grew out of a desire of the Council’s Early Childhood Education Advisory Group to promote some action on leadership development specifically for ECE. Whereas there is a well-developed leadership strategy for the school sector there is no equivalent support for teachers in ECE. Yet it is well established in the literature that an effective professional learning community is most likely to result from leadership that has learning as the central focus. Thus the absence of a cohesive leadership strategy was seen as a significant risk to professional initiatives supporting quality teaching in ECE

News and Correspondence.

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Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay.

Background Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. Methodology and principal findings A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84–100), whiles the sensitivity was 88% (95% CI, 77–95). Conclusion The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure. Author summary Current diagnostic methods to detect M. ulcerans suffer from delayed time-to-results in most endemic countries by the prolonged period of time for the shipment and storage of samples to a distant, centralized laboratory. The M. ulcerans recombinase polymerase amplification assay (Mu-RPA) is a new, rapid diagnostic test developed for the detection of M. ulcerans infection, known commonly as Buruli ulcer, a chronic, debilitating, necrotizing disease of the skin and soft tissues. This assay is suitable for use on a portable detection device, with the potential to be used for quick diagnosis at the point of need, providing timely results to health workers at Buruli ulcer treatment clinics

Acute: high-level programming language design for distributed computation

Existing languages provide good support for typeful programming of standalone programs. In a distributed system, however, there may be interaction between multiple instances of many distinct programs, sharing some (but not necessarily all) of their module structure, and with some instances rebuilt with new versions of certain modules as time goes on. In this paper we discuss programming language support for such systems, focussing on their typing and naming issues. We describe an experimental language, Acute, which extends an ML core to support distributed development, deployment, and execution, allowing type-safe interaction between separately-built programs. The main features are: (1) type-safe marshalling of arbitrary values; (2) type names that are generated (freshly and by hashing) to ensure that type equality tests suffice to protect the invariants of abstract types, across the entire distributed system; (3) expression-level names generated to ensure that name equality tests suffice for type-safety of associated values, e.g. values carried on named channels; (4) controlled dynamic rebinding of marshalled values to local resources; and (5) thunkification of threads and mutexes to support computation mobility. These features are a large part of what is needed for typeful distributed programming. They are a relatively lightweight extension of ML, should be efficiently implementable, and are expressive enough to enable a wide variety of distributed infrastructure layers to be written as simple library code above the byte-string network and persistent store APIs. This disentangles the language runtime from communication intricacies. This paper highlights the main design choices in Acute. It is supported by a full language definition (of typing, compilation, and operational semantics), by a prototype implementation, and by example distribution libraries

Medial gastrocnemius flap for reconstruction of the extensor mechanism of the knee following high-energy trauma. A minimum 5 year follow-up

The purpose of this study was to assess the medium-term results of reconstruction of the extensor mechanism using the medial gastrocnemius while also providing soft tissue coverage.This retrospective review consisted of a consecutive series of four patients (age 28-40 years) with complex high energy traumatic injuries to lower extremity including both soft tissue loss and disruption of the knee extensor mechanism. The medial gastrocnemius rotational flap was used to reconstruct the patellar tendon and restore soft tissue coverage simultaneously. Range of motion and extensor lag; functional recovery was judged by return to work and sports activity. Validated measures included the Oxford Knee Score, Knee Injury and Osteoarthritis Outcome Score, and the modified Cincinnati Score.At the final follow up was 61.5 (57-66) months after reconstruction, the mean SF 12 physical component score ranged from 21.7 to 56.8 with a median of 55.3; the mental component from 42.8 to 60.7 with a median of 58.6. The KSS knee score ranged from 50 to 78 with a median of 68; the function score from 65 to 90 with a median of 85. The Oxford knee score ranged from 22 to 45 with a median of 33.5. The KOOS ranged from 28 to 82.7 with a median of 73.7 and the modified Cincinnati score from 38 to 82 with a median of 76.5. Knee range of motion ranged from 0 to 120°. Of the four patients three returned to working fulltime in their profession and returned to sports, including mountain biking and fitness training.For severe traumatic knee injuries with the combination of soft tissue defects and disruption of the extensor mechanism, the medial gastrocnemius flap provides an excellent reconstructive option to address both problems simultaneously. The results of this small case series support the use of this limb salvage technique

Infection with Mansonella perstans Nematodes in Buruli Ulcer Patients, Ghana.

During August 2010-December 2012, we conducted a study of patients in Ghana who had Buruli ulcer, caused by Mycobacterium ulcerans, and found that 23% were co-infected with Mansonella perstans nematodes; 13% of controls also had M. perstans infection. M. perstans co-infection should be considered in the diagnosis and treatment of Buruli ulcer

Engineering with logic: Rigorous test-oracle specification and validation for TCP/IP and the Sockets API

Conventional computer engineering relies on test-and-debug development processes, with the behavior of common interfaces described (at best) with prose specification documents. But prose specifications cannot be used in test-and-debug development in any automated way, and prose is a poor medium for expressing complex (and loose) specifications. The TCP/IP protocols and Sockets API are a good example of this: they play a vital role in modern communication and computation, and interoperability between implementations is essential. But what exactly they are is surprisingly obscure: their original development focused on “rough consensus and running code,” augmented by prose RFC specifications that do not precisely define what it means for an implementation to be correct. Ultimately, the actual standard is the de facto one of the common implementations, including, for example, the 15 000 to 20 000 lines of the BSD implementation—optimized and multithreaded C code, time dependent, with asynchronous event handlers, intertwined with the operating system, and security critical. This article reports on work done in the Netsem project to develop lightweight mathematically rigorous techniques that can be applied to such systems: to specify their behavior precisely (but loosely enough to permit the required implementation variation) and to test whether these specifications and the implementations correspond with specifications that are executable as test oracles . We developed post hoc specifications of TCP, UDP, and the Sockets API, both of the service that they provide to applications (in terms of TCP bidirectional stream connections) and of the internal operation of the protocol (in terms of TCP segments and UDP datagrams), together with a testable abstraction function relating the two. These specifications are rigorous, detailed, readable, with broad coverage, and rather accurate. Working within a general-purpose proof assistant (HOL4), we developed language idioms (within higher-order logic) in which to write the specifications: operational semantics with nondeterminism, time, system calls, monadic relational programming, and so forth. We followed an experimental semantics approach, validating the specifications against several thousand traces captured from three implementations (FreeBSD, Linux, and WinXP). Many differences between these were identified, as were a number of bugs. Validation was done using a special-purpose symbolic model checker programmed above HOL4. Having demonstrated that our logic-based engineering techniques suffice for handling real-world protocols, we argue that similar techniques could be applied to future critical software infrastructure at design time, leading to cleaner designs and (via specification-based testing) more robust and predictable implementations. In cases where specification looseness can be controlled, this should be possible with lightweight techniques, without the need for a general-purpose proof assistant, at relatively little cost.EPSRC Programme Grant EP/K008528/1 REMS: Rigorous Engineering for Mainstream Systems EPSRC Leadership Fellowship EP/H005633 (Sewell) Royal Society University Research Fellowship (Sewell) St Catharine's College Heller Research Fellowship (Wansbrough), EPSRC grant GR/N24872 Wide-area programming: Language, Semantics and Infrastructure Design EPSRC grant EP/C510712 NETSEM: Rigorous Semantics for Real Systems EC FET-GC project IST-2001-33234 PEPITO Peer-to-Peer Computing: Implementation and Theory CMI UROP internship support (Smith) EC Thematic Network IST-2001-38957 APPSEM 2 NICTA was funded by the Australian Government's Backing Australia's Ability initiative, in part through the Australian Research Council