Affinity adsorption of plasmid DNA

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 ± 0.0013, 0.0095 ± 0.0016, and 0.0080 ± 0.0006 mg, respectively, to 50 μL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 ± 0.0043, 0.0219 ± 0.0035, and 0.0190 ± 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.

Preparation, Analysis and Use of an Affinity Adsorbent for the Purification of GST Fusion Protein

Methods are presented for the preparation, ligand density analysis and use of an affinity adsorbent for the purification of a glutathione S-transferase (GST) fusion protein in packed and expanded bed chromatographic processes. The protein is composed of GST fused to a zinc finger transcription factor (ZnF). Glutathione, the affinity ligand for GST purification, is covalently immobilized to a solid-phase adsorbent (Streamline™). The GST–ZnF fusion protein displays a dissociation constant of 0.6 x10-6 M to glutathione immobilized to Streamline™. Ligand density optimization, fusion protein elution conditions (pH and glutathione concentration) and ligand orientation are briefly discussed.

Plasmid DNA purification via the use of a dual affinity protein

Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the capture and purification of the pDNA. The protein is composed of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent, fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair sequence contained within the target pDNA. A Picogreen™ fluorescence assay and/or anx ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.

Finite volume methods for simulating anomalous transport

In this thesis a new approach for solving a certain class of anomalous diffusion equations was developed. The theory and algorithms arising from this work will pave the way for more efficient and more accurate solutions of these equations, with applications to science, health and industry. The method of finite volumes was applied to discretise the spatial derivatives, and this was shown to outperform existing methods in several key respects. The stability and convergence of the new method were rigorously established.

Study of the novel non-xanthine heterocyclic compound GU 285 as a potent non-selective adenosine receptor antagonist in the rat

The novel pyrazolo[3,4-d]pyrimidine compound GU285 (4-amino-6-alpha-carbamoylethylthio-1- phenylpyrazolo[3,4-d]pyrimidine, CAS 134896-40-5) was examined for its ability (1) to inhibit binding of adenosine (ADO) receptor ligands in rat brain membranes, (2) to antagonise functional responses to ADO agonists in rat right and left atria and coronary resistance vessels, and (3) to reduce the fall in heart rate and arterial blood pressure produced by the ADO A1 agonist N6-cyclopentyladenosine (CPA) in the intact, anaesthetized rat. GU285 competitively inhibited binding of the ADO A1 agonist [3H]-R-N6-phenylisopropyladenosine (R-PIA) yielding a Ki value of 11 (7-18) nmol.l-1 (geometric mean +/- 95% Cl). When assayed against the ADO A2A selective agonist [3H]-2-[p-(2-carboxyethyl)- phenethylamino]-5'-N-ethylcarboxamidoadenosine, (CGS21680), a Ki of 15 (10-24) nmol.l-1 was obtained. In spontaneously beating right atria, GU285 competitively antagonized negative chronotropic effects of R-PIA with a pA2 of 8.7 +/- 0.3 and in electrically paced left atria, GU285 competitively antagonized negative inotropic effects of R-PIA with a pA2 of 9.0 +/- 0.1. In the potassium-arrested, perfused rat heart GU285 (1 mumol.l-1) antagonized only the high sensitivity, ADO A2B mediated component of the biphasic relaxation of the coronary vasculature produced by NECA. The low sensitivity component was unchanged. GU285 (1 mumol.kg-1) antagonized the negative chronotropic and hypotensive effects of the adenosine A1 agonist CPA in anaesthetized rats, producing a 10-fold rightward shift in the dose-response relationship. These data demonstrate that in the rat, GU285 is a potent, non-selective adenosine receptor antagonist that maintains its activity in vivo.

Shifting intimacies

Our world is literally and figuratively turning to ‘dust’. This work acknowledges decay and renewal and the transitional, cyclical natures of interrelated ecologies. It also suggests advanced levels of degradation potentially beyond reparation. Dust exists both on and beneath the border of our unaided vision. Dust particles are predominantly forms of disintegrating solids that often become the substance or catalyst of future forms. Like many tiny forms, dust is an often unnoticed residue with ‘planet-size consequences’. (Hanna Holmes 2001) The image depicts an ethereal, backlit body, continually circling and morphing, apparently floating, suggesting endless cycles of birth, life and death and inviting differing states of meditation, exploration, stillness and play. This never ending video work is taken from a large-scale interactive/media artwork created during a six-month research residency in England at the Institute of Contemporary Art London and at Vincent Dance Theatre Sheffield in 2006. It was originally presented on a raised floor screen made of pure white sand at the ICA in London (see). The project involved developing new interaction, engagement and image making strategies for media arts practice, drawing on the application of both kinetic and proprioceptive dance/performance knowledges. The work was further informed by ecological network theory that assesses the systemic implications of private and public actions within bounded systems. The creative methodology was primarily practice-led which fomented the particular qualities of imagery, generated through cross-fertilising embodied knowledge of Dance and Media Arts. This was achieved through extensive workshopping undertaken in theatres, working ‘on the floor’ live, with dancers, props, sound and projection. And eventually of course, all this dust must settle. (Holmes 2001, from Dust Jacket) Holmes, H. 2001, The Secret Life of Dust: From the Cosmos to the Kitchen Counter, the Big Consequences of Little Things, p.3

Effectiveness of an emergency nurse practitioner service for adults presenting to rural hospitals with chest pain : Protocol for a multicentre, longitudinal nested cohort study

Introduction Chest pain is common in emergency department (ED) patients and represents a considerable burden for rural health services. Health services reforms to improve access to care need appropriately skilled and supported clinicians in the delivery of safe and effective care, including the use of emergency nurse practitioners (ENPs). Despite increasing use of ENPs, little is known about the safety and quality of the service in the rural ED context. The aims of this study are (1) to examine the safety and quality of the ENP service model in the provision of care in the rural environment and (2) to evaluate the effectiveness of the service in the management of patients presenting with undifferentiated chest pain. Methods and analysis This is the protocol for a prospective longitudinal nested cohort study to compare the effectiveness of ENP service with that of standard care. Adults presenting to three rural EDs in Queensland, Australia with a primary presenting complaint of atraumatic chest pain will be eligible for enrolment. We will measure (1) clinician's use of evidence-based guidelines (2) diagnostic accuracy of ECG interpretation for the management of patients with suspected or confirmed ACS (3) service indicators of waiting times, length-of-stay and did-not-wait rates and (4) clinician's diagnostic accuracy as measured by rates of unplanned representation within 7 days (5) satisfaction with care, (6) quality-of-life and (7) functional status. To assess these outcomes we will use a combination of measures collected from routinely collected data, medical record review and questionnaires (with 30-day follow-up). Ethics and dissemination Queensland Health Human Research Ethics Committee (HREC) has approved this protocol. The results will be published in peer-reviewed scientific journals and presented at one or more scientific conferences.