Enhancing methacrylate-monolith-based downstream processes to champion plasmid DNA production

Increasing numbers of preclinical and clinical studies are utilizing pDNA (plasmid DNA) as the vector. In addition, there has been a growing trend towards larger and larger doses of pDNA utilized in human trials. The growing demand on pDNA manufacture leads to pressure to make more in less time. A key intervention has been the use of monoliths as stationary phases in liquid chromatography. Monolithic stationary phases offer fast separation to pDNA owing to their large pore size, making pDNA in the size range from 100 nm to over 300 nm easily accessible. However, the convective transport mechanism of monoliths does not guarantee plasmid purity. The recovery of pure pDNA hinges on a proper balance in the properties of the adsorbent phase, the mobile phase and the feedstock. The effects of pH and ionic strength of binding buffer, temperature of feedstock, active group density and the pore size of the stationary phase were considered as avenues to improve the recovery and purity of pDNA using a methacrylate-based monolithic adsorbent and Escherichia coli DH5α-pUC19 clarified lysate as feedstock. pDNA recovery was found to be critically dependent on the pH and ionic strength of the mobile phase. Up to a maximum of approx. 92% recovery was obtained under optimum conditions of pH and ionic strength. Increasing the feedstock temperature to 80°C increased the purity of pDNA owing to the extra thermal stability associated with pDNA over contaminants such as proteins. Results from toxicological studies of the plasmid samples using endotoxin standard (E. coli 0.55:B5 lipopolysaccharide) show that endotoxin level decreases with increasing salt concentration. It was obvious that large quantities of pure pDNA can be obtained with minimal extra effort simply by optimizing process parameters and conditions for pDNA purification.

Growth medium selection and its economic impact on plasmid DNA production

Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100-1000 μg in 2002 to 500-5000 μg in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5α harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 μg/ml and 6.3 μg/mg dry cell mass respectively) and the lowest was for LB (2.8 μg/ml and 3.3 μg/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 μg/ml and 11.2 μg/mg dry cell mass respectively. However, it is the economic advantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production.

Process engineering aspects of plasmid-based biopharmaceuticals production : tackling the threatening vaccine shortages to prevent global pandemics

Infectious diseases such as SARS, influenza and bird flu have the potential to cause global pandemics; a key intervention will be vaccination. Hence, it is imperative to have in place the capacity to create vaccines against new diseases in the shortest time possible. In 2004, The Institute of Medicine asserted that the world is tottering on the verge of a colossal influenza outbreak. The institute stated that, inadequate production system for influenza vaccines is a major obstruction in the preparation towards influenza outbreaks. Because of production issues, the vaccine industry is facing financial and technological bottlenecks: In October 2004, the FDA was caught off guard by the shortage of flu vaccine, caused by a contamination in a US-based plant (Chiron Corporation), one of the only two suppliers of US flu vaccine. Due to difficulties in production and long processing times, the bulk of the world's vaccine production comes from very small number of companies compared to the number of companies producing drugs. Conventional vaccines are made of attenuated or modified forms of viruses. Relatively high and continuous doses are administered when a non-viable vaccine is used and the overall protective immunity obtained is ephemeral. The safety concerns of viral vaccines have propelled interest in creating a viable replacement that would be more effective and safer to use.

Endotoxin removal : a critical step in vaccine production

A major drawback to the immunological potency of conventional vaccines, resulting in reduced level of immune responses, tissue injury, shock and high cytotoxicity, thus making their applications contraindicated in immunodeficiency diseases, is the presence of high contaminant concentrations in vaccine titers. Vaccine contamination arises from the simultaneous occurrence of competitive pathways resulting in the formation of other bio-products during cellular metabolism aside the pathways necessary for the production of vaccine molecules. One of such vaccine contaminating molecules is endotoxins which are mainly lipopolysaccharides (LPS) complexes found in the membrane of bacterial cell wall. The structural dynamics of these molecules make their removal from vaccine titers problematic, thus making vaccine endotoxin removal a major research endeavour. This presentation will discuss a novel technique for reducing the endotoxin level of vaccines. The technique commences with the disentanglement of endotoxin-vaccine molecular bonding and then capturing the vaccine molecules on an affinity monolith to separate the vaccine molecules from the endotoxins.

Dancing as Knowledge Production/Dancer as Researcher in Practice-led Research

This chapter discusses research undertaken for a PhD in dance, highlighting the oscillating and ambivalent nature of practice-led research methods. Holding an unusual position within the field of practice-led research in dance, the researcher adopted the role of dancer within four creative processes led by four choreographers. Although instigating and leading this research, the author produced new knowledge of dancing practices whilst being directed creatively by the choreographers in the research environment. In line with many definitions of practice-led research, the methods used were emergent from the research arena and responsive to the requirements of the project. Deeply imbricated in the research environment, the researcher used her embodied self as a research tool, as the one who participated, discovered and recorded. This outsider/insider role of researcher/participant produced a series of meta-positions, leading to a mixed-modal outcome that encompassed live performance, a movement treatise and a written thesis.

Bio-hydrogen production from food waste through anaerobic fermentation

In order to protect our planet and ourselves from the adverse effects of excessive CO2 emissions and to prevent an imminent non-renewable fossil fuel shortage and energy crisis, there is a need to transform our current ‘fossil fuel dependent’ energy systems to new, clean, renewable energy sources. The world has recognized hydrogen as an energy carrier that complies with all the environmental quality and energy security, demands. This research aimed at producing hydrogen through anaerobic fermentation, using food waste as the substrate. Four food waste substrates were used: Rice, fish, vegetable and their mixture. Bio-hydrogen production was performed in lab scale reactors, using 250 mL serum bottles. The food waste was first mixed with the anaerobic sewage sludge and incubated at 37°C for 31 days (acclimatization). The anaerobic sewage sludge was then heat treated at 80°C for 15 min. The experiment was conducted at an initial pH of 5.5 and temperatures of 27, 35 and 55°C. The maximum cumulative hydrogen produced by rice, fish, vegetable and mixed food waste substrates were highest at 37°C (Rice =26.97±0.76 mL, fish = 89.70±1.25 mL, vegetable = 42.00±1.76 mL, mixed = 108.90±1.42 mL). A comparative study of acclimatized (the different food waste substrates were mixed with anaerobic sewage sludge and incubated at 37°C for 31days) and non-acclimatized food waste substrate (food waste that was not incubated with anaerobic sewage sludge) showed that acclimatized food waste substrate enhanced bio-hydrogen production by 90 - 100%.

Bayesian networks for information synthesis in complex systems

This thesis introduces a method of applying Bayesian Networks to combine information from a range of data sources for effective decision support systems. It develops a set of techniques in development, validation, visualisation, and application of Complex Systems models, with a working demonstration in an Australian airport environment. The methods presented here have provided a modelling approach that produces highly flexible, informative and applicable interpretations of a system's behaviour under uncertain conditions. These end-to-end techniques are applied to the development of model based dashboards to support operators and decision makers in the multi-stakeholder airport environment. They provide highly flexible and informative interpretations and confidence in these interpretations of a system's behaviour under uncertain conditions.

MIMO enhanced slotted cooperation in urban vehicular networks

The requirement of isolated relays is one of the prime obstacles in utilizing sequential slotted cooperative protocols for Vehicular Ad-hoc Networks (VANET). Significant research advancement has taken place to improve the diversity multiplexing trade-off (DMT) of cooperative protocols in conventional mobile networks without much attention on vehicular ad-hoc networks. We have extended the concept of sequential slotted amplify and forward (SAF) protocols in the context of urban vehicular ad-hoc networks. Multiple Input Multiple Output (MIMO) reception is used at relaying vehicular nodes to isolate the relays effectively. The proposed approach adds a pragmatic value to the sequential slotted cooperative protocols while achieving attractive performance gains in urban VANETs. We have analysed the DMT bounds and the outage probabilities of the proposed scheme. The results suggest that the proposed scheme can achieve an optimal DMT similar to the DMT upper bound of the sequential SAF. Furthermore, the outage performance of the proposed scheme outperforms the SAF protocol by 2.5 dB at a target outage probability of 10-4.

Multi-objective models and techniques for analysing the absolute capacity of railway networks

Railway capacity determination and expansion are very important topics. In prior research, the competition between different entities such as train services and train types, on different network corridors however have been ignored, poorly modelled, or else assumed to be static. In response, a comprehensive set of multi-objective models have been formulated in this article to perform a trade-off analysis. These models determine the total absolute capacity of railway networks as the most equitable solution according to a clearly defined set of competing objectives. The models also perform a sensitivity analysis of capacity with respect to those competing objectives. The models have been extensively tested on a case study and their significant worth is shown. The models were solved using a variety of techniques however an adaptive E constraint method was shown to be most superior. In order to identify only the best solution, a Simulated Annealing meta-heuristic was implemented and tested. However a linearization technique based upon separable programming was also developed and shown to be superior in terms of solution quality but far less in terms of computational time.

Empirical modelling of the relationship between bus and car speeds on signalised urban networks

Vehicle speed is an important attribute for analysing the utility of a transport mode. The speed relationship between multiple modes of transport is of interest to traffic planners and operators. This paper quantifies the relationship between bus speed and average car speed by integrating Bluetooth data and Transit Signal Priority data from the urban network in Brisbane, Australia. The method proposed in this paper is the first of its kind to relate bus speed and average car speed by integrating multi-source traffic data in a corridor-based method. Three transferable regression models relating not-in-service bus, in-service bus during peak periods, and in-service bus during off-peak periods with average car speed are proposed. The models are cross-validated and the interrelationships are significant.

Optimising ketocarotenoid production in potato tubers : effect of genetic background, transgene combinations and environment

Astaxanthin is a high value carotenoid produced by some bacteria, a few green algae, several fungi but only a limited number of plants from the genus Adonis. Astaxanthin has been industrially exploited as a feed supplement in poultry farming and aquaculture. Consumption of ketocarotenoids, most notably astaxanthin, is also increasingly associated with a wide range of health benefits,as demonstrated in numerous clinical studies. Currently astaxanthin is produced commercially by chemical synthesis or from algal production systems. Several studies have used a metabolic engineering approach to produce astaxanthin in transgenic plants. Previous attempts to produce transgenic potato tubers biofortified with astaxanthin have met with limited success. In this study we have investigated approaches to optimising tuber astaxanthin content. It is demonstrated that the selection of appropriate parental genotype for transgenic approaches and stacking carotenoid biosynthetic pathway genes with the cauliflower Or gene result in enhanced astaxanthin content, to give six-fold higher tuber astaxanthin content than has been achieved previously. Additionally we demonstrate the effects of growth environment on tuber carotenoid content in both wild type and astaxanthin-producing transgenic lines and describe the associated transcriptome and metabolome restructuring.

Fine-grained plant classification using convolutional neural networks for feature extraction

We present an overview of the QUT plant classification system submitted to LifeCLEF 2014. This system uses generic features extracted from a convolutional neural network previously used to perform general object classification. We examine the effectiveness of these features to perform plant classification when used in combination with an extremely randomised forest. Using this system, with minimal tuning, we obtained relatively good results with a score of 0:249 on the test set of LifeCLEF 2014.

Reducing nitrous oxide emissions while supporting subtropical cereal production in Oxisols

This is the first study to investigate alternative fertilisation strategies to increase cereal production while reducing greenhouse gas emissions from the most common soil type in subtropical regions. The results of this research will contribute to define future farming practices to achieve global food security and mitigate climate change. The study established that introducing legumes in cropping systems is the most agronomically viable and environmentally sustainable fertilisation strategy. Importantly, this strategy can be widely adopted in subtropical regions since it is economically accessible, requires little know-how transfer and technology investment, and can be profitable in both low- and high-input cropping systems.