Characterization of expiration air jets and droplet size distributions immediately at the mouth opening

Size distributions of expiratory droplets expelled during coughing and speaking and the velocities of the expiration air jets of healthy volunteers were measured. Droplet size was measured using the Interferometric Mie imaging (IMI) technique while the Particle Image Velocimetry (PIV) technique was used for measuring air velocity. These techniques allowed measurements in close proximity to the mouth and avoided air sampling losses. The average expiration air velocity was 11.7 m/s for coughing and 3.9 m/s for speaking. Under the experimental setting, evaporation and condensation effects had negligible impact on the measured droplet size. The geometric mean diameter of droplets from coughing was 13.5m and it was 16.0m for speaking (counting 1 to 100). The estimated total number of droplets expelled ranged from 947 – 2085 per cough and 112 – 6720 for speaking. The estimated droplet concentrations for coughing ranged from 2.4 - 5.2cm-3 per cough and 0.004 – 0.223 cm-3 for speaking.

Size distribution and sites of origin of droplets expelled during expiratory activities

A new Expiratory Droplet Investigation System (EDIS) was used to conduct the most comprehensive program of study to date, of the dilution corrected droplet size distributions produced during different respiratory activities.----- Distinct physiological processes were responsible for specific size distribution modes. The majority of particles for all activities were produced in one or more modes, with diameters below 0.8 µm. That mode occurred during all respiratory activities, including normal breathing. A second mode at 1.8 µm was produced during all activities, but at lower concentrations.----- Speech produced particles in modes near 3.5 µm and 5 µm. The modes became most pronounced during continuous vocalization, suggesting that the aerosolization of secretions lubricating the vocal chords is a major source of droplets in terms of number.----- Non-eqilibrium droplet evaporation was not detectable for particles between 0.5 and 20 μm implying that evaporation to the equilibrium droplet size occurred within 0.8 s.

Development of a Rep-inducible, BBTV-based expression system in banana

Banana bunchy top is regarded as the most important viral disease of banana, causing significant yield losses worldwide. The disease is caused by Banana bunchy top virus (BBTV), which is a circular ssDNA virus belonging to the genus Babuvirus in the family Nanoviridae. There are currently few effective control strategies for this and other ssDNA viruses. “In Plant Activation” (InPAct) is a novel technology being developed at QUT for ssDNA virus-activated suicide gene expression. The technology exploits the rolling circle replication mechanism of ssDNA viruses and is based on a unique “split” gene design such that suicide gene expression is only activated in the presence of the viral Rep. This PhD project aimed to develop a BBTV-based InPAct system as a suicide gene strategy to control BBTV. The BBTV-based InPAct vector design requires a BBTV intergenic region (IR) to be embedded within an intron in the gene expression cassette. To ensure that the BBTV IR would not interfere with intron splicing, a TEST vector was initially generated that contained the entire BBTV IR embedded within an intron in a β-glucuronidase (GUS) expression vector. Transient GUS assays in banana embryogenic cell suspensions indicated that cryptic intron splice sites were present within the IR. Transcript analysis revealed two cryptic intron splice sites in the Domain III sequence of the CR-M within the IR. Removal of the CR-M from the TEST vector resulted in an enhancement of GUS expression suggesting that the cryptic intron splice sites had been removed. An InPAct GUS vector was subsequently generated that contained the modified BBTV IR, with the CR-M (minus Domain III) repositioned within the InPAct cassette. Using transient histochemical and fluorometric GUS assays in banana embryogenic cells, the InPAct GUS vector was shown to be activated in the presence of the BBTV Rep. However, the presence of both BBTV Rep and Clink was shown to have a deleterious effect on GUS expression suggesting that these proteins were cytotoxic at the levels expressed. Analysis of replication of the InPAct vectors by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector through the nicking/ligation activity of BBTV Rep. However, Rep-mediated episomal replicons, indicative of rolling circle replication of the released circularised cassettes, were not observed. The inability of the InPAct cassette to be replicated was further investigated. To examine whether the absence of Domain III of the CR-M was responsible, a suite of modified BBTV-based InPAct GUS vectors was constructed that contained the CR-M with the inclusion of Domain III, the CR-M with the inclusion of Domain III and additional upstream IR sequence, or no CR-M. Analysis of replication by Southern hybridisation revealed that neither the presence of Domain III, nor the entire CR-M, had an effect on replication levels. Since the InPAct cassette was significantly larger than the native BBTV genomic components (approximately 1 kb), the effect of InPAct cassette size on replication was also investigated. A suite of size variant BBTV-based vectors was constructed that increased the size of a replication competent cassette to 1.1 kbp through to 2.1 kbp.. Analysis of replication by Southern hybridisation revealed that an increase in vector size above approximately 1.5 - 1.7 kbp resulted in a decrease in replication. Following the demonstration of Rep-mediated release, circularisation and expression from the InPAct GUS vector, an InPAct vector was generated in which the uidA reporter gene was replaced with the ribonuclease-encoding suicide gene, barnase. Initially, a TEST vector was generated to assess the cytotoxicity of Barnase on banana cells. Although transient assays revealed a Barnase-induced cytotoxic effect in banana cells, the expression levels were sub-optimal. An InPAct BARNASE vector was generated and tested for BBTV Rep-activated Barnase expression using transient assays in banana embryogenic cells. High levels of background expression from the InPAct BARNASE vector made it difficult to accurately assess Rep-activated Barnase expression. Analysis of replication by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector but no Rep-mediated episomal replicons indicative of rolling circle replication of the released circularised cassettes were again observed. Despite the inability of the InPAct vectors to replicate to enable high level gene expression, the InPAct BARNASE vector was assessed in planta for BBTV Rep-mediated activation of Barnase expression. Eleven lines of transgenic InPAct BARNASE banana plants were generated by Agrobacterium-mediated transformation and were challenged with viruliferous Pentalonia nigronervosa. At least one clonal plant in each line developed bunchy top symptoms and infection was confirmed by PCR. No localised lesions were observed on any plants, nor was there any localised GUS expression in the one InPAct GUS line challenged with viruliferous aphids. The results presented in this thesis are the first study towards the development of a BBTV-based InPAct system as a Rep-activatable suicide gene expression system to control BBTV. Although further optimisation of the vectors is necessary, the preliminary results suggest that this approach has the potential to be an effective control strategy for BBTV. The use of iterons within the InPAct vectors that are recognised by Reps from different ssDNA plant viruses may provide a broad-spectrum resistance strategy against multiple ssDNA plant viruses. Further, this technology holds great promise as a platform technology for the molecular farming of high-value proteins in vitro or in vivo through expression of the ssDNA virus Rep protein.

Manufacturing challenges with fabrication of large size mechanical harmonic drives

In truck manufacturing, the exhaust and air inlet pipes are specialized equipment that requires highly skilled, heavy machinery and small batch production methods. This paper describes a project to develop the computer numerically controlled (CNC) pipe bending process for a truck component manufacturer. The company supplies a huge range of heavy duty truck parts to the domestic market and is a significant supplier in Australia. The company has been using traditional methods of machine assisted manual pipe bending techniques. In a drive of continuous improvement, the company has acquired a pre-owned CNC bending machine capable of bending pipes automatically up to 25 bends. However, due to process mismatch, this machine is only used for single bending operation. The researchers studied the bending system and changed the manufacturing process. Using an example exhaust pipe as the benchmark, a significant drop of manufacturing lead time from 70 minutes to 40 minutes for each pipe was demonstrated. There was also a decrease of material cost due to the multiple bends part in one piece without cutting excessive materials for each single bend like it used to be.

Manufacturing challenges with fabrication of large size mechanical harmonic drives

Mechanical harmonic transmissions are relatively new kind of drives having several unusual features. For example, they can provide reduction ratio up to 500:1 in one stage, have very small teeth module compared to conventional drives and very large number of teeth (up to 1000) on a flexible gear. If for conventional drives manufacturing methods are well-developed, fabrication of large size harmonic drives presents a challenge. For example, how to fabricate a thin shell of 1.7m in diameter and wall thickness of 30mm having high precision external teeth at one end and internal splines at the other end? It is so flexible that conventional fabrication methods become unsuitable. In this paper special fabrication methods are discussed that can be used for manufacturing of large size harmonic drive components. They include electro-slag welding and refining, the use of special expandable devices to locate and hold a flexible gear, welding peripheral parts of disks with wear resistant materials with subsequent machining and others. These fabrication methods proved to be effective and harmonic drives built with the use of these innovative technologies have been installed on heavy metallurgical equipment and successfully tested.