Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(−)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (−)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

Thermally evaporated tungsten oxide (WO3) thin films for gas sensing applications

In this thesis, the author proposed and developed gas sensors made of nanostructured WO3 thin film by a thermal evaporation technique. This technique gives control over film thickness, grain size and purity. The device fabrication, nanostructured material synthesis, characterization and gas sensing performance have been undertaken. Three different types of nanostructured thin films, namely, pure WO3 thin films, iron-doped WO3 thin films by co-evaporation and Fe-implanted WO3 thin films have been synthesized. All the thin films have a film thickness of 300 nm. The physical, chemical and electronic properties of these films have been optimized by annealing heat treatment at 300ºC and 400ºC for 2 hours in air. Various analytical techniques were employed to characterize these films. Atomic Force Microscopy and Transmission Electron Microscopy revealed a very small grain size of the order 5-10 nm in as-deposited WO3 films, and annealing at 300ºC or 400ºC did not result in any significant change in grain size. X-ray diffraction (XRD) analysis revealed a highly amorphous structure of as-deposited films. Annealing at 300ºC for 2 hours in air did not improve crystallinity in these films. However, annealing at 400ºC for 2 hours in air significantly improved the crystallinity in pure and iron-doped WO3 thin films, whereas it only slightly improved the crystallinity of iron-implanted WO3 thin film as a result of implantation. Rutherford backscattered spectroscopy revealed an iron content of 0.5 at.% and 5.5 at.% in iron-doped and iron-implanted WO3 thin films, respectively. The RBS results have been confirmed using energy dispersive x-ray spectroscopy (EDX) during analysis of the films using transmission electron microscopy (TEM). X-ray photoelectron spectroscopy (XPS) revealed significant lowering of W 4f7/2 binding energy in all films annealed at 400ºC as compared with the as-deposited and 300ºC annealed films. Lowering of W 4f7/2 is due to increase in number of oxygen vacancies in the films and is considered highly beneficial for gas sensing. Raman analysis revealed that 400ºC annealed films except the iron-implanted film are highly crystalline with significant number of O-W-O bonds, which was consistent with the XRD results. Additionally, XRD, XPS and Raman analyses showed no evidence of secondary peaks corresponding to compounds of iron due to iron doping or implantation. This provided an understanding that iron was incorporated in the host WO3 matrix rather than as a separate dispersed compound or as catalyst on the surface. WO3 thin film based gas sensors are known to operate efficiently in the temperature range 200ºC-500 ºC. In the present study, by optimizing the physical, chemical and electronic properties through heat treatment and doping, an optimum response to H2, ethanol and CO has been achieved at a low operating temperature of 150ºC. Pure WO3 thin film annealed at 400ºC showed the highest sensitivity towards H2 at 150ºC due to its very small grain size and porosity, coupled with high number of oxygen vacancies, whereas Fe-doped WO3 film annealed at 400ºC showed the highest sensitivity to ethanol at an operating temperature of 150ºC due to its crystallinity, increased number of oxygen vacancies and higher degree of crystal distortions attributed to Fe addition. Pure WO3 films are known to be insensitive to CO, but iron-doped WO3 thin film annealed at 300ºC and 400ºC showed an optimum response to CO at an operating temperature of 150ºC. This result is attributed to lattice distortions produced in WO3 host matrix as a result of iron incorporation as substitutional impurity. However, iron-implanted WO3 thin films did not show any promising response towards the tested gases as the film structure has been damaged due to implantation, and annealing at 300ºC or 400ºC was not sufficient to induce crystallinity in these films. This study has demonstrated enhanced sensing properties of WO3 thin film sensors towards CO at lower operating temperature, which was achieved by optimizing the physical, chemical and electronic properties of the WO3 film through Fe doping and annealing. This study can be further extended to systematically investigate the effects of different Fe concentrations (0.5 at.% to 10 at.%) on the sensing performance of WO3 thin film gas sensors towards CO.

Studies on the surface properties of biodegradable polymer carriers in respiratory delivery of drug from Dry Powder Inhaler formulations

Dry Powder Inhaler (DPI) technology has a significant impact in the treatment of various respiratory disorders. DPI formulations consist of a micronized drug (<5ìm) blended with an inert coarse carrier, for which lactose is widely used to date. DPIs are one of the inhalation devices which are used to target the delivery of drugs to the lungs. Drug delivery via DPI formulations is influenced by the physico-chemical characteristics of lactose particles such as size, shape, surface roughness and adhesional forces. Commercially available DPI formulations, which utilise lactose as the carrier, are not efficient in delivering drug to the lungs. The reasons for this are the surface morphology, adhesional properties and surface roughness of lactose. Despite several attempts to modify lactose, the maximum efficient drug delivery to the lungs remains limited; hence, exploring suitable alternative carriers for DPIs is of paramount importance. Therefore, the objective of the project was to study the performance of spherical polymer microparticles as drug carriers and the factors controlling their performance. This study aimed to use biodegradable polymer microspheres as alternative carriers to lactose in DPIs for achieving efficient drug delivery into the lungs. This project focused on fabricating biodegradable polymer microparticles with reproducible surface morphology and particle shape. The surface characteristics of polymeric carriers and the adhesional forces between the drug and carrier particles were investigated in order to gain a better understanding of their influence on drug dispersion. For this purpose, two biodegradable polymers- polycaprolactone (PCL) and poly (DL-lactide-co-glycolide) (PLGA) were used as the carriers to deliver the anti-asthmatic drug - Salbutamol Sulphate (SS). The first study conducted for this dissertation was the aerosolization of SS from mixtures of SS and PCL or PLGA microparticles. The microparticles were fabricated using an emulsion technique and were characterized by laser diffraction for particle size analysis, Scanning Electron Microscopy (SEM) for surface morphology and X-ray Photoelectron Spectroscopy (XPS) to obtain surface elemental composition. The dispersion of the drug from the DPI formulations was determined by using a Twin Stage Impinger (TSI). The Fine particle Fraction (FPF) of SS from powder mixtures was analyzed by High Performance Liquid Chromatography (HPLC). It was found that the drug did not detach from the surface of PCL microspheres. To overcome this, the microspheres were coated with anti-adherent agents such as magnesium stearate and leucine to improve the dispersion of the drug from the carrier surfaces. It was found that coating the PCL microspheres helped in significantly improving the FPF of SS from the PCL surface. These results were in contrast to the PLGA microspheres which readily allowed detachment of the SS from their surface. However, coating PLGA microspheres with antiadherent agents did not further improve the detachment of the drug from the surface. Thus, the first part of the study demonstrated that the surface-coated PCL microspheres and PLGA microspheres can be potential alternatives to lactose as carriers in DPI formulations; however, there was no significant improvement in the FPF of the drug. The second part of the research studied the influence of the size of the microspheres on the FPF of the drug. For this purpose, four different sizes (25 ìm, 48 ìm, 100 ìm and 150 ìm) of the PCL and PLGA microspheres were fabricated and characterized. The dispersion of the drug from microspheres of different sizes was determined. It was found that as the size of the carrier increased there was a significant increase in the FPF of SS. This study suggested that the size of the carrier plays an important role in the dispersion of the drug from the carrier surface. Subsequent experiments in the third part of the dissertation studied the surface properties of the polymeric carrier. The adhesion forces existing between the drug particle and the polymer surfaces, and the surface roughness of the carriers were quantified using Atomic Force Microscopy (AFM). A direct correlation between adhesion forces and dispersion of the drug from the carrier surface was observed suggesting that adhesion forces play an important role in determining the detachment potential of the drug from the carrier surface. However, no direct relationship between the surface roughness of the PCL or PLGA carrier and the FPF of the drug was observed. In conclusion, the body of work presented in this dissertation demonstrated the potential of coated PCL microspheres and PLGA microspheres to be used in DPI formulations as an alternative carrier to sugar based carriers. The study also emphasized the role of the size of the carrier particles and the forces of interaction prevailing between the drug and the carrier particle surface on the aerosolization performances of the drug.

Emergent innovation through the co-evolution of informal and formal media economies

Well-established distinctions between amateur and professional are blurring as the impact of social media, changes in cultural consumption, and crises in copyright industries’ business models are felt across society and economy. I call this the increasingly rapid co-evolution of the formal market and informal household sectors and analyse it through the concept of ‘social network markets’ – individual choices are made on the basis of other’s choices and such networked preferencing is enhanced by the growing ubiquity of social media platforms. This may allow us better to understand sources of disruption and innovation in audiovisual production and distribution in wealthy Western markets which are as significant as those posed by informal practices outside the West. I examine what is happening around the monetization and professionalization of online video (YouTube, for example) and the socialization of professional production strategies (transmedia, for example) as innovation from the margins.

Experimental evidence indicating that mastreviruses probably did not co-diverge with their hosts

Background. Despite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have co-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur. Results. We have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 × 10 -4substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Réunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift. Conclusion. The absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts. © 2009 Harkins et al; licensee BioMed Central Ltd.

A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay

Centre for High-Performance Computing, Rosebank, Cape Town, South Africa Maize streak disease, caused by the A strain of the African endemic geminivirus, maize streak mastrevirus (MSV-A), threatens the food security and livelihoods of subsistence farmers throughout sub-Saharan Africa. Using a well-established transient expression assay, this study investigated the potential of a spliceable-intron hairpin RNA (hpRNA) approach to interfere with MSV replication. Two strategies were explored: (i) an inverted repeat of a 662 bp region of the MSV replication-associated protein gene (rep), which is essential for virus replication and is therefore a good target for post-transcriptional gene silencing; and (ii) an inverted repeat of the viral long intergenic region (LIR), considered for its potential to trigger transcriptional silencing of the viral promoter region. After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicativeform-specific PCR, it was clear that, whilst the hairpin rep construct (pHPrepDI662) completely inhibited MSV replication, the LIR hairpin construct was ineffective in this regard. In addition, pHPrepDI662 inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity. Further investigation by real-time PCR revealed that the pHPrepDI662 inverted repeat was 22-fold more effective at reducing virus replication than a construct containing the sense copy, whilst the antisense copy had no effect on replication when compared with the wild type. This is the first indication that an hpRNA strategy targeting MSV rep has the potential to protect transgenic. © 2011 SGM.

Injury prevention among friends : the benefits of school connectedness

Background: Injury is a leading cause of adolescent death. Risk-taking behaviours, including unsafe road behaviours, violence and alcohol use, are primary contributors. Recent research suggests adolescents look out for their friends and engage in protective behaviour to reduce others’ involvement in risk-taking. A positive school environment, and particularly students’ school connectedness, is also associated with reduced injury-risks. Aim: This study aimed to understand the role of school connectedness in adolescents’ intentions to protect and prevent their friends from involvement in alcohol use, fights, drink driving and unlicensed driving. Method: Surveys were completed by 540 13-14 year old students (49% male). Four sequential logistic regression analyses were conducted to determine whether school connectedness statistically predicted intentions to protect friends from injury-risk behaviours. Gender and ethnicity were entered at step 1, students’ own risk behaviour at step 2, and school connectedness scores at step 3 for all analyses. Results: School connectedness significantly predicted intentions to protect friends from all four injury-risk behaviours, after accounting for the variance attributable to sex, ethnicity and adolescents’ own involvement in injury-risks. Significance: School connectedness is negatively associated with adolescents’ own injury-risk behaviours. This research extends our knowledge of this critical protective factor, as it shows that students who are connected to school are also more likely to protect their friends from alcohol use, violence and unsafe road behaviours. School connectedness may therefore be an important factor to target in school-based prevention programs, both to reduce adolescents’ own injury-risk behaviour and to increase injury prevention among friends.

BabelFuse data fusion unit with precision wireless clock synchronisation

A significant issue encountered when fusing data received from multiple sensors is the accuracy of the timestamp associated with each piece of data. This is particularly important in applications such as Simultaneous Localisation and Mapping (SLAM) where vehicle velocity forms an important part of the mapping algorithms; on fastmoving vehicles, even millisecond inconsistencies in data timestamping can produce errors which need to be compensated for. The timestamping problem is compounded in a robot swarm environment due to the use of non-deterministic readily-available hardware (such as 802.11-based wireless) and inaccurate clock synchronisation protocols (such as Network Time Protocol (NTP)). As a result, the synchronisation of the clocks between robots can be out by tens-to-hundreds of milliseconds making correlation of data difficult and preventing the possibility of the units performing synchronised actions such as triggering cameras or intricate swarm manoeuvres. In this thesis, a complete data fusion unit is designed, implemented and tested. The unit, named BabelFuse, is able to accept sensor data from a number of low-speed communication buses (such as RS232, RS485 and CAN Bus) and also timestamp events that occur on General Purpose Input/Output (GPIO) pins referencing a submillisecondaccurate wirelessly-distributed "global" clock signal. In addition to its timestamping capabilities, it can also be used to trigger an attached camera at a predefined start time and frame rate. This functionality enables the creation of a wirelessly-synchronised distributed image acquisition system over a large geographic area; a real world application for this functionality is the creation of a platform to facilitate wirelessly-distributed 3D stereoscopic vision. A ‘best-practice’ design methodology is adopted within the project to ensure the final system operates according to its requirements. Initially, requirements are generated from which a high-level architecture is distilled. This architecture is then converted into a hardware specification and low-level design, which is then manufactured. The manufactured hardware is then verified to ensure it operates as designed and firmware and Linux Operating System (OS) drivers are written to provide the features and connectivity required of the system. Finally, integration testing is performed to ensure the unit functions as per its requirements. The BabelFuse System comprises of a single Grand Master unit which is responsible for maintaining the absolute value of the "global" clock. Slave nodes then determine their local clock o.set from that of the Grand Master via synchronisation events which occur multiple times per-second. The mechanism used for synchronising the clocks between the boards wirelessly makes use of specific hardware and a firmware protocol based on elements of the IEEE-1588 Precision Time Protocol (PTP). With the key requirement of the system being submillisecond-accurate clock synchronisation (as a basis for timestamping and camera triggering), automated testing is carried out to monitor the o.sets between each Slave and the Grand Master over time. A common strobe pulse is also sent to each unit for timestamping; the correlation between the timestamps of the di.erent units is used to validate the clock o.set results. Analysis of the automated test results show that the BabelFuse units are almost threemagnitudes more accurate than their requirement; clocks of the Slave and Grand Master units do not di.er by more than three microseconds over a running time of six hours and the mean clock o.set of Slaves to the Grand Master is less-than one microsecond. The common strobe pulse used to verify the clock o.set data yields a positive result with a maximum variation between units of less-than two microseconds and a mean value of less-than one microsecond. The camera triggering functionality is verified by connecting the trigger pulse output of each board to a four-channel digital oscilloscope and setting each unit to output a 100Hz periodic pulse with a common start time. The resulting waveform shows a maximum variation between the rising-edges of the pulses of approximately 39¥ìs, well below its target of 1ms.

Navigating the muddy waters of the research into single sex classrooms in co-educational middle years settings

Establishing single sex classes within co-educational sites is an option that Australian schools are again exploring. To date Australia has experienced three ‘waves’ of interest in establishing single sex classes, the first focused on equitable education opportunities for girls (Alloway & Gilbert, 1997), the second centered on boys’ literacy and engagement (Gilbert & Gilbert, 1998) and this current wave focuses on perceived difference between the sexes in co-educational classrooms (Protheroe, 2009; Gurian, Stevens & Daniels, 2009). With the intersection of middle schooling movement, focusing on learner centered classrooms (Pendergast & Bahr, 2010) and current educational agendas aimed at improving student performance and measurable learning outcomes (Ministerial Council on Education, Employment, Training and Youth Affairs, 2008), it is understandable that schools are exploring such student grouping options. However, after thirty years of international research into the efficacy of single sex classes in co-educational settings, the results still remain unclear. This paper seeks to navigate the ‘muddy waters’ of this body of research and suggests a framework to help guide school communities through the decision-making process associated with considering single sex classes.