The potyvirus Papaya ringspot virus (PRSV) is widespread throughout the world in cucurbits (such as zucchini, watermelon, pumpkin etc) and papaya (papaw). There are two serologically indistinguishable strains of PRSV, which can only be differentiated on the basis of host range. PRSV-P is able to infect both papaya and cucurbits whereas PRSV-W only infects cucurbits. Both infections drastically reduce the yield and market quality of the fruit. Australian isolates of PRSV-P and -W are very closely related and there is evidence that PRSV-P arose by mutation from PRSV-W. The aim of this project was to investigate the molecular basis of the host range difference between Australian isolates of PRSV-P and -W. The close relationship between Australian PRSV-P and -W isolates at the molecular level made this an ideal system to investigate molecular host range determinants through the development of full-length infectious cDNA clones. Initially, the complete genomes of PRSV-P and -W were each incorporated into two overlapping clones; one included the CaMV 35S promoter fused to the 5' one third of the PRSV genome and the second included the 3' two thirds of the genome (including a 33 nucleotide poly(A) tail) fused to a CaMV35S terminator. Full-length clones could not be obtained from subcloning of these fragments due to apparent toxicity in E.coli. Several approaches were subsequently undertaken to overcome this problem. In an attempt to prevent transcription of potentially toxic sequences, a plant intron (St-Ls1 IV2 intron) was engineered into the first coding region (P1) of the PRSV-W genome. Although clones were obtained using this strategy these could not be effectively maintained in E.coli. An alternative strategy involved subcloning of the genome into a low copy number vector, pACYC177, to minimise expression of toxic sequences. Again this resulted in clones that produced very small colonies, which were hard to culture and which gave very low plasmid yields. These plasmids were also difficult to maintain in E. coli. A final, successful strategy was developed using overlapping long distance PCR (OE-LD PCR) to generate full-length infectious PCR products of both PRSV-P (rPRSV-P) and -W (rPRSV-W) incorporating a CaMV 35S promoter and terminator. Infectious PCR products of both strains were inoculated onto squash cotyledons in vitro by microprojectile bombardment and subsequently mechanically inoculated to squash with greater than 86% efficiency. RPRSV-P subsequently infected papaya with 96% efficiency while, as expected, rPRSV-W was unable to infect papaya. Once a system for generating infectious clones was developed, both sequence analysis and recombination of infectious clones was utilised to investigate the underlying host range mechanism. The complete genomes of PRSV-P and -W were sequenced and compared to each other and to five full- length sequences of overseas PRSV isolates that were available. Sequence analysis confirmed the close relationship between the Australian PRSV isolates (97.8% nucleotide and 98.4% amino acid identity over the whole genome), supporting the mutation theory between both Australian and Asian P and W pairs. However, there was no consistent amino acid difference over the whole genome that correlated with host range or a single site that could be implicated, suggesting that the mutation and possibly the position of the mutation is different at least between Asian and Australian isolates and potentially differs at each mutation event. To better localise the P/W mutation within the PRSV genome, five different recombinant hybrid PRSVs (rhPRSV1-5) were generated in which 5', middle or 3' regions of the PRSV-P and -W genomes were exchanged. Infectivity of all hybrids was confirmed in squash, however, only hybrids including the 3' third of the PRSV-P genome were able to infect papaya, suggesting that this region encodes the papaya host range determinant. The region implicated encodes the genome-linked protein (VPg), NIa protease, replicase (NIb), coat protein (CP) and 3' UTR. While further identification of the host range determinants was not possible due to time constraints, based on studies with other potyviruses, there is a strong basis for implication of the VPg. Sequence analysis identified only 2 amino acid differences between the VPg of Australian PRSV-P and -W isolates in regions previously implicated in pathogenicity. These will be targeted for mutagenesis in ongoing studies. Identification of the genes/sequences involved in the determination of host range in PRSV will provide valuable information as to the sequence of events that lead to infection and will lead to a better understanding of the significance of changing hosts in the molecular evolution of PRSV, an essential requirement for the development of long-term sustainable control strategies against PRSV.

Malgré l’importance agronomique des cucurbitacées au Venezuela et le fort impact des maladies virales sur la production, le pathosystème viral y a été peu étudié. Cinq virus ont été décrits par des travaux souvent anciens: le cucumovirus Cucumber mosaic virus (CMV), les potyvirus Papaya ringspot virus (PRSV), Zucchini yellow mosaic virus (ZYMV) et Watermelon mosaic virus (WMV), le comovirus Squash mosaic virus (SqMV), et un begomovirus partiellement caractérisé, le Melon chlorotic mosaic virus (MeCMV). Pour lutter contre ces agents pathogènes, il est nécessaire de bien connaître le pathosystème viral présent localement. Nous avons donc caractérisé les principaux virus provoquant des maladies sur ces cultures dans le pays, en vue de comprendre l’évolution du pathosystème et de développer des méthodes de lutte adaptées. Nos études épidémiologiques ont montré que le begomovirus MeCMV représente la principale menace sur melon et pastèque, les potyvirus ZYMV et PRSV étant la principale menace sur courge. Les isolats Vénézuéliens de ZYMV se sont révélés génétiquement homogènes et biologiquement très variables comme cela a été observé dans d'autres régions du monde. La résistance au ZYMV conférée par le gène Zym chez le melon PI 414723 est surmontée par certains isolats, alors que le concombre TGM représente une source stable de résistance au ZYMV. Les types W et P de PRSV sont présents au Venezuela, mais seul le PRSV-W été trouvé sur cucurbitacées cultivées et sauvages. Une autre souche virale, initialement appelée PRSV-T et détectée au Venezuela, constitue une espèce différente du PRSV d’après ses propriétés moléculaires et biologiques établies dans ce travail. / In Venezuela, cucurbits viruses are among de major constraints for cucurbit production. Five viruses have been described infecting cucurbits in the country: Cucumber mosaic virus (CMV, Cucumovirus), Papaya ringspot virus (PRSV, Potyvirus), Zucchini yellow mosaic virus (ZYMV, Potyvirus), Squash mosaic virus (SqMV, Comovirus), and Melon chlorotic mosaic virus (MeCMV, Begomovirus).The current frequency and impact of these viruses is Venezuela is not well known. In this work, the major cucurbit viruses were identified and characterized in order to estimate the viral pathosystem affecting cucurbit production in the country. The begomovirus MeCMV appears to be the major constraint for melon and watermelon production, while the potyviruses ZYMV and PRSV were the most important viruses infecting squash crops in this survey. Molecular characterisation of ZYMV isolates revealed a low genetic diversity of this virus in Venezuela. In contrast, ZYMV isolates were biologically variable as observed in several countries worldwide. Two types of PRSV, P and W, are present in Venezuela. PRSV-W is the only type naturally infecting cucurbits in Venezuela. Another type of PRSV, formerly referred as PRSV-T, was detected. Its molecular and biological characterisation revealed that it is indeed a new species related to but distinct from PRSV. Therefore, the name zucchini tigré mosaic virus (ZTMV) is proposed for this virus.

This work is focused on thermodynamic modelling of isobaric vapour-liquid-liquid equilibrium (VLLE) (homogeneous) and (heterogeneous) for binary, ternary and quaternary systems. This work uses data for organic/aqueous systems; historically these mixtures were used in the production of penicillin and were required to be separated by continuous fractional distillation. Modelling of the separation required phase equilibrium data to be available so that predictions could be made for equilibrium stage temperatures, vapour compositions, liquid compositions and any phase splitting occurring in the liquid phase. Relevant data became available in the literature and work has been carried out to use relevant theories in correlating and predicting as was originally required in the distillation equilibrium stage modelling. All the modelling carried out was at atmospheric pressure. The modelling has been done using an Equation of State, specifically Peng Robinson Styrjek Vera (PRSV), combined with the activity coefficient model UNIversal QUAsi Chemical (UNIQUAC) through Wong Sandler mixing rules (WSMR). The success of all correlations and predictions was justified by minimizing the value of the Absolute Average Deviation (AAD) as defined within the thesis. Initially the integral Area Method and a method called Tangent Plane Intersection (TPI) were used in the prediction of liquid-liquid equilibrium (LLE) binary systems. This work used a modified 2-point search, suggested a 3-point search and has successfully applied both of these methods to predict VLLE for binary systems. It was discovered through the application of the TPI on ternary VLLE systems that the method was strongly sensitive to initial values. This work suggested and tested a Systematic Initial Generator (SIG) to provide the TPI method with realistic initial values close to the real solution and has demonstrated the viability of the SIG on improving the accuracy of the TPI results for the ternary systems investigated. In parallel with the TPI another method the Tangent Plane Distance Function (TPDF) was also investigated. This method is based on the minimisation of Gibbs free energy function related to the Gibbs energy surface. This method consistently showed it was capable of predicting VLLE for both ternary and quaternary systems as demonstrated throughout this work. The TPDF method was found to be computationally faster and less sensitive to the initial values. Some of the methods investigated in this work were also found to be applicable as phase predictors and it was discovered that the TPDF and the SIG methods were successful in predicting the phase regions; however the TPI method failed in identifying the 2 phase region. Applying the techniques described to newly available quaternary data has identified the strengths and weaknesses of the methods. This work has expanded the existing knowledge and developed a reliable model for design, operation and optimisation of the phase equilibria required for prediction in many separation processes. Currently available modelling simulation packages are variable in their predictions and sometimes yield unsatisfactory predictions. Many of the current uses of VLLE models are particularly focused on Hydrocarbon/Water systems at high pressure. The work described in this thesis has demonstrated that an EOS with suitable mixing rules can model and predict data for polar organic liquids at atmospheric and below atmospheric pressure and offers the advantage of using the same modelling equations for both phases.

Biotechnologies potentially have significant benefits for developing countries but many countries lack complete regulatory processes to allow their release. In evaluating the potential benefits of genetically modified crops, one must be able to measure the true cost of regulations in addition to the other costs associated with bringing the crop to market. The objectives of this paper are to (1) identify the direct costs of the regulation of Bt eggplant, Bt rice, ringspot virus resistant (PRSV) papaya and virus resistant tomatoes in the Philippines, and (2) estimate the opportunity cost of time lost in the regulatory process. The study compares the cost of regulations as they differ by factors such as the existence of previous studies on the product or the intention for export or domestic use. It is hypothesized that the costs are greater for products that are intended for export or human consumption or are produced by the private sector. It is also hypothesized that these factors increase the time to complete the regulatory process, therefore increasing the opportunity cost of time. This study evaluates the economic impact of the GMO regulatory process on the change in producer surplus, the net present value and the internal rate return using an economic surplus model. Scientists and other experts in the field of GMOs and regulation were interviewed to obtain the necessary data on the regulatory process. The evaluation was carried out for four different commodities in the Philippines, Bt Rice, Bt Eggplant, PRSV Papaya and MVR Tomato. The results for the open economy model revealed a change in producer surplus, as a result of the GMO research, of $418.3 million for Bt Rice and $353.7 million for PRSV Papaya. The closed economy model of Bt Eggplant has a change in producer surplus of $25.1 million and a change in total surplus of $40.8 million while the result for the change in producer surplus for MVR Tomato is $19.3 million and the change in total surplus is $51.6 million. A sensitivity analysis of the results was then carried out in which the elasticity of supply, the cost of regulation, and the release date were each varied in order to show the welfare impact of such changes. The sensitivity analysis revealed limited changes in surplus when elasticity and regulatory costs were changed. However, changing the date of release or commercialization resulted in monumental changes in surplus. / Master of Science