An improved real-time PCR assay for the detection of Old World screwworm flies

The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle East, the Indian subcontinent, south-east Asia and Papua New Guinea. Although to date Australia remains free of OWS flies, an incursion would have serious economic and animal welfare implications. For these reasons Australia has an OWS fly preparedness plan including OWS fly surveillance with fly traps. The recent development of an improved OWS fly trap and synthetic attractant and a specific and sensitive real-time PCR molecular assay for the detection of OWS flies in trap catches has improved Australia's OWS fly surveillance capabilities. Because all Australian trap samples gave negative results in the PCR assay, it was deemed necessary to include a positive control mechanism to ensure that fly DNA was being successfully extracted and amplified and to guard against false negative results. A new non-competitive internal amplification control (IAC) has been developed that can be used in conjunction with the OWS fly PCR assay in a multiplexed single-tube reaction. The multiplexed assay provides an indicator of the performance of DNA extraction and amplification without greatly increasing labour or reagent costs. The fly IAC targets a region of the ribosomal 16S mitochondrial DNA which is conserved across at least six genera of commonly trapped flies. Compared to the OWS fly assay alone, the multiplexed OWS fly and fly IAC assay displayed no loss in sensitivity or specificity for OWS fly detection. The multiplexed OWS fly and fly IAC assay provides greater confidence for trap catch samples returning negative OWS fly results. © 2014 International Atomic Energy Agency.

Liquid bubble manometer for low differential heads

The development of a highly sensitive liquid bubble manometer which can measure low differential heads to an accuracy of 0.01 mm of water is reported in this paper. The liquid bubble consists of two miscible liquids,benzaldehyde and normal hexane (each of which is immiscible in water) in such a proportion that the bubble density is within ±2 % of the density of water. The movement of the liquid bubble, which occupies the full cross-sectional area of the glass tube containing water in the manometer, is indicative of the applied differential head to a magnified scale. The manometer is found to give excellent results in open channel flow and is recommended for use for differential heads up to 2 cm of water. The manometer is economical, simple in fabrication and with simple modifications the sensitivity of the manometer can be increased to more than 0.01 mm of water.

Tailoring polyolefin properties by metallocene catalysis

This doctoral thesis deals with the syntheses of olefin homo- and copolymers using different kind of metallocene catalyst. Ethene, propene, 1-hexene, 1-hexadecene, vinylcyclohexane and phenylnorbornene were homo- or copolymerized with the catalysts. The unbridged benzyl substituted zirconium dichloride catalysts (1-4), ansa- bridged acenaphtyl substituted zirconium dichloride catalysts, ( 5, 6), rac- and meso-ethylene-bis(1-indenyl)zirconium dichlorides, (rac- and meso-8), rac-ethylene-bis(1-indenyl)hafnium dichloride, ( 12), bis(9-fluorenyl)hafnium dichloride (14 ) enantiomerically pure (R)- phenylethyl[(9-fluorenyl-1-indenyl)]ZrCl2, (11), 14 and asymmetric dimethylsilyl[(3-benzylindenyl-(2-methylbenzen[e]indenyl)] zirconium dichloride, (13), were prepared in our laboratory. Dimethylsilyl-bis(1-indenyl)zirconium dichloride, (9), isopropylidene(9-fluorenyl-cyclopentadienyl)zirconium dichloride, (10), and were obtained commercially. The solid-state structures of the catalysts rac- and meso-1 were determined by X-ray crystallography. Computational methods were used for the structure optimization of the catalyst rac- and meso-1 in order to compare the theoretical calculations with the experimental results. Polymerization experiments were conducted in a highly purified autoclave system using low pressures (< 5 bar) of gaseous monomers. The experiments were designed to attain the optimal catalytic activity and a uniform copolymer composition. The prepared homo- and copolymers were characterized by the gel permeation chromatography, GPC, differential scanning calorimetry, DSC, nuclear magnetic resonance, NMR, and Fourier transform infrared spectrometry, FTIR . Molar mass (Mw, Mn), molar mass distribution (Mw/Mn), tacticity, comonomer content, melting temperature, glass transition temperature, and end group structures and content were determined. A special attention was paid on the correlation of the polymer properties with the catalyst structures and polymerization conditions. An intramolecular phenyl coordination was found in phenyl substituted benzyl zirconocenes 1-3 explaining the decreased activity of the catalysts. Novel copolymers poly(propene-co-phenylnorbornene) and poly(propene co-vinylcyclohexane), were synthesized and high molar mass poly(ethene-co-1-hexene) and poly(ethene-co-1-hexadecene) copolymers with elastic properties were prepared. Activation of a hafnocene catalyst was studied with UV-Vis spectrometry and activation process for the synthesis of ultra high molar mass poly(1-hexene) was found out.

A note on non-separable solutions of linear partial differential equation

A method has been presented for constructing non-separable solutions of homogeneous linear partial differential equations of the type F(D, D′)W = 0, where D = ∂/∂x, D′ = ∂/∂y, Image where crs are constants and n stands for the order of the equation. The method has also been extended for equations of the form Φ(D, D′, D″)W = 0, where D = ∂/∂x, D′ = ∂/∂y, D″ = ∂/∂z and Image As illustration, the method has been applied to obtain nonseparable solutions of the two and three dimensional Helmholtz equations.

Validation of a new spot form of net blotch differential set and evidence for hybridisation between the spot and net forms of net blotch in Australia

A recently developed spot form of blotch differential set of 16 barley lines was tested for reaction response to 60 Pyrenophora teres f. maculata isolates from geographically disperse barley crops of Australia. Twelve barley lines (Arimont, Barque, Chebec, CI5286, CI5791, CI9214, CII6150, Dairokkaku, Esperance Orge 289, Galleon, Keel, Skiff, Torrens and TR250) provided differential response between the isolates. The susceptible controls Gairdner and Kombar provided indication of isolate virulence or avirulence. Abundant pathogenic diversity was revealed with 33 designated pathotypes, some of which related to geographic region. AFLP analysis also revealed abundant diversity with each of the isolates representing a unique genotype and one isolate that contained both AFLP bands unique to P. teres f. maculata and P. teres f. teres, the cause of spot form and net form of net blotch respectively, suggesting that sexual recombination between the net form and spot form isolates may have occurred naturally in the field.

Development of a Rapid Multiplex PCR Assay To Genotype Pasteurella multocida Strains by Use of the Lipopolysaccharide Outer Core Biosynthesis Locus

Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the "gold standard." The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains.

Renormalization methods in KAM theory

It is well known that an integrable (in the sense of Arnold-Jost) Hamiltonian system gives rise to quasi-periodic motion with trajectories running on invariant tori. These tori foliate the whole phase space. If we perturb an integrable system, the Kolmogorow-Arnold-Moser (KAM) theorem states that, provided some non-degeneracy condition and that the perturbation is sufficiently small, most of the invariant tori carrying quasi-periodic motion persist, getting only slightly deformed. The measure of the persisting invariant tori is large together with the inverse of the size of the perturbation. In the first part of the thesis we shall use a Renormalization Group (RG) scheme in order to prove the classical KAM result in the case of a non analytic perturbation (the latter will only be assumed to have continuous derivatives up to a sufficiently large order). We shall proceed by solving a sequence of problems in which theperturbations are analytic approximations of the original one. We will finally show that the approximate solutions will converge to a differentiable solution of our original problem. In the second part we will use an RG scheme using continuous scales, so that instead of solving an iterative equation as in the classical RG KAM, we will end up solving a partial differential equation. This will allow us to reduce the complications of treating a sequence of iterative equations to the use of the Banach fixed point theorem in a suitable Banach space.

Boundedness of weakly singular integral operators on domains

The monograph dissertation deals with kernel integral operators and their mapping properties on Euclidean domains. The associated kernels are weakly singular and examples of such are given by Green functions of certain elliptic partial differential equations. It is well known that mapping properties of the corresponding Green operators can be used to deduce a priori estimates for the solutions of these equations. In the dissertation, natural size- and cancellation conditions are quantified for kernels defined in domains. These kernels induce integral operators which are then composed with any partial differential operator of prescribed order, depending on the size of the kernel. The main object of study in this dissertation being the boundedness properties of such compositions, the main result is the characterization of their Lp-boundedness on suitably regular domains. In case the aforementioned kernels are defined in the whole Euclidean space, their partial derivatives of prescribed order turn out to be so called standard kernels that arise in connection with singular integral operators. The Lp-boundedness of singular integrals is characterized by the T1 theorem, which is originally due to David and Journé and was published in 1984 (Ann. of Math. 120). The main result in the dissertation can be interpreted as a T1 theorem for weakly singular integral operators. The dissertation deals also with special convolution type weakly singular integral operators that are defined on Euclidean spaces.

Methods to improve gene signal: Application to cDNA microarrays

Microarrays are high throughput biological assays that allow the screening of thousands of genes for their expression. The main idea behind microarrays is to compute for each gene a unique signal that is directly proportional to the quantity of mRNA that was hybridized on the chip. A large number of steps and errors associated with each step make the generated expression signal noisy. As a result, microarray data need to be carefully pre-processed before their analysis can be assumed to lead to reliable and biologically relevant conclusions. This thesis focuses on developing methods for improving gene signal and further utilizing this improved signal for higher level analysis. To achieve this, first, approaches for designing microarray experiments using various optimality criteria, considering both biological and technical replicates, are described. A carefully designed experiment leads to signal with low noise, as the effect of unwanted variations is minimized and the precision of the estimates of the parameters of interest are maximized. Second, a system for improving the gene signal by using three scans at varying scanner sensitivities is developed. A novel Bayesian latent intensity model is then applied on these three sets of expression values, corresponding to the three scans, to estimate the suitably calibrated true signal of genes. Third, a novel image segmentation approach that segregates the fluorescent signal from the undesired noise is developed using an additional dye, SYBR green RNA II. This technique helped in identifying signal only with respect to the hybridized DNA, and signal corresponding to dust, scratch, spilling of dye, and other noises, are avoided. Fourth, an integrated statistical model is developed, where signal correction, systematic array effects, dye effects, and differential expression, are modelled jointly as opposed to a sequential application of several methods of analysis. The methods described in here have been tested only for cDNA microarrays, but can also, with some modifications, be applied to other high-throughput technologies. Keywords: High-throughput technology, microarray, cDNA, multiple scans, Bayesian hierarchical models, image analysis, experimental design, MCMC, WinBUGS.

On Orlicz-Sobolev capacities

This thesis consists of three articles on Orlicz-Sobolev capacities. Capacity is a set function which gives information of the size of sets. Capacity is useful concept in the study of partial differential equations, and generalizations of exponential-type inequalities and Lebesgue point theory, and other topics related to weakly differentiable functions such as functions belonging to some Sobolev space or Orlicz-Sobolev space. In this thesis it is assumed that the defining function of the Orlicz-Sobolev space, the Young function, satisfies certain growth conditions. In the first article, the null sets of two different versions of Orlicz-Sobolev capacity are studied. Sufficient conditions are given so that these two versions of capacity have the same null sets. The importance of having information about null sets lies in the fact that the sets of capacity zero play similar role in the Orlicz-Sobolev space setting as the sets of measure zero do in the Lebesgue space and Orlicz space setting. The second article continues the work of the first article. In this article, it is shown that if a Young function satisfies certain conditions, then two versions of Orlicz-Sobolev capacity have the same null sets for its complementary Young function. In the third article the metric properties of Orlicz-Sobolev capacities are studied. It is usually difficult or impossible to calculate a capacity of a set. In applications it is often useful to have estimates for the Orlicz-Sobolev capacities of balls. Such estimates are obtained in this paper, when the Young function satisfies some growth conditions.

Evaluation of a multiplex PCR to identify and serotypeActinobacillus pleuropneumoniaeserovars 1, 5, 7, 12 and 15

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and Impact of the Study A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.

Stochastic Filtering

The stochastic filtering has been in general an estimation of indirectly observed states given observed data. This means that one is discussing conditional expected values as being one of the most accurate estimation, given the observations in the context of probability space. In my thesis, I have presented the theory of filtering using two different kind of observation process: the first one is a diffusion process which is discussed in the first chapter, while the third chapter introduces the latter which is a counting process. The majority of the fundamental results of the stochastic filtering is stated in form of interesting equations, such the unnormalized Zakai equation that leads to the Kushner-Stratonovich equation. The latter one which is known also by the normalized Zakai equation or equally by Fujisaki-Kallianpur-Kunita (FKK) equation, shows the divergence between the estimate using a diffusion process and a counting process. I have also introduced an example for the linear gaussian case, which is mainly the concept to build the so-called Kalman-Bucy filter. As the unnormalized and the normalized Zakai equations are in terms of the conditional distribution, a density of these distributions will be developed through these equations and stated by Kushner Theorem. However, Kushner Theorem has a form of a stochastic partial differential equation that needs to be verify in the sense of the existence and uniqueness of its solution, which is covered in the second chapter.

First report of Cotton leafroll dwarf virus in Thailand using a species-specific PCR validated with isolates from Brazil

Partial virus genome sequence with high nucleotide identity to Cotton leafroll dwarf virus (CLRDV) was identified from two cotton (Gossypium hirsutum) samples from Thailand displaying typical cotton leaf roll disease symptoms. We developed and validated a PCR assay for the detection of CLRDV isolates from Thailand and Brazil.

Genome-wide association analysis and differential expression analysis of resistance to Sclerotinia stem rot in Brassica napus

Brassica napus is one of the most important oil crops in the world, and stem rot caused by the fungus Sclerotinia sclerotiorum results in major losses in yield and quality. To elucidate resistance genes and pathogenesis-related genes, genome-wide association analysis of 347 accessions was performed using the Illumina 60K Brassica SNP (single nucleotide polymorphism) array. In addition, the detached stem inoculation assay was used to select five highly resistant (R) and susceptible (S) B. napus lines, 48 h postinoculation with S. sclerotiorum for transcriptome sequencing. We identified 17 significant associations for stem resistance on chromosomes A8 and C6, five of which were on A8 and 12 on C6. The SNPs identified on A8 were located in a 409-kb haplotype block, and those on C6 were consistent with previous QTL mapping efforts. Transcriptome analysis suggested that S. sclerotiorum infection activates the immune system, sulphur metabolism, especially glutathione (GSH) and glucosinolates in both R and S genotypes. Genes found to be specific to the R genotype related to the jasmonic acid pathway, lignin biosynthesis, defence response, signal transduction and encoding transcription factors. Twenty-four genes were identified in both the SNP-trait association and transcriptome sequencing analyses, including a tau class glutathione S-transferase (GSTU) gene cluster. This study provides useful insight into the molecular mechanisms underlying the plant's response to S. sclerotiorum.