In the first phase of this study, the diversity of methanogenic bacteria in avian ceca was found to be minimal. Based on 16S rDNA clone libraries, a common phylotype, designated CH101, ranged between 92.86 to 100 % of the total clones whereas less than 1% of the other phylotypes were found. On the basis of the sequence identity, all of the sequences, except sequence CH1270, are related from 98.97 to 99.45% to 16S rDNA Methanobrevibacter woesei GS. Sequence CH1270 is 97.62% homologous to the sequence identified to uncultured archaeon clone ConP1-11F. Clearly, the predominant methanogen found to reside in the chicken ceca was M. woesei. By using a MPN enumeration method, methanogen counts were found to be in the range of 6.38 to 8.23 log10 organisms per gram wet weight. The 16S rDNA copy number per gram wet weight in the samples was between log10 5.50 and 7.19. The second phase of the study was conducted to observe the effects of selected nitrocompounds and two different feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Initially, one of the three nitrocompounds was added to incubations containing cecal contents from laying hens supplemented with either alfalfa or layer feed. Both feed materials influenced volatile fatty acids (VFA) production and also fostered methane production in the incubations although methane was lower (P < 0.05) in incubations with added nitrocompound, particularly nitroethane. Secondly, nitroethane was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either alfalfa or layer feed. Unlike cecal contents, layer feed significantly (P < 0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that nitroethane impedes methane production, especially in incubations of chicken cecal contents. The final phase of this study was carried out to determine the methanogenic establishment in the chicken ceca by the cultural method with the quantitative PCR. The results suggested that methanogens colonized in chicken ceca at a few days after birth. Litter and house flies could be potential sources for methanogenic colonization in broiler chicks.

Rapid diagnosis of infectious disease and timely initiation of proper clinical antibiotic treatment is the determinant in obtaining the optimal clinical outcomes and reducing emergences of multidrug-resistant organisms. In particular, acute infections require the detection to be accomplished in limited time with high sensitivity due to the low concentration of organisms causing the infections. Real-time Polymerase Chain Reaction can provide quantitative identification of specific genetic materials and has revolutionized clinical microbiology laboratory diagnosis. It is becoming a standard for infectious disease detection. However, most real-time PCR instruments on the market are bulky, fragile and costly due to their delicate optical components, which restricted their use to point-of-care application. Modern microfluidic and sensing technology provide a transition from benchtop real-time PCR to miniaturizable, robust, and portable real-time PCR devices to achieve rapid, low-cost, and efficient point-of-care diagnosis. In this work, an innovative electrokinetic PCR (EK-PCR) platform that combines AC electrothermal flow (ACEF) and Joule heating induced temperature gradient to implement thermal cycling for DNA amplification is discussed. In addition, in situ electrochemical sensing is incorporated in the EK-PCR chamber for real-time monitoring of the DNA concentration toward quantification of the initial copies of the DNA template. EK-PCR can improve the energy efficiency with minimized total thermal mass and remain high amplification efficiency. More importantly, it represents a highly integrated strategy for portable point-of-care devices.

Smoking is a worldwide problem and it is the second major cause of death. People often try to quit, but few succeed mainly because of withdrawal symptoms such as irritability, anxiety, increased appetite, hyperventilation and difficulty concentrating. The overall aim of this project was to study neurochemical changes in the brain following sensitization to nicotine which could give more information about what causes an individual to go from using drugs to abusing the drugs. Therefore, we investigated messenger ribonucleic acid (mRNA) expression of several genes known to be involved in the mesolimbic dopamine pathway in the nucleus accumbens, caudate putamen, prefrontal cortex and medial prefrontal cortex using real-time polymerase chain reaction (real-time PCR). The results showed that in the nucleus accumbens, mRNA expression of gamma-aminobutyric acid (GABA) Aα1 subunit receptor and GABA transporter 3 (GAT-3) were significantly increased following nicotine administration, while in the caudate putamen no difference in expression was observed. In prefrontal cortex, the expression of adrenergic subunit receptor α2A was significantly increased following hexamethonium administration. In medial prefrontal cortex a significant decrease of expression of GAT-1 was shown following nicotine and hexamethonium administration, while a decrease of CART expression only was shown following nicotine administration. Overall, these changes in the GABA system may help to explain the mechanism of nicotine sensitization.

No description available.

Shade tree incorporation is beneficial in coffee cropping systems under sub-optimal conditions. This study was performed in lowland Costa Rica, at a 12-year-old experimental coffee farm. The main objective was to compare the effect of a nitrogen fixing shade tree, Erythrina poeppigiana, on nutrient availability in the rhizosphere of coffee under conventional fertilization. Accumulation of nutrients (mineral N, available P, and exchangeable base cations) in rhizosphere relative to bulk soil was greater under shade than full sun. Low nitrate availability in rhizosphere soil of full sun coffee was explained by root-induced acidification relative to bulk soil, as abundance of ammonia-oxidizing bacteria (AOB), which mediate nitrification, were positively correlated with pH. Organic fertilization enhanced AOB abundance and altered soil bacterial community structure relative to conventional fertilization. This study indicates clear effects of shade-tree presence on nutrient availability at the micro-scale, management of which is critical for stability of coffee agroforestry systems.

Shade tree incorporation is beneficial in coffee cropping systems under sub-optimal conditions. This study was performed in lowland Costa Rica, at a 12-year-old experimental coffee farm. The main objective was to compare the effect of a nitrogen fixing shade tree, Erythrina poeppigiana, on nutrient availability in the rhizosphere of coffee under conventional fertilization. Accumulation of nutrients (mineral N, available P, and exchangeable base cations) in rhizosphere relative to bulk soil was greater under shade than full sun. Low nitrate availability in rhizosphere soil of full sun coffee was explained by root-induced acidification relative to bulk soil, as abundance of ammonia-oxidizing bacteria (AOB), which mediate nitrification, were positively correlated with pH. Organic fertilization enhanced AOB abundance and altered soil bacterial community structure relative to conventional fertilization. This study indicates clear effects of shade-tree presence on nutrient availability at the micro-scale, management of which is critical for stability of coffee agroforestry systems.

Ongoing work in the laboratory of Professor Chris Backhouse is aimed at developing a portable and inexpensive lab on a chip instrument. A system capable of molecular biology protocols including sample preparation (SP), polymerase chain reaction (PCR), and melting curve analysis (MCA) would meet the requirements for point of care genetic analysis. The SP, PCR, and MCA modules were designed and tested on a standalone basis and then integrated for analysis of raw clinical samples. An automated XY stage was developed for magnetic bead-based DNA purification. In addition, a LED/CCD-based optical detection module was employed for real time PCR and MCA. Data analysis algorithms and protocols were implemented to remove noise and interpret data. This work culminated in proof of principle on-chip SP-PCR-MCA to detect ß2m DNA from human buccal cells in a modular and inexpensive system. / Biomedical Engineering

The use of PCR as a tool for the diagnostic virology and viral research laboratories has greatly increased in recent years, however the use of conventional PCR and amplicon detection systems can be a complex and relatively slow process that increases the risk of amplicon carry-over contamination. Many conventional PCR systems are unsuited for, or unable to perform as accurate diagnostic and quantitative tools because viruses are present in such a diverse variety of patient tissues and in a broad range of concentrations. Traditional viral culture, while still the gold standard for the detection of many viruses, is lengthy, expensive and often subjective. In addition, successful isolation of infectious virus is variable and dependent upon appropriate cell lines, lengthy incubations and careful transport and storage of clinical specimens. Many of the disadvantages arising from the use of traditional assays for the detection of viruses have been overcome by the development of real-time PCR. The technology has continued to develop due to the introduction of several commercial thermal cycling platforms and the appearance of numerous specific and non-specific fluorogenic chemistries. For the purpose of this thesis, human virology was sectioned into three diagnostic divisions containing the synthetic viruses, the well characterised viruses and the new or emerging viruses. This thesis proposes the hypothesis that real-time PCR could greatly improve upon traditional techniques for the detection, quantitation and characterisation of the members of these three divisions in both research and diagnostic environments. Conventional competitive quantitative PCR assays and a non-oligoprobe real-time PCR assay were constructed to detect novel synthetic gene therapy vectors developed from retroviruses. When compared to oligoprobe-based real-time PCR, it was clear that conventional molecular assays, whilst improving upon traditional methods of viral culture and immunofluorescence, were slower, more complex, less versatile and were hindered by a limited dynamic range. Synthetic control templates were developed and an improved method of assaying these template preparations was devised. The controls were used to precisely optimise each assay, create quality assurance reagents and to construct external standard curves permitting the absolute quantitation of viral templates. Real-time PCR achieved several significant goals during the studies performed for this thesis. The new assays detected human enterovirus (HEV) and the emerging pathogen, human metapneumovirus (hMPV) which were both responsible for seasonal outbreaks of serious disease that would otherwise have gone undiagnosed. These data led to the first description of hMPV outside of the Netherlands, as well as the first description of two validated rapid diagnostic RT-PCR assays which permitted the definitive classification of hMPV as a global pathogen of children and adults. Building upon its detection, an extensive molecular epidemiological study permitted the description of subtle differences between Australian and the more recently described international hMPV strains resulting in the classification of two distinct types of hMPV (A and B) and within these, four subtypes (A1, A2, B1 and B2). Real-time PCR rapidly detected, quantitated and genotyped herpes simplex viruses in a single reaction and determined the successful delivery of human and non-human genes by novel retroviral vectors in less time than any other phenotype detection assay. Additionally, these studies produced quantitative data which permitted the rapid calculation of transduction efficiency. Real-time PCR was able to quickly assess the efficiency of the PCR either in response to the titration of individual reaction components or as a result of amplification modifiers present within specimen extracts. The use of nucleotide sequencing studies ideally complemented earlier diagnostic studies of HEV and permitted the discrimination of pathogenic enterovirus 71. This thesis demonstrated that real-time PCR is more able to accommodate the demanding aspects of viral research and diagnostics than any other single method, and is now in a position to replace many of the traditional techniques still used by laboratories unfamiliar with the benefits of real-time PCR. The assays, techniques, reagents and publications resulting from these studies have benefited several areas of viral research and diagnostics and have improved the understanding of the role of real-time PCR in virology and of the technique in general, among the greater scientific community whilst successfully addressing the proposed hypothesis.

Transfection efficiencies of several polymeric gene carriers were compared and correlated quantitatively to the amounts of cellular accumulation of plasmid DNA and to the expression of mRNA by quantitative real time PCR. Three cationic methacrylate polymer systems with similar chemical structure were used in this study, namely: poly(dimethylamino)ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA copolymer and PEO-b-poly(diethylamino)ethyl methacrylate (PEO-b-PDEA) copolymer. Despite their similar chemical structures, their transfection efficiencies were significantly different. PEO-b-PDEA copolymer was significantly less efficient as gene carrier compared to both PDMA and PEO-b-PDMA systems. Results from quantitative real-time polymerase chain reaction (real-time PCR), cytotoxicity and Zeta potential measurements showed correlations between the physical properties of the polymers and the efficiencies of cellular uptake of the transgene and transfections. In the case of PEO-b-PDEA system, cytotoxicity was due primarily to the excess polymers that did not participate in the DNA binding. In addition, the inability of the polymer/DNA complexes to interact with cell effectively was identified as the main barrier for high efficiency of transfection. This study demonstrated that the use of quantitative real-time PCR in combination with other physical characterization techniques can provide greater insights into the transfection barrier at different cellular levels. / Singapore-MIT Alliance (SMA)

Determination of quantitative expression levels of alternatively spliced isoforms provides an important approach to the understanding of the functional significance of each isoform. Real-time PCR using exon junction overlapping primers has been shown to allow specific detection of each isoform. However, this design often suffers from severe cross amplification of sequences with high homology at the exon junctions. We used human GFRα2b as a model to evaluate the specificity of primers substituted with locked nucleic acids (LNAs). We demonstrate here that single LNA substitutions at different positions of 3’ terminus could improve the discrimination of the primers against GFRα2a template, a highly homologous isoform. While LNA substitutions of GFRα2b primer at the residues possessing different sequences as GFRα2a has limited improvement in specificity, two consecutive LNA substitutions preceding the different sequences has dramatically improved the discrimination by greater than 100,000-fold compared to the non-substituted primer. Thus, LNA when substituted at certain residues can allow the discrimination of highly homologous sequences. / Singapore-MIT Alliance (SMA)