Transport stress research has shown correlations among stress, morbidity, and mortality in calves subjected to the traditional U.S. market system, indicating the possibility of compromised immune function. The objective of this study was to determine if expression of specific immune and inflammatory response genes differed between calves that were subjected to either an acute stress (AS, handled and weaned for 1.5 h) or a chronic stress (CS, weaned, handled and transported for 3 to 4 d). Two groups of forty calves, Bos taurus (n = 20) and crossbred calves (n = 20), weighing 181 kg to 250 kg were used in each of two trials. Jugular veni-puncture blood samples (9 ml) were collected from AS calves 1.5 h after the start of handling and separation from their dam. Samples were collected from CS calves during processing after arrival at a north Texas feed lot. RNA for gene expression analysis was extracted from peripheral blood leukocytes obtained from blood samples by a filtration method. During the second trial, the filtrate was centrifuged for measurement of plasma cortisol. A diagonal covariance mixed model ANOVA was used to determine effects of treatment, breed, and breed by treatment interaction on cortisol concentrations. Expression values for each gene were analyzed using linear models that considered the effects of treatment (AS and CS) and breed (Bos taurus and crossbred calves) comparing each trial separately. Mean plasma cortisol concentrations did not differ between AS (16.40 +/- 1.08 ng/ml) and CS calves (18.06 +/- 1.14 ng/ml) (P > 0.296). The interaction of effects was detected for 2 genes in Trial 1, and 3 genes in Trial 2 (P < 0.029). Breed was influential for 5 genes in both Trial 1 and 2 (P < 0.046). Significant differences were found in relative quantification for 30 genes in Trial 1 and 36 genes in Trial 2, in which CS calves had greater expression than AS calves (P < 0.047). Fifteen of those genes were common between the two trials with mean treatment differences of RQ values from the 15 genes ranging from 0.309 to 913.19, excluding outliers. Similar elevated cortisol concentrations in both the AS and CS calves indicated that both groups experienced significant stress. However, changes in gene expression differences were greater in the calves subjected to CS, indicating that gene expression may be more useful than cortisol for identifying detrimental long-term stress.

Rhodococcus fascians, a plant pathogenic actinomycete with a very broad host range, causes leafy galls and other malformations. The plant hormone, group, the cytokinins has been implicated in the alteration of host morphology. The aim of this project was to gain insight into the interaction of the cytokinin biosynthetic, isopentenyltransferase (IPT), cytokinin activating ( LOG (The Lonely Guy)) and the cytokinin metabolic, cytokinin oxidase/dehydrogenase (CKX) gene families of both Pisum sativum and R. fascians during infection of the plant. R. fascians colonisation and infection of pea were examined using scanning electron microscopy (SEM) and light microscopy. The expression of genes related to cytokinin biosynthesis, activation and metabolism were isolated and assessed with polymerase chain reaction (PCR) and real time-quantitative PCR (RT-qPCR) analysis. Primers were designed to discriminate between pea genes and R. fascians genes. In addition, the response of the pea cotyledons to R. fascians was measured through chlorophyll estimation and the expression of the transporter genes, sucrose transporter (SUT) and amino acid permease (AAP) which were assayed through RT-qPCR. The pea response regulators were monitored as an indirect measure of the level of endogenous cytokinins in pea. Two R. fascians strains, the avirulent strain 589 and the virulent strain 602, were selected for this project based on their virulence and similar growth rate under identical conditions. The virulence of R. fascians virulent strain 602 was also confirmed through Koch's postulates. The phenotypic alterations in the pea infected with the virulent strain 602 included stunted growth, multiple shoots, small leaves, thickened primary roots and reduced secondary root growth. Delayed senescence of shoots and dark green, intact cotyledons were also observed. Microscopic analyses revealed epiphytic colonisation by both the avirulent strain 589 and the virulent strain 602 in pea cotyledons, roots, shoots and leaves and endophytic colonisation in the seed coat from the time of seed inoculation to 45 days post inoculation (dpi). The expression of R. fascians genes was relatively high at 5 and 9 dpi in pea cotyledons and at 15 and 25 dpi in roots and shoots of pea infected with the virulent strain 602. The expression of RfIPT, RfLOG and RfCKX was not detected both in the control pea and the pea infected with the avirulent strain 589. The cytokinin biosynthesis, metabolism and response regulator (RR) multi-gene families of PsIPTs, PsLOGs, PsCKXs and PsRRs revealed differential and tissue-specific expression patterns. The expression of PsIPTs and PsLOGs was induced immediately after inoculation with the R. fascians virulent strain 602 in the cotyledon but not in roots and shoots, and the expression level reduced at later growth stages. The PsCKXs and PsRRs expression level increased with the growth of the host infected with the virulent strain 602. In pea infected with the avirulent strain 589 the expression of PsIPTs, PsLOGs and PsCKXs gene family members generally increased after 25 dpi in cotyledons, roots and shoots, whereas PsRRs expression was low at all time points. The up-regulation of PsIPTs and PsLOGs immediately after inoculation in cotyledons and at 15 dpi in roots and shoots by R. fascians virulent strain leads to elevated cytokinins which is reflected by the up-regulation of PsRRs. The plant responds to elevated cytokinin by producing phenotypic changes including shoot malformations. The plant activates its cytokinin homeostasis mechanism due to change in cytokinin level which is indicated by up-regulation of PsCKXs. Generally, the expression of the PsRRs was also up-regulated over time following infection by the R. fascians virulent strain. This indicates the presence of biologically active cytokinins in the host which maintain the symptoms. The outcome due to the avirulent strains indicates that, even though PsIPTs and PsLOGs are up-regulated at later growth stages (25 to 35 dpi), expression of PsCKX gene families were varied (either up-regulated or down regulated after 25 dpi). However, PsRRs expression was down-regulated suggesting low cytokinins levels in tissues which may be due to the activation of homeostatic mechanisms of the plant to reduce the level of biologically active cytokinins. The chlorophyll content increased in cotyledons infected with the virulent strain 602 and PsSUTs and PsAAPs expression pattern in pea cotyledon and shoot infected with the virulent strain 602 indicates that R. fascians converts the infected tissue into a sink for their establishment and growth.

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The advent of microarray technology, allowing measurement of gene expression levels for thousands of genes in parallel, has made possible experiments designed to investigate the genetic control of variation in gene expression level (described in the literature as ???genetical genomics??? or ???eQTL??? experiments). Published results from these studies, in yeast and in mice, show that genetic variation is an important factor in gene regulation, and furthermore that individual polymorphisms modify the expression level of many genes. The concern of this thesis is the bioinformatic analyses of the expression level and genotype data sets that are the raw material for these studies. In particular this thesis addresses the two issues of detection of artefactual effects, and maximizing the information that can be extracted from the data. It is shown that while a polymorphism affecting the expression of many genes may be readily detected, care must be taken to determine whether the detected effect is genuinely one of genetic control of expression level, rather than the effect of correlations in measured expression level not of genetic cause. A significance test is devised to distinguish between these cases. The detection of artefactual correlation is explored further in the reanalysis of the published data from a large yeast study. A critique is given of the permutation method used to ascribe genetic control as the cause of inter gene expression level correlation. The presence of some degree of artefactual correlation is shown, and novel methods are presented for identifying such artefacts. To extend the analyses that may be applied to eQTL data, an algorithm is presented for determining secondary eQTLs for gene expression level (as opposed to a single primary QTL), along with a significance test for the putative QTL found. The technique is demonstrated on a large public data set. In addition to the use for which they are intended, the data sets generated for eQTL studies provide opportunities for additional analyses. In this thesis a method is developed for calculating a genome wide map of meiotic recombination frequency from the genotype data for multiple segregant strains. The method is demonstrated on the published genotype data generated for a large yeast eQTL study.

Typescript (photocopy) / 194 leaves, [19] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1981

Typescript (photocopy) / Appendix in end pocket / vi, 99 leaves, [17] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1981

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