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Recently, it has been shown that genetic variation that perturbs the regulation of gene expression is widespread in eukaryotic genomes. Regulatory variation (RV) is expected to be an important driver of phenotypic differences, evolutionary change, and susceptibility to complex genetic diseases. Because trans-acting regulators of gene expression control mRNA levels of multiple genes simultaneously, we hypothesise that RV that affects these components will have a shared-influence upon the expression levels of multiple genes. Since genes are regulated in trans by combinations of basal and tissue specific factors, we further hypothesise that RV in these components may have different effects in each tissue. We used microarrays to identify 755 genes that were affected by RV in at least one of the brain, kidney and liver of two inbred mouse strains, C57BL/6J and DBA/2J. Just 2% were affected in all three tissues, suggesting that the influence of RV is predominantly tissue specific. To study shared-RV, we measured the expression levels of these 755 genes in the same 3 tissues from a panel of recombinant inbred mice, and identified groups of correlated genes that are putatively under the influence of shared trans-acting RV. Using methods that we developed for studying the effects of RV in multiple tissues, we identified 212 genes that are correlated in all three tissues, which include 10 groups of at least 3 genes. We developed a novel method called coherency analysis to show that RV consistently affected the expression levels of these groups of genes in different genetic backgrounds. Strikingly, the relative up- or down-regulation of genes in each group was markedly different in the three tissues of the same mouse, suggesting that the influence of RV itself is not tissue specific as previously expected, but that RV can influence genes with differing outcomes in each tissue. These observations are compatible with RV affecting combinations of basal and tissue specific regulatory factors. This is the first cross-tissue investigation into the influence of shared-RV in multiple tissues, which has important implications in humans, where access to the phenotypically relevant tissue may be necessarily limited.

Recently, it has been shown that genetic variation that perturbs the regulation of gene expression is widespread in eukaryotic genomes. Regulatory variation (RV) is expected to be an important driver of phenotypic differences, evolutionary change, and susceptibility to complex genetic diseases. Because trans-acting regulators of gene expression control mRNA levels of multiple genes simultaneously, we hypothesise that RV that affects these components will have a shared-influence upon the expression levels of multiple genes. Since genes are regulated in trans by combinations of basal and tissue specific factors, we further hypothesise that RV in these components may have different effects in each tissue. We used microarrays to identify 755 genes that were affected by RV in at least one of the brain, kidney and liver of two inbred mouse strains, C57BL/6J and DBA/2J. Just 2% were affected in all three tissues, suggesting that the influence of RV is predominantly tissue specific. To study shared-RV, we measured the expression levels of these 755 genes in the same 3 tissues from a panel of recombinant inbred mice, and identified groups of correlated genes that are putatively under the influence of shared trans-acting RV. Using methods that we developed for studying the effects of RV in multiple tissues, we identified 212 genes that are correlated in all three tissues, which include 10 groups of at least 3 genes. We developed a novel method called coherency analysis to show that RV consistently affected the expression levels of these groups of genes in different genetic backgrounds. Strikingly, the relative up- or down-regulation of genes in each group was markedly different in the three tissues of the same mouse, suggesting that the influence of RV itself is not tissue specific as previously expected, but that RV can influence genes with differing outcomes in each tissue. These observations are compatible with RV affecting combinations of basal and tissue specific regulatory factors. This is the first cross-tissue investigation into the influence of shared-RV in multiple tissues, which has important implications in humans, where access to the phenotypically relevant tissue may be necessarily limited.

Recently, it has been shown that genetic variation that perturbs the regulation of gene expression is widespread in eukaryotic genomes. Regulatory variation (RV) is expected to be an important driver of phenotypic differences, evolutionary change, and susceptibility to complex genetic diseases. Because trans-acting regulators of gene expression control mRNA levels of multiple genes simultaneously, we hypothesise that RV that affects these components will have a shared-influence upon the expression levels of multiple genes. Since genes are regulated in trans by combinations of basal and tissue specific factors, we further hypothesise that RV in these components may have different effects in each tissue. We used microarrays to identify 755 genes that were affected by RV in at least one of the brain, kidney and liver of two inbred mouse strains, C57BL/6J and DBA/2J. Just 2% were affected in all three tissues, suggesting that the influence of RV is predominantly tissue specific. To study shared-RV, we measured the expression levels of these 755 genes in the same 3 tissues from a panel of recombinant inbred mice, and identified groups of correlated genes that are putatively under the influence of shared trans-acting RV. Using methods that we developed for studying the effects of RV in multiple tissues, we identified 212 genes that are correlated in all three tissues, which include 10 groups of at least 3 genes. We developed a novel method called coherency analysis to show that RV consistently affected the expression levels of these groups of genes in different genetic backgrounds. Strikingly, the relative up- or down-regulation of genes in each group was markedly different in the three tissues of the same mouse, suggesting that the influence of RV itself is not tissue specific as previously expected, but that RV can influence genes with differing outcomes in each tissue. These observations are compatible with RV affecting combinations of basal and tissue specific regulatory factors. This is the first cross-tissue investigation into the influence of shared-RV in multiple tissues, which has important implications in humans, where access to the phenotypically relevant tissue may be necessarily limited.

Phosphite has been used to effectively control the soil borne plant pathogen Phytophthora cinnamomi in many horticultural crops, forest trees and natural ecosystems. However, the molecular mechanisms behind phosphite action on this pathogen are poorly understood. Several studies have shown that phosphite inhibits growth and zoospore production of P. cinnamomi and in addition induces significant physiological and metabolic changes in the mycelium. As an approach to understanding the mechanisms and relevance of these changes in the pathogen, the effect of phosphite on gene expression was investigated using microarray analysis. To construct the microarray, RNA was extracted from phosphite-treated (40 ug/ml) mycelium of P. cinnamomi isolate MP 80. The chosen phosphite concentration inhibited the mycelial growth by 70% but provided sufficient mycelium for RNA extractions after 4 days growth at 25C. The mRNA was reverse transcribed into cDNA and cloned into lambda to construct a library consisting of 2 million pfu of which 80 % were recombinant phage. The inserts were sequenced for a random selection of clones from the library. The nucleotide sequences generated revealed a range of different P. cinnamomi genes being expressed and demonstrated that the cDNA library provided a good representation of the transcripts expressed in P. cinnamomi. The types of genes found to be expressed in the mycelium of P. cinnamomi included genes encoding GTP binding proteins involved in vesicle transport, structural proteins involved in maintaining cell membrane integrity,elicitors, phosphatases and ribosomal proteins. Over nine thousand cDNA transcripts were randomly selected from the cDNA library and prepared by PCR amplification and purification for microarray construction. Custom made cDNA arrays containing 9216 cDNA transcripts were constructed and probed with RNA from untreated mycelium and mycelium grown in medium with 40 ug/ml phosphite. Two genes, EF-1 alpha and cinnamomin gene, identified by qRT-PCR as being constitutively expressed were also positioned on the arrays as positive controls. In the process of identifying constitutively expressed genes, qRT PCR revealed that phosphite down-regulated a gene encoding ubiquitin-conjugating enzyme, a component of the ubiquitin/proteasome pathway involved in the removal of abnormal and short lived-regulatory proteins and rate limiting enzymes. From the arrays a further seventy-two transcripts with altered patterns in gene expression (fold change > 2) were identified. The majority of the cDNA transcripts spotted on the array were down-regulated with changes in gene expression ranging from 2- to 3.5-fold. Thirty-two cDNA transcripts were up-regulated with changes in gene expression ranging from 2- to 16-fold. Characterisation by sequencing revealed that the most highly induced transcripts coded for ADP-ribosylation factors, an ABC cassette transporter and a glycosyl transferase. A transcript encoding a vitamin B6 biosynthesis protein was also identified as up-regulated by 2.9-fold. In contrast, the down-regulated transcripts coded for cellulose synthase I, annexin, glutamine synthetase, metallothionein and an alternative oxidase. The results are discussed in terms of possible roles and mechanism(s) of phosphite action within the mycelium of P.cinnamomi. This work is the first comprehensive screen for phosphite regulated-gene expression in P. cinnamomi and represents a significant step towards an understanding of the mode of action of phosphite on this organism. This thesis provides valuable information on the molecular interaction between phosphite and P. cinnamomi, which in future studies may stimulate the discovery of novel methods and cellular targets for the control of plant pathogenic Oomycetes.

RhoA is a small GTPase that acts as a molecular switch to control a variety of signal transduction pathways in eukaryotes. From an initial established role in the regulation of the actin cytoskeleton, RhoA has now been implicated in a range of functions that include gene transcription and regulation of cell morphology. In earlier studies from this laboratory that employed differential display and in situ hybridisation, RhoA was indicated as being up-regulated during the stages of early heart development in the developing chick embryo. Given the important effects of RhoA on both gene expression and morphology in other systems, it was hypothesised that RhoA plays a central role in the molecular mechanisms controlling cardiogenesis. This thesis describes investigations undertaken to elucidate the role of RhoA in these processes. As an initial approach to corroborate the earlier gene expression findings and provide further evidence for a role in tissue developmental mechanisms, RhoA proteins levels in the developing chick embryo were analysed using immunocytochemistry. These experiments demonstrated that RhoA is most abundant in heart-forming regions, findings compatible with the earlier gene expression studies and the proposed role of this protein in early heart development. Preliminary studies from this laboratory had also suggested that chick RhoA is expressed as different length mRNA transcripts that vary only in the 3' untranslated region (UTR). This thesis presents additional evidence for the existence of these different RhoA transcripts from experiments using Northern hybridisation and RT-PCR analyses. These analyses also serve to demonstrate that the second shortest RhoA transcript (designated RhoA2) is the most abundant transcript in developing heart tissue, in contrast to the situation in other embryonic tissues, findings that could be taken to suggest a possible role for this 3'UTR in developmental mechanisms that is yet to be elucidated. One potentially informative approach for testing the function of a protein in a biological system is to inhibit its expression and/or activity and observe the changes induced. The effects of inhibiting RhoA in early heart development and early organogenesis in the chick embryo model were investigated using small interfering RNAs (siRNA). Reduction in RhoA expression by siRNA treatment, as confirmed by real-time PCR, resulted in loss of heart tube fusion and abnormal head development, the former result providing further direct evidence of a role for RhoA in heart developmental processes. In order to investigate the function of RhoA specifically during the process of cardiomyocyte differentiation, an inducible model of cardiomyogenesis, P19CL6 cells, was used in combination with over-expression of different forms of mouse RhoA. The striking result from these investigations was that over-expression of the dominant negative mutant of mouse RhoA (mRhoAN19) prevented the differentiation of induced P19CL6 cells to the cardiomyocyte phenotype, results consistent with an essential role for RhoA in this cellular transition. The mechanism by which RhoA mediates its different cellular functions is unclear, however some studies have implicated RhoA in the regulation of transcription factors. To investigate such a mechanism as a possible explanation for the requirement of RhoA in cardiomyocyte differentiation, the P19CL6 inducible cell system over-expressing different forms of RhoA was analysed through real-time PCR to quantify the levels of transcription of genes known to play an important role in early heart development. These investigations indicated that RhoA inhibition causes an accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac-cx-actin. The expression of a protein is controlled by, among other factors, regulatory proteins that control transcription. To investigate factors in heart that potentially regulate RhoA expression at the molecular level, the chick RhoA gene organisation was analysed. The gene was shown to contain three introns that interrupt the protein coding sequence and at least one intron in the 5'UTR. Comparative RhoA gene studies indicated both an almost identical organisation and coding sequence of the chick, mouse and human RhoA genes, indicative of strict conservation of this gene during evolution. The putative promoter region of RhoA was predicted by computer analyses and tested for promoter activity using luciferase reporter analyses in non-differentiated and differentiated cardiomyocytes, using the inducible P19CL6 cell system. These investigations served to define a putative core promoter region that exhibited significantly higher promoter activity in differentiated cardiomyocytes than in non-differentiated cells, and other elements upstream of this core region that appear to be required for transcriptional regulation of RhoA. The majority of the consensus transcription factor sites identified in this putative promoter have been previously implicated in either heart development and/or organogenesis. These results therefore provide further, although indirect, evidence for an important role for RhoA in the molecular mechanisms controlling both cardiogenesis and embryogenesis in general. In summary, this thesis provides novel information on the role of RhoA in the processes of cardiogenesis and provides a firm foundation for continuing investigations aimed at elucidating the molecular basis of this contribution.

This study examines the differences in the beliefs of primary (elementary) class teachers about the role of expression in visual arts education. The focus is on the relationship between primary teachers implicit theories, and expression in their visual arts lessons. Expression here, refers to the manifestation of an individual students interests or views through visual images. Students' expression of their personal responses through visual images, is a central intended outcome of the visual arts curriculum in Western Australia (Curriculum Council, 1998).

Background and objectives: The most widely accepted tooth movement model is defined by the pressure-tension hypothesis. An orthodontic force applied to a tooth generates areas of compression and tension in the periodontal ligament (PDL), which are transmitted to the alveolar bone. Areas of tissue exposed to tensile strain undergo bone deposition, whereas areas of tissue exposed to compressive strain undergo bone resorption. We propose that human PDL cells in monolayer culture exposed to tensile mechanical strain would express multiple genes involved in osteogenesis. Materials and Methods: Human PDL cells were isolated and cultured from premolar teeth that were extracted for orthodontic reasons. These cells were plated on control and experimental Uniflex[TM] plates. Using a Flexercell FX4000 strain unit, PDL cells on experimental plates were exposed to a 12% uni-axial cyclic strain for 6 seconds out of every 90 seconds over a 24 hour period. RNA was extracted from the PDL cells at 6 hours, 12 hours and 24 hours. The differential expression of 78 genes implicated in osteoblast differentiation and bone metabolism was analysed using real-time reverse transcriptase polymerase chain reaction (RT-PCR) array technology. Results: Of the 78 genes tested, sixteen genes showed statistically significant (p<0.05) changes in expression in response to the mechanical strain regime. Eight genes were up-regulated (ALPL, BMP2, BMP6, COL2A1, ICAM1, PHEX, SOX9, and VEGFA) and eight genes were down-regulated (ANXA5, BMP4, COL11A1, COL3A1, EGF, ITGB1, MSX and SMAD1). Conclusions: This study has demonstrated that cultured human PDL cells express multiple osteogenic genes under tensile strain, which suggests that PDL cells may have a potential role in osseous remodeling during tooth movement. Key Words: Tooth movement, human PDL cells, tensile mechanical strain, osteogenic genes, real-time RT-PCR array, and Flexercell FX4000.

The focus of this dissertation was leptin and the leptin receptor, and the role of these genes (OB and OB-R) in the development of obesity and type 2 diabetes in humans and Psammomys obesus, a polygenic rodent model of obesity and type 2 diabetes. Studies in humans showed that circulating leptin concentrations were positively associated with adiposity, and independently associated with circulating insulin and triglyceride concentrations. Analysis of two leptin receptor sequence polymorphisms in a Caucasian Australian population and a population of Nauruan males, with very high prevalence rates of obesity, showed no associations between sequence variation within the OB-R gene and obesity- or diabetes-related phenotypic measures. In addition, these two OB-R polymorphisms were not associated with longitudinal changes in body mass or composition in either of the populations examined. A unique analysis of the effects of multiple gene defects in the Nauruan population, demonstrated that the presence of sequence alterations in both the OB and OB-R genes were associated with insulin resistance. Psammomys obesus is regarded as an excellent rodent model in which to study the development of obesity and type 2 diabetes in humans. Examination of circulating leptin concentrations in Psammomys revealed that, as in humans, leptin concentrations were associated with adiposity, and independently associated with circulating insulin concentrations. This animal model was utilised to examine expression of OB-R, and the regulation of expression of this gene after dietary manipulation. OB-R is known to have several isoforms, and in particular, OB-RA and OB-RB gene expression were examined. OB-RB is the main signalling isoform of the leptin receptors. It has a long intracellular domain and has previously been shown to play an important role in energy balance and body weight regulation in rodents and humans. OB-RA is a much shorter isoform of OB-R, and although it lacks the long intracellular domain necessary to activate the JAK/STAT pathway, OB-RA is also capable of signalling, although to a lesser degree than OB-RB. OB-RA is found to be expressed almost ubiquitously throughout the body, and this isoform may be involved in transport of leptin into the cell, although its role remains unclear. OB-RA and OB-RB were both found to be expressed in a large number of tissues in Psammomys obesus. Interestingly, obese Psammomys were found to have lower levels of expression of OB-RA and OB-RB in the hypothalamus, compared to lean animals. This finding raises the possibility that decreased leptin signalling in the brain of obese, hyperleptinemic Psammomys obesus may contribute to the leptin resistance previously described in this animal model. However, the primary defect is unclear, as alternatively, increased circulating leptin concentrations may lead to down-regulation of leptin receptors. The effect of fasting on leptin concentrations and gene expression of OB-RA and OB-RB was also examined. A 24-hour fast resulted in no change in body weight, but a reduction in circulating leptin concentrations, and an increase in hypothalamic OB-RB gene expression in lean Psammomys. In obese animals, fasting again did not alter body weight, but resulted in an increase in both circulating leptin concentrations and hypothalamic OB-RB gene expression. In the liver, fasting resulted in a large increase in OB-RA gene expression in both lean and obese animals. These results highlighted the fact that regulation of leptin receptor gene expression in polygenic models of obesity and type 2 diabetes is complex, and not solely under the control of circulating leptin concentrations. Sucrose-feeding is an established method of inducing obesity and type 2 diabetes in rodents, and this experimental paradigm was utilised to examine the effects of longer term perturbations of energy balance on the leptin signalling pathway in Psammomys obesus. Addition of a 5% sucrose solution to the diet of lean and obese Psammomys resulted in increased body weight in both groups of animals, however only obese Psammomys showed increased fat mass and the development of type 2 diabetes. The changes in body mass and composition with sucrose-feeding were accompanied by decreased circulating leptin concentrations in both groups of animals, as well as a range of changes in leptin receptor gene expression. Sucrose-feeding increased hypothalamic OB-RB gene expression in obese Psammomys only, while in the liver there was evidence of a reduction in OB-RA and OB-RB gene expression in both lean and obese animals. The direct effects of sucrose on the leptin signalling pathway are unclear, however it is possible to speculate that the effect of sucrose to decrease leptin concentrations may have been involved in the exacerbation of obesity and the development of type 2 diabetes in obese Psammomys, From these studies, it appears that sequence variation in the OB and OB-R genes is unlikely to be a major factor in the etiology of obesity in human populations. The ability to examine regulation of expression of these genes in Psammomys obesus, however, has demonstrated that the effects of nutritional modifications on leptin receptor gene expression need closer attention. The role of the OB and OB-R genes in metabolism and the development of type 2 diabetes also warrants further examination, with particular attention on the differential effects of dietary modifications on leptin receptor gene expression across a range of tissues.

Gene expression and its regulation is a highly coordinated system, involved in many biological processes such as cell growth, division and differentiation. Transcriptional regions, involved in gene regulation, consist of a heterogeneous collection of smaller regulatory elements. In some cases, co-regulated genes contain a common set of transcription factor binding sites (TFBS). Analysis of promoter regions is the major approach in understanding the transcriptional regulatory mechanisms. It is also useful for interpretation of mammalian gene expression studies, where co-expressed genes may share motifs representing putative TFBS. Motif identification also has the advantage that it can predict control regions in genes that have not been measured experimentally. However, a common problem is incomplete genomic sequence for the experimental species of interest. The approach here is to identify and use orthologous gene promoter sequences from a related and well-characterised species. The primary aim of this study was to identify and predict regulatory TFBS in species where promoter sequence does not exist or is incomplete. The MEME programme was employed for the motif prediction step. The predicted elements were subsequently compared to known TFBS using TRANSFAC and JASPAR databases for identification. A methodology based on relative entropy was used. The validity of the method was confirmed as the predicted motifs in the training set were the expected sites involved in regulation of muscle development. The technique was applied to two data sets, generated from expressed sequence tag (EST) clustering analysis and microarray experiments. All data sets, software and results are available on the accompanying CD. Bovine expression data was analysed for cardiac-specific expression using two separate approaches, combining bovine library EST frequency and human gene expression ratios. For each approach, the orthologous human and bovine promoter sequences were analysed for common motifs. Across all comparisons, 37% of motifs were identified as known TFBS using the TRANSFAC and JASPAR databases. As the human comparison had more promoter sequences available, this was the main limiting factor for the corresponding bovine analysis, rather than cross-species divergence or accuracy of gene expression measurement. Results from this study demonstrate that using promoter sequences from a related species is a viable approach when studying gene expression in species with limited amount of genomic sequence. As the bovine genome becomes more complete, it can in turn serve as the reference genome for other agriculturally important ruminants, such as sheep, goat and deer. The second application concerned in silico analysis of gene regulation patterns in response to stimuli. Recently it has been shown that a mutation in the bone morphogenetic receptor IB leads to an increased ovulation rate in sheep. The objective of this study was to analyse gene expression patterns in cultured cells in response to four members of the BMP family, i.e. BMP2, BMP4, BMP6 and BMP7 and the control TGFβ. Microarray data was provided by J. Young. Twelve highly upregulated genes were stimulated by all BMPs, seven of which are known BMP target genes. Analysis of the predicted motifs identified four elements that may be involved in the regulation process. Cross-species comparison for one of the genes, ID1, showed high conservation of one of the motifs across 11 mammalian genomes. This particular motif had not been identified as a known binding site. In summary, the analysis of the expression data suggest an extension of the list of BMP targets. The proposed method is relatively robust when sufficiently co-expressed (co-regulated) sequences can be identified, whether from the same or another species.