The subject of this thesis is the molecular genetic study of idiopathic hyperphosphatasia. The work in this thesis describes linkage analysis, mutation screening of candidate genes and the functional analysis of mutant proteins expressed in patients with a clinical diagnosis of idiopathic hyperphosphatasia. Idiopathic hyperphosphatasia is an autosomal recessive bone disease characterized by excessive bone resorption and bone formation. Affected children are normal at birth but develop deformities of long bones, kyphosis and acetabular protrusion with increasing severity as they pass through adolescence. There is considerable variability in phenotype, with some cases diagnosed in infancy and others in later childhood. A genome-wide search of a New Zealand family affected by idiopathic hyperphosphatasia suggested linkage to a locus on the long arm of chromosome 8 (8q24). The gene TNFRSF11B encoding osteoprotegerin (OPG), which lies within 8q24, was an obvious candidate gene given the critical role of OPG in regulating osteoclast development. Mutation screening of this gene indicated an apparent disease-causing mutation in exon 3 in affected individuals of the New Zealand family. Subsequently eight families, recruited by the members of the Idiopathic Hyperphosphatasia Collaborative Group in Turkey, Germany, Argentina and the United kingdom were also screened for mutations in the TNFRSF11B gene. Recombinant wild-type and mutant OPG CDNAs were expressed in human epithelial kidney cells, and secreted OPG was collected form the conditioned medium. In vitro measurements of osteoclastic bone resorption showed that wild type OPG suppressed bone resorption, whereas the mutant forms did not, confirming them to be inactivating mutations.

The subject of this thesis is the molecular genetic study of idiopathic hyperphosphatasia. The work in this thesis describes linkage analysis, mutation screening of candidate genes and the functional analysis of mutant proteins expressed in patients with a clinical diagnosis of idiopathic hyperphosphatasia. Idiopathic hyperphosphatasia is an autosomal recessive bone disease characterized by excessive bone resorption and bone formation. Affected children are normal at birth but develop deformities of long bones, kyphosis and acetabular protrusion with increasing severity as they pass through adolescence. There is considerable variability in phenotype, with some cases diagnosed in infancy and others in later childhood. A genome-wide search of a New Zealand family affected by idiopathic hyperphosphatasia suggested linkage to a locus on the long arm of chromosome 8 (8q24). The gene TNFRSF11B encoding osteoprotegerin (OPG), which lies within 8q24, was an obvious candidate gene given the critical role of OPG in regulating osteoclast development. Mutation screening of this gene indicated an apparent disease-causing mutation in exon 3 in affected individuals of the New Zealand family. Subsequently eight families, recruited by the members of the Idiopathic Hyperphosphatasia Collaborative Group in Turkey, Germany, Argentina and the United kingdom were also screened for mutations in the TNFRSF11B gene. Recombinant wild-type and mutant OPG CDNAs were expressed in human epithelial kidney cells, and secreted OPG was collected form the conditioned medium. In vitro measurements of osteoclastic bone resorption showed that wild type OPG suppressed bone resorption, whereas the mutant forms did not, confirming them to be inactivating mutations.

The architecture of epithelial monolayers is critically influenced by the adhesion-mediating signalling receptor, E-cadherin. E-cadherin has long been known to be fundamental for maintaining cell-cell cohesion and cell-cell junctional integrity, and this relies on its close association with the actin cytoskeleton. The exact underlying molecular mechanism of Ecadherin-actin cooperation at contacts is, however, poorly understood. Analysing the many effector proteins that mediate the cadherin-actin interaction is the first step toward a clearer understanding of this interaction. The interaction between E-cadherin and actin is complex. This is likely due to the dynamic nature of intercellular contacts. A pertinent example of the dynamic evolution of cell-cell contacts is seen when cell contacts mature in relation to one another over a period of time. Here, initial discontinuous contacts are replaced by continuous linear contacts and cadherin and actin morphologies undergo a concomitant transformation. In this study I have identified a molecular pathway that is necessary for this intercellular contact maturation process. I now report that Myosin VI critically regulates the morphogenesis of epithelial cellcell contacts. When epithelial monolayers mature in culture, Myosin VI is recruited to cell contacts as they become linear and cohesive, where it forms a biochemical complex with Ecadherin. Myosin VI is necessary for strong cadherin adhesion, for cells to form cohesive linear contacts, and for the integrity of the apical junctional complex. Importantly, I found that vinculin mediates this effect of Myosin VI. In the absence of Myosin VI, vinculin is lost from cell-cell contacts and is unable to interact with E-cadherin. Further, vinculin is able to rescue contact integrity even in the absence of Myosin VI, thus identifying vinculin as a downstream effector of Myosin VI necessary for the biogenesis of intercellular contacts. Together this shows that the actin effector proteins, Myosin VI and vinculin, form a molecular apparatus that generates cohesive cell-cell contacts in cultured mammalian epithelia. From a broader perspective, this work emphasises the dynamic nature of cadherinactin interactions at cell-cell contacts and its regulation by the recruitment of specific actin effectors in a time-and context-dependent manner.

The subject of this thesis is the molecular genetic study of idiopathic hyperphosphatasia. The work in this thesis describes linkage analysis, mutation screening of candidate genes and the functional analysis of mutant proteins expressed in patients with a clinical diagnosis of idiopathic hyperphosphatasia. Idiopathic hyperphosphatasia is an autosomal recessive bone disease characterized by excessive bone resorption and bone formation. Affected children are normal at birth but develop deformities of long bones, kyphosis and acetabular protrusion with increasing severity as they pass through adolescence. There is considerable variability in phenotype, with some cases diagnosed in infancy and others in later childhood. A genome-wide search of a New Zealand family affected by idiopathic hyperphosphatasia suggested linkage to a locus on the long arm of chromosome 8 (8q24). The gene TNFRSF11B encoding osteoprotegerin (OPG), which lies within 8q24, was an obvious candidate gene given the critical role of OPG in regulating osteoclast development. Mutation screening of this gene indicated an apparent disease-causing mutation in exon 3 in affected individuals of the New Zealand family. Subsequently eight families, recruited by the members of the Idiopathic Hyperphosphatasia Collaborative Group in Turkey, Germany, Argentina and the United kingdom were also screened for mutations in the TNFRSF11B gene. Recombinant wild-type and mutant OPG CDNAs were expressed in human epithelial kidney cells, and secreted OPG was collected form the conditioned medium. In vitro measurements of osteoclastic bone resorption showed that wild type OPG suppressed bone resorption, whereas the mutant forms did not, confirming them to be inactivating mutations.

The subject of this thesis is the molecular genetic study of idiopathic hyperphosphatasia. The work in this thesis describes linkage analysis, mutation screening of candidate genes and the functional analysis of mutant proteins expressed in patients with a clinical diagnosis of idiopathic hyperphosphatasia. Idiopathic hyperphosphatasia is an autosomal recessive bone disease characterized by excessive bone resorption and bone formation. Affected children are normal at birth but develop deformities of long bones, kyphosis and acetabular protrusion with increasing severity as they pass through adolescence. There is considerable variability in phenotype, with some cases diagnosed in infancy and others in later childhood. A genome-wide search of a New Zealand family affected by idiopathic hyperphosphatasia suggested linkage to a locus on the long arm of chromosome 8 (8q24). The gene TNFRSF11B encoding osteoprotegerin (OPG), which lies within 8q24, was an obvious candidate gene given the critical role of OPG in regulating osteoclast development. Mutation screening of this gene indicated an apparent disease-causing mutation in exon 3 in affected individuals of the New Zealand family. Subsequently eight families, recruited by the members of the Idiopathic Hyperphosphatasia Collaborative Group in Turkey, Germany, Argentina and the United kingdom were also screened for mutations in the TNFRSF11B gene. Recombinant wild-type and mutant OPG CDNAs were expressed in human epithelial kidney cells, and secreted OPG was collected form the conditioned medium. In vitro measurements of osteoclastic bone resorption showed that wild type OPG suppressed bone resorption, whereas the mutant forms did not, confirming them to be inactivating mutations.

The architecture of epithelial monolayers is critically influenced by the adhesion-mediating signalling receptor, E-cadherin. E-cadherin has long been known to be fundamental for maintaining cell-cell cohesion and cell-cell junctional integrity, and this relies on its close association with the actin cytoskeleton. The exact underlying molecular mechanism of Ecadherin-actin cooperation at contacts is, however, poorly understood. Analysing the many effector proteins that mediate the cadherin-actin interaction is the first step toward a clearer understanding of this interaction. The interaction between E-cadherin and actin is complex. This is likely due to the dynamic nature of intercellular contacts. A pertinent example of the dynamic evolution of cell-cell contacts is seen when cell contacts mature in relation to one another over a period of time. Here, initial discontinuous contacts are replaced by continuous linear contacts and cadherin and actin morphologies undergo a concomitant transformation. In this study I have identified a molecular pathway that is necessary for this intercellular contact maturation process. I now report that Myosin VI critically regulates the morphogenesis of epithelial cellcell contacts. When epithelial monolayers mature in culture, Myosin VI is recruited to cell contacts as they become linear and cohesive, where it forms a biochemical complex with Ecadherin. Myosin VI is necessary for strong cadherin adhesion, for cells to form cohesive linear contacts, and for the integrity of the apical junctional complex. Importantly, I found that vinculin mediates this effect of Myosin VI. In the absence of Myosin VI, vinculin is lost from cell-cell contacts and is unable to interact with E-cadherin. Further, vinculin is able to rescue contact integrity even in the absence of Myosin VI, thus identifying vinculin as a downstream effector of Myosin VI necessary for the biogenesis of intercellular contacts. Together this shows that the actin effector proteins, Myosin VI and vinculin, form a molecular apparatus that generates cohesive cell-cell contacts in cultured mammalian epithelia. From a broader perspective, this work emphasises the dynamic nature of cadherinactin interactions at cell-cell contacts and its regulation by the recruitment of specific actin effectors in a time-and context-dependent manner.

In maize, a loss-of-function mutation in a MEI2-like gene, terminal ear1 (te1), leads to morphological defects able to be traced back to the shoot apical meristem. One MEI2-like gene has been identified in maize, while six have been identified in rice and nine in Arabidopsis thaliana. In this thesis, a programme of reverse genetic analysis has been designed to investigate if Arabidopsis genes most closely aligned in parsimony trees with TE1, TERMINAL EAR-LIKE 1 (TEL1), TERMINAL EAR-LIKE 2 (TEL2), perform the same function as TE1. The expression pattern of TEL1 and TEL2 genes is restricted to the Shoot Apical Meristem (SAM) and the Root Apical Meristem (RAM) suggesting these genes are important in meristem maintenance or function. Results of the molecular genetic analysis of TEL genes in Arabidopsis support models in which these genes help maintain cells in a pluripotent state. For the first part of the thesis, analysis of lines carrying single knockouts of TEL1 and TEL2 and double knockout lines reveals a slightly accelerated rate of organogenesis, consistent with these genes normally acting to inhibit terminal differentiation pathways. Plants grown on medium containing gibberellic acid and sucrose, at higher than normal concentrations, present a further accelerated rate of organogenesis. As the second part of the thesis, in situ and promoter/reporter GUS fusion analyses indicate TEL1 is preferentially expressed in both the root and shoot apical meristems. Deletion analysis using GFP reporter constructs show that 5' sequences are sufficient to drive quiescent centre (QC) expression in the root while additional sequences are required for central zone (CZ) expression in the SAM. Physiological studies indicate expression of TEL1 in the root is sensitive to the hormones, auxin, gibberellic acid and zeatin, when added at physiological concentrations. To confirm the auxin effect, GFP expression is no longer visible after 12 hours of exposure to auxin transport inhibitors in plants containing GFP under the control of the TEL1 promoter, suggesting, in common with other QC markers, that TEL expression is sensitive to auxin levels. Analysis of mutant plants with altered root patterning suggests QC specific expression of TEL1 requires early acting genes, such as PLETHORA 1 and 2, but does not depend on later acting genes such as SCARECROW or SHORTROOT.

Content removed from thesis due to copyright restrictions: Eaton, C. J., I. Jourdain, et al. (2008). "Functional analysis of a fungal endophyte stress-activated MAP kinase." Current Genetics 53(3): 163-174. Scott, B. and C. J. Eaton (2008). "Role of reactive oxygen species in fungal cellular differentiations." Current Opinion in Microbiology 11(6): 488-493. / In the mutually beneficial association between the fungal endophyte Epichloë festucae and perennial ryegrass, fungal growth is highly regulated and coordinated with that of the host. This implies there must be signalling between the fungus and its host to maintain this close association. Recent work has shown a novel role for reactive oxygen species (ROS) in this symbiotic maintenance, with multiple components of the superoxideproducing NADPH oxidase (Nox) complex being essential for normal association. However, the mechanism by which the Nox complex is regulated is unclear. To identify potential regulators of the E. festucae Nox complex, comparisons were made with well-characterised mammalian systems. This search identified three candidate regulators: a stress activated MAP kinase, sakA, and the p21-activated kinases, pakA and pakB. To investigate if these genes were involved in symbiotic maintenance, replacement mutants were generated by homologous recombination. In culture analysis revealed that the ?sakA mutant was hypersensitive to a range of stresses, whereas the pak mutants were hypersensitive to cell wall stress-inducing agents and displayed altered growth and morphology. Examination of perennial ryegrass infected with these mutants revealed drastically altered plant interaction phenotypes for the ?sakA and ?pakA mutants in comparison to the wild-type strain. ?sakA-infected plants were stunted and displayed striking changes in development, with the base of tillers showing loss of anthocyanin pigmentation and disorganisation of host cells below the meristem, resulting in swollen bases. Plants infected with the ?pakA mutant were severely stunted, had no more than two tillers and senesced soon after planting. In contrast, plants infected with the ?pakB mutant were similar to wild-type, with only slight deregulation of growth in planta. Examination of ROS in culture revealed that ?sakA and ?pakA displayed elevated levels of both superoxide and hydrogen peroxide. ROS levels were also elevated around ?sakA hyphae in planta. These results support roles for SakA and PakA in Nox regulation. This work highlights the fine balance between mutualism and antagonism, and provides insight into the molecular basis for mutualism.

Preeclampsia is a serious multisystem complication of late pregnancy with adverse effects for mothers and babies. Currently this disorder is diagnosed from clinical observations occurring late in the disease process. Unknown factors in the maternal circulation, possibly released by the preeclamptic placenta, have been linked to the pathophysiological changes characteristic of the disorder. The research in this thesis used proteomic techniques to identify putative preeclampsia biomarkers from two sources: secreted from a placental cell line undergoing differentiation, and directly sampled from the serum and plasma of women with late-onset preeclampsia. The first part of this research examined the secreted proteome of a placental choriocarcinoma cell line (BeWo) undergoing forskolin-mediated differentiation. Development of serum-free culture techniques enabled analysis of these secreted proteins by two-dimensional gel electrophoresis (2DE). Statistical testing revealed the significant involvement of seven spots during this differentiation model, with VE-cadherin and matrix metalloproteinase 2 among the proteins identified. In the second part of this research, maternal serum and plasma proteins were compared from women with preeclampsia and healthy pregnant women. Serum samples were analyzed using 2DE, and plasma was subjected to difference gel electrophoresis (DIGE). Bioinformatic analysis of both datasets identified multiple spot clusters able to classify samples according to disease state. Five of these serum proteins were differentially regulated in preeclampsia, including two isoforms of apolipoprotein E whose isoform-specific expression was confirmed using western blots. Analysis of plasma from preeclamptic women identified six proteins, again including apolipoprotein E. Proteins from both studies are linked to preeclampsia pathophysiology through lipid transport, complement, and retinol transport systems. The culture methods and secreted proteomic techniques developed in this work have uncovered proteins in a placental cell line and maternal serum and plasma that are associated with preeclampsia. These methods can be extended to any system where secreted proteins are of interest. The differentially regulated proteins found in this study provide an important first step towards developing effective biomarkers for diagnosing and/or predicting preeclampsia.

Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.