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.

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.

The bacterium Helicobacter pylori is a human pathogen that infects a large proportion of the world's population and is associated with serious diseases such as gastric ulcers and adenocarcinoma. The motility of this organism, by virtue of sheathed polar flagella is essential to colonisation and persistence in the human host. The sequencing of the H. pylori genome in 1996 identified homologues of the majority of the flagellar genes found in S. enterica serovai typhimurium. These included genes encoding the flagellum ATPase, FliI and FliH a presumptive inhibitor, the primary focus of this study. Sequencing did not originally identify an H. pylori homologue of the flagellar chaperone FliJ, and this is also considered in this study. Bioinformatic analysis and modeling suggests a structural and functional relationship between FliI and homologues such as F1-ATPase α- and β-subunit. In particular, residues 2-91 of FliI resemble the N-terminal domain of the F1-ATPase α- and β-subunits. Biochemical analyses reported in this thesis showed that a truncated FliI-(2- 91) protein was folded, although the N-terminal 18 residues were likely unstructured. Furthermore, deletion mutagenesis showed that this disordered segment of the protein mediates interaction with FliH and very likely forms an amphipathic α-helix upon forming of the FliI-FliH complex. The scanning mutagenesis of this interaction segment of FliI identified a cluster of conserved hydrophobic residues that was critical for the interaction with FliH. Thus, the interaction between FliI and FliH has similarities to the interaction between the N-terminal α-helix of the α-subunit and the globular domain of the δ-subunit of the F1-ATPase. This similarity suggests that FliH, by analogy with the δ-subunit of the F1-ATPase, may function as a molecular stator of the flagellum. The findings presented above have been published (96). The function of a putative H. pylori FliJ homologue, HP0256, was also investigated by knock-out mutagenesis. Disruption of this gene does not abolish flagellar assembly, however further research continued beyond this thesis showed that the knock-out mutant results in impaired motility.

No description available.

Origins and dispersal of the sweet potato and bottle gourd in Oceania :|bimplications for prehistoric human mobility : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand

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.

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.

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.