published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Paediatrics / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

No description available.

This study was undertaken to characterize the nod and sdh operons of Bradyrhizobium japonicum and Sinorhizobium meliloti. Ten putative B. japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. The mutants were found to have higher nodY expression than the wild-type in the presence of genistein. The increased sensitivity of all mutants to genistein was more apparent under suboptimal inducer concentration (0.1muM) and/or temperature (15°C). The expression of nodY gene induction was determined for five strains (Bj30050, 53, 56, 57, 58) under different temperature and inducer conditions. These five strains were also found to produce more lipochitooligosaccharide than the wild-type, at both 25°C and 15°C. Three of the ten mutant strains (including Bj30056 and 57) were unable to fix nitrogen with soybeans grown at optimal temperatures. Based on nodY gene expression and symbiotic phenotype the B. japonicum mutants were classified into three groups. / A molecular genetic approach was taken to investigate the regulation of expression of succinate dehydrogenase (SDH) in S. meliloti. The sdhCDAB genes encoding SDH were shown by RT-PCR to be co-transcribed and thus constitute an operon. The transcriptional start site and putative promoter region of the first gene in the operon, sdhC , were identified by 5'-RACE and DNA sequence analysis. Transcriptional lacZ fusions to sdhC indicated that expression of the operon is regulated by carbon source in the growth medium but not by growth phase. The highest expression of the sdh operon was observed in cells grown with acetate, arabinose and glutamate, as sole carbon sources, and the lowest expression was observed in cells grown with glucose and pyruvate as sole carbon sources. / Also presented is the isolation and characterization of the first defined sdh mutant in a rhizobial species. The mutants helped demonstrate that the total lack of SDH activity would be lethal to S. meliloti cells. Symbiotic phenotype of the mutants indicated that SDH is required for N2-fixation.

In the N2 fixing legume symbiosis, mineral nitrogen (N) not only decreases N2 fixation, but also delays and inhibits the formation and development of nodules. The purposes of this thesis were to elucidate the role of signaling in the mineral N effects on nodulation and nitrogen fixation in soybean [Glycine max (L.) Merr.] and to attempt to find ways to overcome this inhibition. The responses of soybean plants, in terms of daidzein and genistein synthesis and exudation, to different mineral N levels were studied. Daidzein and genistein distribution patterns varied with plant organs, mineral N levels, and plant development stages. Mineral N inhibited daidzein and genistein contents and concentrations in soybean root and shoot extracts, but did not affect root daidzein and genistein excretion in the same way. In both synthesis and excretion, daidzein and genistein were not affected equally by mineral N treatments. Variability existed among soybean cultivars in the responses of root daidzein and genistein contents and concentrations to mineral N levels. The amount of daidzein and genistein excreted by soybean roots did not always correspond to the daidzein and genistein contents and concentrations inside the roots. On the Bradyrhizobium japonicum side, nod gene expression was inhibited by mineral nitrogen. Genistein was used to pre-incubate B. japonicum cells or was applied directly into the plant growing medium. The results showed that genistein manipulation increased nodule weight and nodule nitrogen fixation under greenhouse conditions, but interactions existed among soybean cultivars, genistein concentrations and nitrate levels. Similar results were found under field conditions. Soybean yield was increased on sandy-loam soil by preincubation of B. japonicum cells with genistein. Addition of genistein beginning at the onset of nitrogen fixation also improved soybean nodulation and nitrogen fixation. Soybean cultivars had different sensitivities to genistein additi / Other studies also show that temperature affected genistein and daidzein content and concentration in soybean roots. The effect of temperature varied among soybean cultivars. Some PGPR strains can mitigate the negative effects of nitrate on soybean nodulation and nitrogen fixation, however, this is influenced by soybean genotype. Applying PGPR together with genistein preincubation of B. japonicum cells improved soybean nodulation and increased yield. The level of improvement varied among soybean cultivars and PGPR strains. Preincubation of B. japonicum cells with genistein improved strain competitiveness under greenhouse, but not field conditions. / Overall, these findings suggested that both plant-to-Bradyrhizobium and Bradyrhizobium-to-plant signals play important roles in the effects of mineral N on nodulation and N fixation. Signal manipulation could partially overcome the inhibitory effects of mineral N on soybean- B. japonicum N fixation symbiosis.

Stat6 is a protein that activates the transcription of IL-4-stimulated genes. Amino acids critical for Stat6 function were examined in a mutational analysis of the Src homology (SH2) domain of the Stat6 protein. One mutation, substitution of two Alanines for Valine and Threonine in the N-terminal portion of the SH2 domain, produced a constitutively active form of the molecule that did not require IL-4 for activation. This mutant was named Stat6VT. Mice expressing Stat6VT in lymphocytes were generated, and it was found that approximately 10% of the population of Stat6VT mice, a lympho-proliferative disorder (LPD) occurred. In this study, we are examining genes that have a possible role in the development of this proliferative condition. Specifically, we examined the expression levels of Tiam1, Tacstdl, and Gfi-1 and Gfi-1B (genes known to regulate cellular proliferation and survival) in wildtype, normal Stat6VT and Stat6VT/LPD splenocytes by RT-PCR. Tiam1 results were inconclusive, and Tacstdl was not expressed at levels different from those seen in controls. Interestingly, Gfi-1 B, the homolog of Gfi-1, was expressed at increased levels in a specific subpopulation of cells from Stat6VT/LPD mice. Taken together, these data suggest that in cells expressing a constitutively active Stat6, increased expression of Gfi-1B may play a role in the mechanism of lymphoma development. / Department of Biology

Many environmental stresses result in increased generation of active oxygen species (AOS) in plant cells, leading to the induction of protective mechanisms. In this study, signalling components linking AOS perception to downstream responses were examined, with particular emphasis on H₂O₂ signalling. All AOS investigated had an early [Ca²⁺]_cyt peak in common, but differed in other aspects of their Ca²⁺ signatures, indicating that the plant is able to discriminate between different types of AOS. An early event in AOS signal transduction may involve changes in the cellular redox balance as reduction of glutathione levels prior to stress application increased the height of the first [Ca²⁺]_cyt peak. Inhibiting or enhancing the height of the H₂O₂-triggered Ca²⁺ signature lead to inhibition or enhancement of GST1 and APX1 induction, respectively, demonstrating that the Ca²⁺ signature is required for induction of genes encoding antioxidant enzymes. OX1, encoding a putative ser/thr kinase, was shown to be involved in signal transduction in response to H₂O₂-generating stresses. Transcript levels of OX1 were increased upon treatment with H₂O₂ and a range of abiotic and biotic stresses as well as ABA, all of which have been shown to result in H₂O₂ accumulation. Inhibition of stress-induced [Ca²⁺]_cyt elevations inhibited OX1 induction, placing the OX1 kinase downstream of Ca²⁺ in the signalling chain. OX1 is required for full activation of AtMPKS and AtMPK6 in response to ozone fumigation, indicating that OX1 functions upstream of these MAP kinases. An ox1 null-mutant displayed enhanced susceptibility to infection with a virulent Peronospora parasitica isolate as well as reduced induction of several defence genes. In addition, the ox1 mutant exhibited shorter root hairs and an early flowering phenotype. AOS treatment induced several genes encoding AtERF transcription factors, but did not have an effect on other members of this family. Induction occurred in an ethylene-independent but Ca²⁺-dependent manner.

Notch signalling plays a key role in physiological development and tumourigenesis. The recent discovery and characterisation of Notch ligand Delta-like 4 (D114), which is predominantly expressed in endothelial cells, have underscored the role of Notch signalling in angiogenesis. This thesis investigates the regulation and function of D114-Notch signalling in angiogenesis and tumourigenesis. First, the D114-Notch pathway interacted with the cellular hypoxia-sensing pathway. In human umbilical vein endothelial cells (HUVECs), D114 overexpression repressed hypoxic induction, and the repression was mediated by Notch target gene Hey2. In the breast cancer cell line MCF7, hypoxia induced Notch target gene Hey1 and ligand Jagged2 via hypoxia inducible factor 1. The hypoxic induction of Hey1 was also dependent on Notch signalling. Second, D114 expression in HUVECs was up-regulated by several pathways. Notch signalling, activated by receptor overexpression, ligand stimulation or cell-cell contact, induced D114 expression. Treatment with vascular endothelial growth factor (VEGF) or hypoxia also induced D114 expression via Notch signalling. In addition, VEGF promoted Notch signalling and D114 expression in tumours. Third, D114 expression in HUVECs was up-regulated by co-culturing with cancer cells. B16 mouse melanoma cells and human breast cancer cell lines induced D114 expression in HUVECs via Notch signalling. B16 cells also induced D114 expression via soluble factors independent of Notch signalling. Finally, D114-Notch signalling regulated tumour growth in vivo. D114 overexpression in cancer cell lines activated Notch signalling in the stroma of xenograft tumours, and promoted the growth of human U87 (glioblastoma) and PC3 (prostate cancer) xenografts. In addition, D114-overexpressing U87 tumours were resistant to anti- VEGF treatment during later stages; however, they did respond to anti-Notch treatment. Altogether, the D114-Notch pathway is tightly regulated and plays an important role in physiological and tumour angiogenesis. Inhibiting this pathway may be a viable therapy for cancers resistant to VEGF inhibition.