A stable intracellular ionic environment is necessary for hepatocytes to function normally. Thus, during hypotonic shock or L-alanine uptake, hepatocytes swell and then exhibit a regulatory volume decrease (RVD), which comprises an increase in K$\sp+$ conductance (G$\sb{\rm K}$), an increased K$\sp+$ efflux, and a hyperpolarization of transmembrane potential (V$\sb{\rm m}$). Since hepatocyte intracellular Cl$\sp-$ has been demonstrated to distribute passively with V$\sb{\rm m}$, this study is designed to test the hypothesis that the hypotonic shock- or L-alanine uptake-induced hyperpolarization of V$\sb{\rm m}$ might provide an electromotive force for the efflux of hepatocyte intracellular Cl$\sp-$, which in turn would contribute osmotically to the RVD in hepatocytes. Double-barreled ion-selective microelectrodes were used to measure the changes of hepatocyte transmembrane potential, intracellular ionic activities (especially intracellular Cl$\sp-$ activity, (a$\sp{\rm i}\sb{\rm Cl}$)), and intracellular water volume during either anisotonic stress or L-alanine uptake. Hepatocyte V$\sb{\rm m}$ hyperpolarized, (a$\sp{\rm i}\sb{\rm Cl}$) decreased, intracellular K$\sp+$ activity (a$\sp{\rm i}\sb{\rm K}$) decreased, and intracellular water volume increased during hyposmotic stress. When perfused with L-alanine, hepatocyte V$\sb{\rm m}$ exhibited a transient depolarization followed by repolarization and then underwent a constant hyperpolarization. Meanwhile, hepatocyte intracellular Na$\sp+$ activity (a$\sp{\rm i}\sb{\rm Na}$) increased, a$\sp{\rm i}\sb{\rm K}$ & a$\sp{\rm i}\sb{\rm Cl}$ decreased, and intracellular water volume increased. In both hypotonic shock and L-alanine uptake conditions, the decreased a$\sp{\rm i}\sb{\rm K}$ could be attributed to cell swelling. However, the decrease in a$\sp{\rm i}\sb{\rm Cl}$ was greater than could be accounted for by cell swelling. When the change of V$\sb{\rm m}$ was inhibited by K$\sp+$ channel blockers, the change of a$\sp{\rm i}\sb{\rm Cl}$ was also inhibited. Based on the measured a$\sp{\rm i}\sb{\rm Cl}$, Cl$\sp-$ was always at its electrochemical equilibrium in all of the control and experimental conditions. The conclusions of this study emphasize the passive distribution of hepatocyte intracellular Cl$\sp-$ with the changes of V$\sb{\rm m}$ induced by hypotonic stress and L-alanine uptake. Thus, the data strongly support the idea that the hypotonic shock- or L-alanine uptake-induced hyperpolarization of V$\sb{\rm m}$ provides electromotive force for the efflux of hepatocyte intracellular Cl$\sp-$. This could contribute to hepatocyte volume regulation during both hypotonic shock and organic solute transport.

As nitrogen (N) is a critical nutrient for plant growth, the development of synthetic N fertilisers dramatically changed agricultural production in the twentieth century. Improvement in N use efficiency (NUE) has been a focus of breeding for grain crop species, since protein is an important component of the harvested product. The study of NUE in sugarcane has lagged behind grain crops, mainly because N is not a component of sucrose, the primary product of the traditional sugarcane industry. Recently, improvement in NUE has become a focus of sugarcane breeding, due largely to environmental concerns regarding pollution from high N fertilisation, and the increasing cost of N fertilisers. This thesis aimed to gain an initial understanding of the genetic basis for variability in NUE in sugarcane. This was achieved through: (i) the screening of 168 sugarcane genotypes under limiting and non-limiting N supply in two glasshouse experiments; (ii) the mapping of marker-trait associations (MTA) for biomass and physiological traits under limiting and non-limiting N supply in a sugarcane mapping population; (iii) the analysis of expression of candidate genes encoding enzymes involved in the central processes of N assimilation and remobilisation in plants; and (iv) the mapping of candidate genes in a sugarcane genetic map. Genetic variation was identified for growth traits as well as physiological traits including %N, internal NUE (iNUE, g dry weight g-1 N) and leaf glutamine synthetase (GS) activity in a sugarcane mapping population. These traits were also analysed for linkage with genetic markers. Genetic variation in the screened genotypes was higher under limiting N supply, a finding that was reflected by the fact that marker-trait associations (MTA) for increases in iNUE were not identified under non-limiting N supply in the commercial parent of the mapping population. Contrary to findings in grain crop species, there was no link between GS activity and other traits, either through phenotypic correlations or co-location of MTA. The expression of candidate genes encoding GS, nitrate reductase (NR) and alanine amintotransferase (AlaAT) was quantified with Sequenom™ MassARRAY technology. Plants were grown under growth-limiting N supply, non-limiting N supply, or a N-pulse treatment, which consisted of growth-limiting N supply followed by non-limiting N supply 24 hours prior to sampling. Two genes, scAlaAT.d and scGS1.a, encoding AlaAT and GS respectively, were identified as non-responsive to changes in N supply, whereas scAlaAT.a, scGS1.b and scGS1.c had significantly (p<0.05) increased expression under a N-pulse, indicating an important role for these genes in the response of sugarcane to a sudden increase in N availability. The location of candidate genes associated with variation in NUE in a sugarcane genetic map were sought through restriction fragment length polymorphism (RFLP) markers. Twenty-two probes were screened, of which two generated single-dose markers, allowing the mapping of a single allele of scAspAT, encoding aspartate aminotransferase, and two alleles of scGS2, encoding plastidic GS. Because of the economic and environmental consequences of inefficient N fertiliser application, the development of sugarcane cultivars with improved NUE is essential. Since variation for NUE exists, especially in unimproved sugarcane varieties, this may be achieved through traditional breeding methods by screening existing breeding populations under limiting N supply. Additionally, an improved understanding of the genetic basis of variation for NUE in sugarcane should be pursued by further analysis of candidate gene response to changing N availability by screening widely varying cane species for differences in gene expression, enzyme activity and metabolite profiles. The further addition of candidate gene locations to sugarcane genetic maps will aid both future marker-assisted selection in breeding, and a fundamental understanding of genetic control of NUE variation. Through the development of sugarcane cultivars with improved NUE and an enhanced knowledge of the genetic control underpinning sugarcane N physiology, concerns regarding high N fertiliser applications may be mitigated and sustainability ensured.

Bibliography: leaves 151-166. This thesis describes the development of two novel and complementary analytical approaches for assaying cyanoalanine non-protein amino acids. These assays are used to determine the distribution of these compounds both within and between plants and to identify accessions of common vetch which contain low levels of the cyanoalanine non-protein amino acids in germplasm collections. These analytical tools are used to correlate toxicity observed in animal feeding experiments with the cyanoalanine content. This thesis covers also the first report of the use of diffuse reflectance using dispersive infrared spectrometry for the "in situ" quantification of specific organic components from plant tissue as well as the first use of micellar electrokinetic chromatography for the quantitative analysis of 9-fluorenylmethyl chloroformate (FMOC) derivatised and non-derivatised components of extracts from plant material.

Some pathogenic bacteria derive significant amounts of iron heme from their hosts. In this study we investigated SiaA, a heme binding protein from Streptococcus pyogenes. The wildtype methionine79 putative axial ligand was mutated to alanine. SiaA M79A was expressed in E. coli in three production runs, lysed by sonication or French press, and purified by fast protein liquid chromatography (FPLC). Nickel affinity FPLC was found to give much purer SiaA when 30 mM imidazole was added to the binding buffer. The protocol using extensive sonication resulted in SiaA weighing 30464 Da. The protocol using French press resulted in SiaA weighting 33358 Da. Despite the difference in masses, the two forms of SiaA interacted with heme similarly.

Defining the intrinsic properties of amino acids which dictate the formation of helices, the most common protein secondary structure element, is an essential part of understanding protein folding. Pauling and co-workers initially predicted helical peptide folding motifs in the absence of solvent, suggesting that in vacuo studies may potentially discern the role of solvation in protein structure. Ion mobility-mass spectrometry (IMMS) combines a gas-phase ion separation based on collision cross-section (apparent surface area) with time-of-flight MS. The result is a correlation of collision cross-section with mass-to-charge, allowing detection of multiple conformations of the same ion. Most gas-phase peptide ions assume a compact, globular state that minimizes exposure to the low dielectric environment and maximizes intramolecular charge solvation. Conversely, a small number of peptides adopt a more extended (β-sheet or α-helix) conformation and exhibit a larger than predicted collision cross-section. Collision cross-sections measured using IM-MS are correlated with theoretical models generated using simulated annealing and allow for assignment of the overall ion structural motif (e.g. helix vs. chargesolvated globule). Here, two series of model peptides having known solution-phase helical propensities, namely Ac-(AAKAA)nY-NH2 (n = 3, 4, 5, 6 and 7) and Ac-Y(AEAAKA)nF-NH2 (n = 2, 3, 4, and 5), are investigated using IM-MS. Both protonated ([M + H]+) and metalcoordinated ([M + X]+ where X = Li, Na, K, Rb or Cs) species were analyzed to better understand the interplay of forces involved in gas-phase helical structure and stability. The data are analyzed using computational methods to examine the influence of peptide length, primary sequence, and number of basic (Lys, K) and acidic (Glu, E) residues on anhydrous ion structure.

Ce mémoire présente trois approches différentes vers la synthèse du 3–(trans–2–nitrocyclopropyl)alanine, un intermédiaire synthétique de la hormaomycine. Cette molécule naturelle démontre d’intéressantes activités biologiques et pharmacologiques. Il est intéressant de souligner que ce dérivé donne facilement accès au 3–(trans–2–aminocyclopropyl)alanine, unité centrale de la bélactosine A. Ce composé naturel possédant lui aussi d’intéressantes propriétés biologiques, plusieurs études relationnelles structures-activités menant à des dérivés plus actifs de cette molécule ont été entreprises, démontrant l’intérêt toujours présent de synthétiser de façon efficace et optimale ces dérivés cyclopropaniques. Une méthodologie développée au sein de notre groupe de recherche et basée sur une réaction de cyclopropanation intramoléculaire diastéréosélective sera mise à profit afin d’élaborer une nouvelle voie de synthèse aussi élégante qu’efficace à la construction du 3–(trans–2–nitrocyclopropyl) alanine. En utilisant un carbène de rhodium généré soit par la dégradation d’un dérivé diazoïque, soit par la formation d’un réactif de type ylure d’iodonium, une réaction de cyclopropanation diastéréosélective permettra la formation de deux autres centres contigus et ce, sans même utiliser d’auxiliaire ou de catalyseur énantioenrichis. Ensuite, un réarrangement intramoléculaire précédant deux réactions synchronisées d’ouverture de cycle et de décarboxylation permettront l’obtention du composé d’intérêt avec un rendement global convenable et en relativement peu d’étapes. De cette manière, la synthèse formelle de la bélactosine A et de l’hormaomycine a été effectuée. Cette synthèse se démarque des autres par l’utilisation d’une seule transformation catalytique énantiosélective. / This master’s thesis presents three different approaches toward the synthesis of 3–(trans–2–nitrocyclopropyl)alanine, a key constituent of the natural product hormaomycin. This unusual compound demonstrates interesting biological and pharmaceutical activity. It is noteworthy that this unique amino acid can be readily converted to the corresponding 3–(trans–2–aminocyclopropyl)alanine, the central core of belactosin A, a natural compound exhibiting interesting biological properties. Efficient syntheses of these aminocyclopropane derivatives are of current interest since several structure-activity relationships in syntheses of belactosin A and hormaomycin analogues are currently under study in an effort to discover enhanced biological activity. A methodology developed in our research group based on a diastereoselective intramolecular cyclopropanation reaction will be used to elaborate a unique and elegant pathway to the synthesis of the 3–(trans–2–nitrocyclopropyl)alanine. By using a rhodium carbene generated either by the degradation of a diazoic derivative or by the formation of the corresponding iodonium ylide, a diastereoselective cyclopropanation reaction can be applied in the concerted elaboration of two chiral centers needed in the desired aminocyclopropanes, avoiding in this way the utilisation of chiral reagents. Following this key sequence, an intramolecular rearrangement followed by synchronous ring–opening/decarboxylation reactions will permit a convenient formation of the desired product in an acceptable overall yield and in few ensuing steps. In this manner, the formal synthesis of the hormaomycin and the belactosin A can be achieved. This synthesis is unique since it involves only one asymmetric step in the whole synthetic process.

No description available.

Some pathogenic bacteria derive significant amounts of iron heme from their hosts. In this study we investigated SiaA, a heme binding protein from Streptococcus pyogenes. The wildtype methionine79 putative axial ligand was mutated to alanine. SiaA M79A was expressed in E. coli in three production runs, lysed by sonication or French press, and purified by fast protein liquid chromatography (FPLC). Nickel affinity FPLC was found to give much purer SiaA when 30 mM imidazole was added to the binding buffer. The protocol using extensive sonication resulted in SiaA weighing 30464 Da. The protocol using French press resulted in SiaA weighting 33358 Da. Despite the difference in masses, the two forms of SiaA interacted with heme similarly.

Bibliography: leaves 151-166. / xi, 166 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the development of two novel and complementary analytical approaches for assaying cyanoalanine non-protein amino acids. These assays are used to determine the distribution of these compounds both within and between plants and to identify accessions of common vetch which contain low levels of the cyanoalanine non-protein amino acids in germplasm collections. These analytical tools are used to correlate toxicity observed in animal feeding experiments with the cyanoalanine content. This thesis covers also the first report of the use of diffuse reflectance using dispersive infrared spectrometry for the "in situ" quantification of specific organic components from plant tissue as well as the first use of micellar electrokinetic chromatography for the quantitative analysis of 9-fluorenylmethyl chloroformate (FMOC) derivatised and non-derivatised components of extracts from plant material. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Science, 1997. / Includes bibliographical references (leaves 151-166).