Kidney Induction: control by Notch, Wnt and GDNF/Ret signalling

The permanent mammalian kidney (metanephros) develops as a result of complex reciprocal tissue interactions between a ureteric epithelium and the renal mesenchyme. The overall goal of the research in this thesis was to gain data that will eventually help in elucidating the formation of congenital renal malformations. The experiments in my thesis aimed to reveal the mechanisms by which Notch, Wnt and GDNF/Ret signalling pathways regulate the development of functional kidney. The function of Notch pathway was studied by a transgenic mouse model, where it was shown that overactivation of Notch signalling disturbs kidney development and alters the expression of Gdnf and Ret/GFRa1. This indicates that Notch signalling interplays with GDNF/Ret in the regulation of the primary ureteric budding and its subsequent branching. The data also suggested that strict spatio-temporal regulation of these two pathways is required for determination of ureteric tip-identity, which appeared to be crucial for the branch formation. The function of Wnt signalling in the ureteric morphogenesis was studied by in vivo and in vitro methods to show that a canonical pathway is required for ureteric branching. Stabilisation and deletion of the canonical pathway mediator, b-catenin specifically in the ureteric epithelium result in renal aplasia/hypodysplasia. These defects originate from severe blockage of ureteric branching due to the disrupted Ret signalling. Consequently, ureteric tip specific markers are lost and ureteric stalk identity is expanded throughout the whole epithelium. Thus, the data demonstrates that the Wnt/b-catenin pathway plays an essential role in the patterning and branching of the ureteric epithelium. A novel in vitro method was generated and utilised in nephron induction studies to reveal the mechanisms through which nephrogenesis is induced. Transient GSK3 inhibition results in stabilisation of b-catenin in the isolated renal mesenchyme, which efficiently triggers nephron formation. Also genetic stabilisation of b-catenin specifically in the mesenchyme results in spontaneous nephrogenesis. The results show that activation of the canonical Wnt pathway is sufficient to initiate nephrogenesis, and suggest that this pathway mediates the nephron induction in murine kidney mesenchymes. Taken together, this thesis demonstrates Notch and Wnt signalling pathways as novel regulators of ureteric branching morphogenesis, and that activation of the canonical Wnt pathway is sufficient for nephron induction. The studies also indicate that the Notch and Wnt pathways cross-talk with GDNF/Ret signalling in the patterning of ureteric epithelium.

Preliminary investigation into the induction of reproduction in Ulva spp. in Southeast Queensland for mass cultivation purposes

Ulva is a common component of marine intertidal flora in Australia with many species frequently observed along the Queensland coastline. Three species of Ulva, U. lactuca, U. intestinalis and U. prolifera were found to naturally occur at the Bribie Island Research Centre (BIRC) in Southeast Queensland. Studies were undertaken to establish the most optimal conditions for growing Ulva in the BIRC laboratory. These tests were conducted in order to condition the algal material prior to the sporulation studies, offering more controlled material to assess treatment effects conclusively, and helping eliminate other potentially confounding environmental factors. Results showed that a stocking density of between 5-20 grams of Ulva per litre along with the addition of the soluble fertiliser Aquasol at a rate of 87 mg/L of seawater was ideal for achieving a desired doubling of growth per week. In the wild the formation of Ulva fragments occurs naturally in the ocean through wave and storm action. This breakage can trigger a survival response mechanism which stimulates the algae to form and release gametes. By chopping the tissue, this process could be artificially simulated in the laboratory and creating a simple and easy way to produce new individuals. Studies performed into inducing sporulation in Ulva through a combination of fragmentation and renewal of medium at BIRC showed that sporulation can be successfully induced in all three species of Ulva through these methods, however it was found to be to a degree that would not meet the demands of commercial production with on average a rate of only 33% achieved. While the current study did not find a method suitable for a commercial application the results presented here contribute to increasing our understanding about Ulva reproduction and set a platform for future work in to cultivating Ulva within Southeast Queensland.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a modelantigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.

Hormonal induction of riboflavin carrier protein in the chicken oviduct and liver: a comparison of kinetics and modulation

Estrogen (E) induction of riboflavin carrier protein (RCP) in the chicken oviduct and liver was investigated to compare and contrast the kinetics, hormonal specificity and modulation of its elaboration in the 2 steroid-responsive tissues. During primary stimulation, continued daily E administration to immature female chicks elicited, after an initial lag, rapid growth and RCP content of the oviduct; neither progesterone (P) nor testosterone (T) could substitute for E in this respect. Furthermore, P given along with E curtailed tissue growth and its RCP content, whereas E + T had a synergistic effect on tissue growth only. During secondary stimulation, E administration steeply enhanced both tissue weight and RCP content without any lag. Interestingly, P (but not T) could substitute for E in augmenting magnum RCP concentration to a comparable extent while a concomitant effect on tissue growth was less marked. In contrast, hepatic induction of RCP was absolutely E-specific during both primary and secondary stimulations. Secondary stimulation with either E or P of E-primed birds enhanced the rates of RCP synthesis in the oviduct relative to that of total protein, whereas in the liver only E was effective in this regard. The absolute rate of E-induced RCP synthesis in both the steroid-stimulated tissues was significantly higher than that of general protein elaboration.

Induction of carnitine acetyltransferase by clofibrate in rat liver

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine -acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.

Modified current source inverter fed induction motor drive with reduced torque pulsations

A new technique for reducing the torque pulsations in a conventional current source inverter fed induction motor drive is presented. This does not attempt to improve the current waveforms, but modifies the airgap MMF directly. This is based on the use of a motor with two sets of balanced phase windings, with a 30 electrical degree phase difference between them, and each set being fed from a conventional current source inverter. The two inverters are further connected in series so that they can operate from the same current source. As a consequence of this arrangement, the voltage rating of the components of each inverter is reduced, along with reduced torque ripple. This scheme has been experimentally verified and compared with the performance of a conventional scheme.

Supermodels: sorghum and maize provide mutual insight into the genetics of flowering time

Nested association mapping (NAM) offers power to dissect complex, quantitative traits. This study made use of a recently developed sorghum backcross (BC)-NAM population to dissect the genetic architecture of flowering time in sorghum; to compare the QTL identified with other genomic regions identified in previous sorghum and maize flowering time studies and to highlight the implications of our findings for plant breeding. A subset of the sorghum BC-NAM population consisting of over 1,300 individuals from 24 families was evaluated for flowering time across multiple environments. Two QTL analysis methodologies were used to identify 40 QTLs with predominately small, additive effects on flowering time; 24 of these co-located with previously identified QTL for flowering time in sorghum and 16 were novel in sorghum. Significant synteny was also detected with the QTL for flowering time detected in a comparable NAM resource recently developed for maize (Zea mays) by Buckler et al. (Science 325:714-718, 2009). The use of the sorghum BC-NAM population allowed us to catalogue allelic variants at a maximal number of QTL and understand their contribution to the flowering time phenotype and distribution across diverse germplasm. The successful demonstration of the power of the sorghum BC-NAM population is exemplified not only by correspondence of QTL previously identified in sorghum, but also by correspondence of QTL in different taxa, specifically maize in this case. The unification across taxa of the candidate genes influencing complex traits, such as flowering time can further facilitate the detailed dissection of the genetic control and causal genes.

Modelling temperature, photoperiod and vernalization responses of Brunonia australis (Goodeniaceae) and Calandrinia sp (Portulacaceae) to predict flowering time

Crop models for herbaceous ornamental species typically include functions for temperature and photoperiod responses, but very few incorporate vernalization, which is a requirement of many traditional crops. This study investigated the development of floriculture crop models, which describe temperature responses, plus photoperiod or vernalization requirements, using Australian native ephemerals Brunonia australis and Calandrinia sp. A novel approach involved the use of a field crop modelling tool, DEVEL2. This optimization program estimates the parameters of selected functions within the development rate models using an iterative process that minimizes sum of squares residual between estimated and observed days for the phenological event. Parameter profiling and jack-knifing are included in DEVEL2 to remove bias from parameter estimates and introduce rigour into the parameter selection process. Development rate of B. australis from planting to first visible floral bud (VFB) was predicted using a multiplicative approach with a curvilinear function to describe temperature responses and a broken linear function to explain photoperiod responses. A similar model was used to describe the development rate of Calandrinia sp., except the photoperiod function was replaced with an exponential vernalization function, which explained a facultative cold requirement and included a coefficient for determining the vernalization ceiling temperature. Temperature was the main environmental factor influencing development rate for VFB to anthesis of both species and was predicted using a linear model. The phenology models for B. australis and Calandrinia sp. described development rate from planting to VFB and from VFB to anthesis in response to temperature and photoperiod or vernalization and may assist modelling efforts of other herbaceous ornamental plants. In addition to crop management, the vernalization function could be used to identify plant communities most at risk from predicted increases in temperature due to global warming.

Regulation of strawberry growth and development

Strawberries (Fragaria sp.) are adapted to diverse environmental conditions from the tropics to about 70ºN, so different responses to environmental conditions can be found. Most genotypes of garden strawberry (F. x ananassa Duch.) and woodland strawberry (F. vesca L.) are short-day (SD) plants that are induced to flowering by photoperiods under a critical limit, but also various photoperiod x temperature interactions can be found. In addition, continuously flowering everbearing (EB) genotypes are found. In addition to flowering, axillary bud differentiation in strawberry is regulated by photoperiod. In SD conditions, axillary buds differentiate to rosette-like structures called "branch crowns", whereas in long-day conditions (LD) they form runners, branches with 2 long internodes followed by a daughter plant (leaf rosette). The number of crown branches determines the yield of the plant, since inflorescences are formed from the apical meristems of the crown. Although axillary bud differentiation is an important developmental process in strawberries, its environmental and hormonal regulation has not been characterized in detail. Moreover, the genetic mechanisms underlying axillary bud differentiation and regulation of flowering time in these species are almost completely unresolved. These topics have been studied in this thesis in order to enhance strawberry research, cultivation and breeding. The results showed that 8-12 SD cycles suppressed runner initiation from the axillary buds of the garden strawberry cv. Korona with the concomitant induction of crown branching, and 3 weeks of SD was sufficient for the induction of flowering in the main crown. Furthermore, a second SD treatment given a few weeks after the first SD period can be used to induce flowering in the primary branch crowns and to induce the formation of secondary branches. Thus, artificial SD treatments effectively stimulate crown branching, providing one means for the increase of cropping (yield) potential in strawberry. It was also shown by growth regulation applications, quantitave hormone analysis and gene expression analysis that gibberellin (GA) is one of the key signals involved in the photoperiod control of shoot differentiation. The results indicate that photoperiod controls GA activity specifically in axillary buds, thereby determining bud fate. It was further shown that chemical control of GA biosynthesis by prohexadione-calcium can be utilized to prevent excessive runner formation and induce crown branching in strawberry fields. Moreover, ProCa increased berry yield up to 50%, showing that it is an easier and more applicable alternative to artificial SD treatments for controlling strawberry crown development and yield. Finally, flowering gene pathways in Fragaria were explored by searching for homologs of 118 Arabidopsis thaliana flowering-time genes. In total, 66 gene homologs were identified, and they distributed to all known flowering pathways, suggesting the presence of these pathways also in strawberry. Expression analysis of selected genes revealed that the mRNA of putative floral identity gene APETALA1 accumulated in the shoot apex of the EB genotype after the induction of flowering, whereas it was absent in vegetative SD genotype, indicating the usefulness of this gene product as the marker of floral initiation. The present data enables the further exploration of strawberry flowering pathways with genetic transformation, gene mapping and transcriptomics methods.

Microprocessor-Based Field-Oriented Control of A CSI-Fed Induction Motor Drive

This paper describes the method of field orientation of the stator current vector with respect to the stator, mutual, and rotor flux vectors, for the control of an induction motor fed from a current source inverter (CSI). A control scheme using this principle is described for orienting the stator current with respect to the rotor flux, as this gives natural decoupling between the current coordinates. A dedicated microcomputer system developed for implementing this scheme has been described. The experimental results are also presented.

Induction of riboflavin-carrier protein in the immature male rat by estrogen: kinetic and hormonal specificity

The kinetics of estrogen-induced accumulation of riboflavin-carrier protein in the plasma was investigated in immature male rats using a specific and sensitive homologous radio-immunoassay procedure developed for this purpose. Following a single injection of the steroid hormone, plasma riboflavin-carrier protein levels increased markedly after an initial lag period of approximately 24 h, reaching peak levels around 96 h and declining thereafter. A 1.5 fold amplification of the inductive response was evident on secondary stimulation with the hormone. The magnitude of the response was dependent on hormonal dose, whereas the initial lag phase and the time of peak riboflavin-carrier protein induction were unaltered within the range of the steroid doses (0.1–10 mg/ kg body wt.) tested. Simultaneous administration of progesterone did not affect either the kinetics or the maximum level of the protein induced. The hormonal specificity of this induction was further adduced by the effect of administration of antiestrogens viz., En and Zu chlomiphene citrates, which effectively curtailed hormonal induction of the protein. That the induction involvedde novo-protein synthesis was evident from the complete inhibition obtained upon administration of cycloheximide. Passive immunoneutralization of endogenous riboflavin-carrier protein with antiserum to the homologous protein terminated pregnancy in rats confirming the earlier results with antiserum to chicken riboflavin-carrier protein.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Inhibition of cortisol-mediated induction of hepatic tryptophan-2,3-dioxygenase by norepinephrine

Norepinephrine inhibits cortisol-mediated induction of hepatic tryptophan pyrrolase in rats. During cold exposure the stabilization of this enzyme appears to occur by an interaction of corticoids and norepinephrine on the induction process.

Low temperature exposure of root system and inflorescence affected flowering and fruit set in 'Chardonnay' grapevines (Vitis vinifera)

The mechanisms by which low temperature affects flowering and fruit set of grapevines are poorly understood, as is the specific response of the grapevine root system and inflorescence to low temperature effects that reduce fruit set. This study aimed to determine the responses of the root system and inflorescence of the grapevine 'Chardonnay' to low temperature (10 degrees C) during flowering, and considered the possible mechanisms of low temperature effects on those parts. Temperature treatments of 10 degrees C or 20 degrees C were imposed to potted 'Chardonnay' grapevines in a glasshouse for up to two weeks during the early stages of flowering. When the root system alone was exposed to 10 degrees C (with the rest of the plant at 20 degrees C) during flowering, the number of attached berries and percentage fruit set were significantly reduced by 50 % than when the root system alone was exposed to 20 degrees C. Whereas, exposure of the inflorescence alone to 10 degrees C (with the rest of the plant at 20 degrees C) delayed flowering, allowed rachis to grow longer, and increased both the number of attached berries (from 22 to 62 per vine) and fruit set (from 8 % to, 20 %), than when the inflorescence alone was exposed to 20 degrees C. This study will enhance our understanding of the possible mechanisms of low temperature effects on grapevine fruit set and productivity.