Dissertations / Theses: 'Fields of Research – 270000 Biological Sciences'

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Delmiglio, Catia. "The incidence and phylogenetic analysis of viruses infecting New Zealand's native grasses." Thesis, University of Auckland, 2008. http://hdl.handle.net/2292/3364.

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Grasses form the basis for the meat, dairy, wool and deer industries, which contribute to nearly 50% of New Zealand exports, and are also an important component of natural ecosystems. Worldwide >100 plant viruses infect grass species and even mild and symptomless infections can adversely effect plant populations through reduced reproductive rates and greater susceptibility to environmental extremes. The only previously published study on viruses in New Zealand’s natural grasslands found that cereal viruses have invaded the native grass flora of the South Island. This research provided an extensive survey of New Zealand native grasses, showing that barley yellow dwarf virus diseases (BYDV, Luteoviridae) and Cocksfoot mottle virus (CoMV, Sobemovirus) are widespread in the North and South islands of New Zealand. Significant findings include seven new virus hosts amongst the New Zealand native flora, the first report of BYDV-PAS in New Zealand, detection in Hierochloe redolens of a novel virus in the Luteoviridae family (proposed name BYDV-To), and in Festuca novae-zelandiae a novel dsRNA virus possibly belonging to the Partitiviridae family. New virus host reports in New Zealand include CoMV in Poa anceps, P. cita, F. novae-zelandiae, and Chionochloa rubra; BYDV-PAV and BYDV-PAS in Microlaena stipoides and Dichelacne crinita; BYDV-MAV in P. cita, F. novae-zelandiae and H. redolens; and CYDV-RPV in P. cita and M. stipoides. Molecular techniques for virus detection and identification were developed or improved during this study. Phylogenetic analyses of viral coat protein sequences from native and exotic grass species indicate either frequent or recent virus movement into native ecosystems, and multiple virus introduction events in New Zealand. The likely origins of the virus species are discussed. Two CoMV variants were identified, one of which caused severe necrosis in susceptible cocksfoot cultivars. Reciprocal aphid transmission of BYDV-PAV using cereals and native grasses showed that although transmission to natives was low, the efficiency of transmission from natives to cereals was comparable to that between cereal species, suggesting virus adaptation to the cereal host species. The findings from this study are discussed in respect to disease management and bio-security in New Zealand, and recommendations are made for future research.
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Franicevic, Simon Carl. "Biological control of Botrytis cinerea and Sclerotinia sclerotiorum on kiwifruit." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/1971.

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Botrytis cinerea and Sclerotinia sclerotiorum are the two most serious pathogens on kiwifruit in New Zealand. Because of the pesticide regulations in some of the countries to which New Zealand exports fruit, total protection from Botrytis stem end rot with current dicarboximide fungicides is not possible. The aim of this thesis was to investigate biological control measures for Botrytis stem end rot and Sclerotinia diseases of kiwifruit. More than 1000 microorganisms, isolated from the leaves and flowers of kiwifruit during spring and autumn, and selected from BCAs reported to be effective against B. cinerea and./or S. sclerotiorum, were tested in vitro for their antagonistic ability against B. cinerea and S. sclerotiorum. Successful antagonists were those that, in dual culture on agar plates, produced a zone of inhibition, an area of browning of the pathogens, or grew rapidly over the pathogens and inhibited their growth. The fifty most promising isolates from the initial screen were tested on fruit for their ability to reduce Botrytis and Sclerotinia fruit rots. Mature kiwifruit were artificallv wounded and dual inoculated with a spore suspension of one of the fifty test organisms and either a conidial suspension of B. cinerea or a mycelial suspension of S. sclerotiorum. Following 8-12 weeks incubation in a cool store, fruit were assessed for Botrytis or Sclerotinia induced rot. Isolates of Bacillus spp., Epicoccum purpurascens, Pseudomonas sp. and Trichoderma. spp. reduced, Botrytis fruit rot from 92% (inoculated control) to 0%.Isolates of Alternaria spp., pestalotia sp. and a non-sporulating isolate also reduced the number of fruit rotting to some extent. Similarly, isolates of Bacillus spp., E purpurascens and Trichoderma spp. reduce d Sclerotinia fruit rot from 100% (inoculated control) to 0%. Isolates of Alternaria spp., Myrothecium verrucaria and Pestalotia sp. were also successful at reducing the level of Sclerotinia fruit rot. It was considered undesirable if potential biological control agents (BCAs) were able to colonize kiwifruit that were to be marketed for human consumption. In order to determine if microorganisms, shown to be effective in preventing Botrytis or Sclerotinia fruit rot, were capable of themselves colonizing fruit, isolations were made from fruit dual inoculated with B. cinerea, S. sclerotiorum and/or one of several BCAs. Strains of the BCAs Bacillus spp., Pseudomonas sp. and E. purpurascens were not found to be saprophytic on fruit. Isolates of Alternaria sp., Bacillus sp., E purpurascens, pestalotia sp., Pseudomonas sp. and T. harzianum significantly inhibited germination and germ tube elongation of B. cinerea conidia in vitro in a nutrient solution, over a 24 h period. For example, the presence of Alternaria alternata A6 spores in a nutrient solution reduced germination of B. cinerea conidia from 100% to 20%. The presence of E purpurascens A77 spores inhibited B. cinerea conidial germ tube elongation from >840 pm (in control conidia) to 27 µm. The presence of any one of the BCAs tested prevented germination of B. cinerea conidia in a non-nutrient water solution, in comparision to germination of up to 86% in controls. A spore or cell suspension of each of the isolates Bacillus sp.M60, E. purpurascens A77 and T. harzianum C65 were spray inoculated onto kiwifruit blossoms produced in vivo in the glasshouse, immediately prior to inoculation of the blossoms with a condial suspension of B. cinerea. Application of the BCAs were completely effective in preventing colonization of blossoms by B- cinerea conidia. The effectiveness of each of the isolates E. purpurascens A77,T. harzianum C65 and either Bacillus sp.M60 or M53 to reduce the viability of sclerotia of B. cinerea and S. sclerotiorum was tested in soil punnets. A spore or cell suspension of each respective BCA was applied to the surface of replicated punnets that were seeded with either B. cinerea or S. sclerotiorum. Following 8 weeks incubation, punnets were harvested and viability of sclerotia assessed. T. harzianum C65 and Bacillus sp. M60 significantly reduced the viability of B. cinerea sclerotia from 8 sclerotia/punnet (control) to 4 sclerotia/punnet. T. harzianum C65 and E. purpurascens A77 caused a significant reduction in apothecia production of S. sclerotiorum, from 2.7 apothecia/punnet (control) to 0.7 apothecia/punnet. Bacillus sp.M8 and E purpurascens A77 were tested for their ability to reduce Botrytis stem end rot and Sclerotinia field rot in a kiwifruit orchard. The isolates tested did not successfully reduce either disease. Possible explanations for this are discussed. In order to monitor the survival of particular isolates of BCAs in the field, a technique was developed to distinguish between individual strains of a BCA species. The polymerase chain reaction (PCR) was utilized to identify DNA polymorphisms within the genome of T. harzianum C65, in comparison with other strains of Trichoderma spp.. A sequence of polymorphic DNA was cloned, sequenced and used as a hybridization probe in southern blotting to enable T. harzianum c65 to be distinguished from other strains of Trichoderma spp.. From the results obtained in this study, it was considered that Bacillus M60, E purpurascens 477 and Pseudomonas M30 were the best isolates for the biological control of Botrytis stem end rot on kiwifruit. Further work to enable application of these isolates as postharvest BCAs is discussed. Of the isolates tested in this study, T. harzianum C65 was considered the best isolate for use against Sclerotinia diseases on kiwifruit. Methods of selecting more effective BCAs against S. sclerotiorum are discussed.

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Kay, Stuart James. "The biological control of sapstain of Pinus radiata with microorganisms." Thesis, University of Auckland, 1995. http://hdl.handle.net/2292/2474.

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A total of six hundred and sixteen fungal and two hundred and thirty two bacterial isolates were obtained either from the sapwood of Pinus radiata or from other sources, including UV mutagenesis. All isolates were screened on Pinus radiata wood chips for their survival and colonisation attributes. Of these isolates, two hundred and eighty two failed to grow or caused permanent deep seated discolourations or decay and were eliminated from the study. The remaining five hundred and sixty six isolates were assessed for their antagonistic ability against sapstain. In a dual screen on Pinus radiata wood chips, one hundred and twelve fungal and four bacterial isolates inhibited the growth of the known sapstain fungus, Ophiostoma piceae. In a second biological control screen, on Pinus radiata wood blocks, isolates of Gliocladium viride, Gliocladium roseum, Trichoderma hamatum, Trichoderma harzianum, Trichoderma sp., Trichothecium roseum and an isolate of the Thelephoraceae proved inhibitory to the sapstain isolates Ophiostoma piceae and Sphaeropsis sapinea providing between 94 and 100% control. These isolates were considered for further examination in the field. The remaining isolates provided poor or inconsistent inhibition or were mould fungi and, therefore, not suitable for direct application. All fungal and bacterial isolates that had shown inhibitory ability in the initial biological control screen and the remaining non-staining bacteria were examined for their ability to produce non-volatile metabolites that were inhibitory to sapstain. The bacterial isolates were examined in a preliminary dual plate screen in which 91 isolates were identified as producing inhibitory compounds. The best of these bacterial isolates were screened, with the fungal isolates, in a non-volatile metabolite trial utilising filter sterilised culture filtrates. Isolates of Bacillus sp., Fusarium solani, Gliocladium roseum, Gliocladium virens, Trichoderma harzianum, Trichoderma sp., Trichoderma viride and Trichothecium roseum were found to be significantly inhibitory to the growth of Ophiostoma piceae at concentrations of 50% or less. However, the filtrates did not provide adequate sapstain control, when tested on Pinus radiata wood block, to prompt consideration for further examination in the field. Studies are currently examining several of these isolates for the production of biologically active compounds. The six most promising isolates, from the wood chip and wood block trials, were tested in the field for their ability to control sapstain on unseasoned Pinus radiata sapwood and/or peeled logs. These were Gliocladium viride (FK75), Trichoderma hamantum (FK561), Trichoderma harzianum (FK228), Trichoderma sp. (FK247), Trichothecium roseum (FK238) and an isolate of the Thelephoraceae (FK33). The fungi were prepared as mycelial/spore homogenates. For application to the timber, the homogenates were mixed with 0.2% Alcosorb gel, producing 108 cfu/ml suspensions, these suspensions were applied by dipping. Diluted homogenates, 108 cfu/ml, were applied as spray treatments to the logs. All of the biological control agent treatments reduced the level of sapstain on either the logs or timber with Trichoderma harzianum (FK228), Trichoderma sp. (FK247) and Trichothecium roseum (FK238) providing control equivalent to that of the fungicides NP-1 and Diffusol for portions of the trial. Trichoderma sp. (FK247) and Trichothecium roseum (FK238) gave sapstain control in excess of 90% for the first 30 days of the timber trial equalling the control provided by NP-1 and Diffusol. In another trial, Trichoderma harzianum (FK228) was more effective than NP-1, providing 60% sapstain control, after six months, on the internal tissue of Pinus radiata logs. The six isolates selected for the field trials were examined in additional studies. In a dual inoculation study, Trichoderma sp. (FK247) exhibited localised antibiotic ability causing the lysis of mycelium of sapstain fungi. There was no evidence of mycoparasitic action by any of the six isolates. Trichoderma harzianum (FK228), Trichoderma sp. (FK247) and Trichothecium roseum (FK238) were observed to degrade cellulose. However, neither these nor the other isolates caused a significant change in the mechanical properties of Pinus radiata timber when compared to untreated controls. Decreasing pH or the addition of nitrate were identified as having potential for the promotion of biological control agent growth. The potential of mixed biological control agent inoculations was also examined. While these results are preliminary, they are extremely encouraging and provide a basis from which future studies can develop.

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Waghorn, Tania Susanne. "Molecular and Ecological Aspects of Heliothis Armigera." Thesis, University of Auckland, 1999. http://hdl.handle.net/2292/522.

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The species status and host range of Heliothis armigera was investigated. DNA profiling, mitochondrial DNA sequencing, RAPD's, along with traditional morphological and crossing methods were used to investigate micro, macro and mega-population structuring. Thirty-six new host records were added, of which a number are important common weeds and crops. Mortality due to parasitoids and fungal infections were quantified on many host species. Genetic analysis of the COII and the AT-rich regions of the mitochondrial DNA showed very high levels of variation, as did the DNA profiling using the probes (CA)n and 33.15. The morphological analyses also showed variation, but to a lesser degree and without statistical significance. The variation found at all levels and in all aspects is discussed with respect to caterpillar host-plants and geographical location. All host-plant populations of caterpillars showed very high levels of genetic variability. However, the population of caterpillars found on Sulla (Hedysarium coronarium) was significantly more variable than those found on Lotus and Lucerne when compared using DNA profiling. The sequences obtained from the two mtDNA regions also showed considerable variation, a great percentage of which was uninformative. This variation did not allude to any structuring of caterpillar populations with respect to host-plant or geographical location. H. armigera is genetically a very variable species which does not equate with any population structuring present in the host-plant or geographical populations investigated here. This study has greatly increased the general understanding of this insect, and has elucidated a portion of the genetic makeup, but not helped in the development any new control methods.

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Haw, James. "Effects of Argentine Ant (Linepithema Humile) on Arthropod Fauna in New Zealand Native Forest." Thesis, University of Auckland, 2006. http://hdl.handle.net/2292/625.

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Although Argentine ant(Linepithema humile), a highly invasive ant species, has been in New Zealand for at least l4 years, little is known about their ecology and potential for invasion. Increasing spread and establishment of populations throughout New Zealand is disturbing because of the devastating impacts documented on native invertebrate biodiversity overseas. The primary aim of this study was to determine the impacts of Argentine ants on arthropods in native forest habitats in west Auckland. Pitfall traps at invaded and uninvaded sites were used to quantify ant and non-ant arthropod faunas. Argentine ants did not adversely affect native host ant communities. Moreover, two ant species appeared to be resistant to invasion. Argentine ant invasion reduced the abundance of a few orders of invertebrates while several taxa were more abundant in the presence of Argentine ants. Distribution and foraging activity of Argentine ant populations were monitored in this study from 2000-2003. Also, rate of spread was investigated to evaluate whether native forest habitats would be at risk from invasion. Measurements of foraging ant trails on monitored tree trunks revealed seasonal distribution patterns involving high activity in summer/autumn and low activity in winter/early spring. Argentine ants were found to be established primarily along the edge of the forest and did not invade into the interior of the forest during the study period. An Argentine ant poisoning operation on Tiritiri Matangi Island in January 2001 provided the opportunity to document the results of the eradication trial. In addition, pitfall traps placed at two treated sites and one untreated site were used to compare pre-poison and post-poison effects on ant and non-ant invertebrate communities. Fipronil baiting at 0.01% effectively reduced Argentine ants at the study sites and very few ants were observed in both tree count and pitfall trap recordings two months after poisoning. The invasion of Argentine ants on Tiritiri Matangi Island decimated native host ants and no recovery was detected throughout the study. Several groups of invertebrates appeared to benefit from the removal of Argentine ants while a few showed no detectable changes. Conservation implications resulting from the findings of this study are discussed Also, potential future research involving Argentine ants are outlined.

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Beck, Nancy Gunther. "Lepidopterous pests on vegetable brassicas in Pukekohe, New Zealand: their seasonality, parasitism, and management." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/1982.

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The vegetable brassicas of cabbage, broccoli, and cauliflower are grown in Pukekohe for the Auckland fresh-produce markets. These brassicas are attacked by three major lepidopterous pests: diamondback moth (DBM) (Plutella xylostella (L)) (Yponomeutidae), white butterfly (WB) (Pieris rapae (L.)) (Pieridae), and soybean looper (SBL) (Thysanoplusia orichalcea (F.)) (Noctuidae). Current grower strategy to combat these pests is calendar-scheduled insecticide applications. The goal of this thesis is to develop pest management alternatives. The seasonality of these three pests is discussed. DBM and WB are each under biological control by a larval and a pupal parasitoid, but this natural control is not sufficient to allow economic harvests in cabbage and was not synchronized. No parasitoids of SBL were found. The importation of additional natural enemies is discussed. A scouting system of the percent of cabbage plants infested coupled with an action threshold of. 15%-20% infested plants, resulted in good yields in field trials and allowed up to a 50% reduction in insecticide applications over the growth period when compared to a 14-day calendar schedule. Implementation of the 15% infested threshold in commercial cabbage fields resulted in up to an 83% reduction in insecticide applications with no yield decrease in quality or quantity. Application of this 15% infested plant threshold to broccoli and cauliflower decreased insecticide applications by 40% and 17%, respectively. Study of larval biology indicated that all of the lepidopterans preferentially fed on leaves; timing of the first insecticide application in broccoli and cauliflower to coincide with floret initiation decreased insecticide applications by 80% and 67%, respectively. Laboratory and field trials comparing DBM oviposition preference, larval survivability, and parasitism rates between cabbage, broccoli, and cauliflower are discussed. Knowledge of lepidopterous pest seasonality and biology, linked to careful timing of insecticide applications to coincide with threshold levels of pests, can take full advantage of natural enemies and reduce insecticide input in the vegetable brassicas of cabbage, broccoli, and cauliflower with no decrease in crop quality.

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Ochieng'-Odero, James Patrick. "Aspects of the life cycle, biological performance and quality of the black lyre leafroller 'Cnephasia' jactatana (Walker)." Thesis, University of Auckland, 1988. http://hdl.handle.net/2292/2480.

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The thesis answers the general question of whether the quality of artificially reared insect species should be based on performance tests for intended use or whether quality should be based on a more holistic biological approach. The empirical research is carried out using the lepidopteran leafroller 'Cnephasia' jactatana (Walker). The thesis defines biological performance and quality in terms of the success of an insect population in survival and reproduction and regards the laboratory environment as an artificial habitat that insects must colonise in order to survive and reproduce. Changes in biological performance that occurred during 12 successive generations of laboratory rearing were due to selection, acclimatisation and domestication and not adaptation. Artificial colonisation is theoretically successful within a limited range of environmental factors. As the inherent genetic variability of the founder population determines the resilience of the population to changes in performance, the ranges of environmental factors during colonisation should be wide to 'capture' much of the variability. Using body size (weight) as an aspect of overall quality, the thesis presents evidence that the final instar larva of C. jactatana has a threshold mechanism (larval critical weight, LCW) that determines pupal and adult size. There is a proportionate decrease in weight from the maximum weight that a larva attains in the final instar (LMW) to pupa ( described as constant DP ) and to adult (DA). There is a direct relation between the latent feeding period (period between attaining an LCW and LMW), LMW, pupal and adult size, and the reproductive performance (fecundity ). Within the experimental conditions diet quality, temperature, photoperiod and artificial selection had no effect on the larval critical weight, DP or DA, the larval threshold mechanism in C. jactatana is probably a mechanical trigger that initiates pupation. Diet quality, temperature and thermophotoperiods affected pupal size, adult size and reproductive performance. Photoperiod had no significant effects on size and reproductive performance. Positive assortative selections for slow development and low pupal weight significantly decreased pupal and adult size, and reproductive performance. Selection for fast development and heavy pupal weight for three generations had no significant effect on size or reproductive performance. Larval critical weight is demonstrated as useful to define quality indices and predict the performance of laboratory reared insects. The general conclusion of the thesis is that insect quality should be defined more in terms of the success in survival and colonising ability rather than solely on the success for 'intended role' or 'fitness for use'.

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Paulin, M. G. (Michael Geoffrey). "A mathematical and comparative study on cerebellar control of vestibular reflexes." Thesis, University of Auckland, 1985. http://hdl.handle.net/2292/2041.

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The first aim of this thesis is an introduction to some basic aspects of multivariate control theory which are relevant to the question of how the brain controls movements. A regulator is a device which forces a system to follow a specified trajectory in the presence of perturbations which might cause it to diverge from that trajectory. Regulation involves constructing an additional control input which depends upon the difference between the actual system state and the desired state. This requires the construction of a state estimate from raw data about system input and output. For effective state estimation, the sensor input gain to the state estimator needs to be time-varying. Under certain assumptions, the appropriate input gain can be specified analytically. The feedback regulation signal can then be constructed as a function of the state estimate. For effective regulation, the gain of the feedback function has to vary during maneuvers. Under certain assumptions an appropriate feedback gain can be specified analytically. The state observer input gain equations have a simple relationship to the feedback gain equations, so that gain specification is essentially the same task in each case. Cerebellar research has been dominated for the past 25 years by the theories of James Albus and David Marr. These mathematicians proposed similar models in which certain synapses in the cerebellar cortex are continuously modified by experience in such a way that movements which are consistently repeated under a given set of circumstances come to be performed automatically by the cerebellum. Much experimental work has focussed on the role of the vestibulo-cerebellum in fine control and learning of the vestibulo-ocular reflex. The state of the art along this line is formally described by Fujita's adaptive filter model of the cerebellar cortex. In chapter 4 it is shown that a basic feature of Fujita's model is inconsistent with available evidence. The 'Tensorial theory of brain function' is discussed in chapter 5. This is a novel theory of brain function which has been used in an attempt two explain cerebellar function. The attempt is a failure, based on sophistocated misconceptions and flawed by poor reasoning and clumsy analysis. The approach serves to confuse rather than clarify the question of cerebellar function. The final chapter of the first part of the thesis presents a basis for a new approach to cerebellar function based on the engineering theory of control of multivariate dynamical systems. It is proposed that the cerebellum is involved in movement regulation by controlling the gains of brainstem motor pathways, and in mapping the animal's environment by controlling the gains of sensory inputs to the midbrain. While learning undoubtedly does occur in the cerebellar cortex, this is not specifically a 'learning device', as commonly conceived. The second part of the thesis is concerned with the development and application of a method of system identification for characterising the dynamics of the vestibulo-ocular reflex and its components in an elasmobranch. The chosen method involves pulse-rate modulated bilateral electrical stimulation of the horizontal semicircular canal ampullary nerves. This produces a synthetic vestibulo-ocular reflex in a stationary preparation. The stimulus pattern is a pseudorandom binary sequence of pulse rates, so that cross-correlation of the stimulus pattern with the response signal gives a Unit Impulse Response dynamic signature for the system. Computer software for signal generation, recording, analysis and display was written by the author. The identification system was applied first to characterise the dynamics of the eye movement response to horizontal canal ampullary nerve stimulation, and compare this to the dynamics of the eye motor plant alone. The eye motor preparation acts as a first order low-pass filter with a time constant of about 0.2 seconds (16°C), while the ampullary preparation acts as a second order low-pass filter with a dominant time constant of about 0.75 seconds (16°C). Central pathways of the elasmobranch vestibulo-ocular reflex extend the time constant of the motor plant by a factor of 3-4, as in other animals. Eye movements predicted by fitted linear models accurately mimic eye movements recorded during experiments, suggesting both that central pathways of the reflex operate normally during this somewhat un-naturally evoked response and that the identification procedure is effective. Furthermore, combination of the ampullary nerve to eye movement transfer function obtained in this study, with head rotation to ampullary nerve transfer functions obtained by other workers, gives a consistent picture of elasmobranch vestibulo-ocular reflex function predicting compensatory eye movements in the band 0.2 - 4.0 Hz., and perhaps higher. The identification method has also been applied to produce models of vestibulocerebellar Purkinje cell dynamics during electrically evoked vestibular eye movements. Linear identification gives a poor characterisation of Purkinje cell activity during the high frequency vestibulo-ocular reflex. This is incompatible with linear phase-compensator models of the cerebellar cortex, but consistent with the reflex gain modulation theory of cerebellar function advocated in the first part of the thesis.

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Whittaker, David J. "Ethylene Biosynthetic Genes in Actinidia Chinensis." Thesis, University of Auckland, 1997. http://hdl.handle.net/2292/2169.

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Actinidia chinensis, a diploid relative of kiwifruit, has valuable fruit characteristics, and varieties with superior flavour and marketable size have recently been selected in a classical breeding programme. However, the marketability of the fruit of A. chinensis and many other species of Actinidia is limited by poor fruit storage properties. Pioneering work in tomato has demonstrated that fruit ripening and senescence can be very effectively delayed by down-regulating genes required for biosynthesis of the phytohormone ethylene. The goal of this work was to isolate genes for ethylene biosynthesis in A. chinensis, characterise their expression, and to generate transgenic plants containing T-DNA constructs designed for ethylene downregulation. A small cDNA library was constructed from RNA isolated from the ripe fruit of A. chinensis. The library was screened for genes encoding each of the enzymes in the ethylene biosynthetic pathway, by probing with PCR products amplified from kiwifruit cDNA and with a cDNA clone previously isolated from kiwifruit. Three distinct cDNA clones encoding S-adenosyl-L-methionine (SAM) synthetase (ACSAM1, ACSAM2 and ACSAM3) were isolated from the library, together with two distinct 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase cDNA clones (ACAO1 and ACAO2). No ACC synthase cDNAs were detected in the library, indicating low transcript abundance. However, a partial ACC synthase cDNA (ACAS1) was amplified from ripe fruit using PCR techniques, and subsequently cloned in a plasmid vector. Phylogenetic analysis of SAM synthetase protein sequences from A. chinensis and other plant species indicates bifurcation of angiosperm SAM synthetase sequences into two main branches; ACSAM3 was assigned to a different branch from ACSAM1 and ACSAM2. The peptide sequence of ACAS1 shows higher homology to several auxin-inducible ACC synthase peptides than the product of the ethylene-inducible ACC synthase gene which is predominantly transcribed in ripening tomato fruit. RNAse protection assays were employed to estimate the relative transcript levels of each of the ethylene biosynthetic genes isolated from A. chinensis during ethylene-induced, post-harvest fruit ripening, and in immature fruit and floral samples. The response of the mature fruit to exogenous ethylene indicated a clear separation of ethylene sensitivity and ethylene production in A. chinensis. The application of exogenous ethylene correlated with increased transcript levels for all three SAM synthetase genes (ACSAM1, ACSAM2 and ACSAM3) and for the ACC oxidase gene family. Transcription of the ACC synthase gene ACAS1 was not affected by exogenous ethylene, but transcript levels increased during subsequent ethylene biosynthesis, consistent with this being a controlling step for the onset of ethylene production. One or more ACC oxidase transcripts increased significantly both prior to and during ethylene production. Only one of the SAM synthetase transcripts (ACSAM3) was induced during the late ethylene burst, and these transcripts were also abundant in floral tissues and young fruit. A role for SAM synthetase genes in the methionine salvage pathway is discussed. The expression patterns for ACAS1 and the ACC oxidase gene family arc consistent with the consensus view that the rate of ethylene biosynthesis in plant tissues is dependent on both ACC synthase and ACC oxidase activity levels. Therefore, with the aim of down-regulating ethylene biosynthesis in A. chinensis, expression cassettes containing ACAS1 and ACAO1 cDNAs, each controlled by a d35S promoter, were inserted in tandem into the Agrobacterium binary vector pCGN1549, in both the sense and antisense orientations. Leaf tissue from the ‘Earligold’ variety of A. chinensis was transformed with the resulting binary vectors, and transgenic plants were regenerated. PCR and Southern analysis indicated intact T-DNAs were integrated in at least half of the transformed plants, and Northern analysis detected mRNAs from one of the transgenes transcribed from both the sense and antisense constructs. No decrease in wound-induced ethylene biosynthesis was detected in the leaves of a small sample of these transgenic plants, and a larger number of transformants are now being grown for phenotypic screening. Down-regulation of ethylene biosynthesis may improve the storage properties and/or the shelf life of transgenic A. chinensis plants and may provide insights into the roles of ethylene in fruit ripening.
Appendix 1 restricted at the request of the author

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Millar, Craig D. (Craig Donald). "A molecular and evolutionary study of skua breeding systems." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/2269.

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The skua (Family Stercorariidae) are a group of large, gull-like, predatory seabirds. Two skua species are found in the Antarctic region; the south polar skua (Catharacta maccormicki) and the brown skua (C. lonnbergi). The breeding distribution of the former, is restricted to the Antarctic continent and nearby islands, while the latter has a circumpolar distribution which extends northward from the Antarctic Peninsula and includes many of the Southern Ocean islands. The south polar skua is strictly monogamous, while in contrast, a number of populations of brown skua are comprised in part of communally breeding groups. The brown skua represents the only known example of a communally breeding seabird. In every skua species, breeding females are on average larger and heavier than males. However, in most skua species this dimorphism is relatively small and is of only limited use in sexing individuals. The discovery of sex-specific fragments in the DNA fingerprints of the south polar skua is reported. The multilocus probe pV47-2 hybridised to Hae III restriction fragments which were present exclusively in females and therefore presumably W-linked. The presence of these sex-specific fragments were used to identify female adults and chicks. In addition, the use of these fragments as potentially informative maternal markers is discussed. The parentage of the 13 families from two populations from Ross Island, Antarctica, determined by DNA fingerprinting, revealed a single instance of extra-pair paternity and a single instance of a chick which was parented by neither resident adult. The most likely explanation for the latter is the 'adoption' of a chick from a neighbouring territory. Similarly, DNA fingerprinting was used to assign the sex of individuals of brown skua from a population which breeds on the Chatham Islands, New Zealand. A large proportion of the Chatham Islands population breed in communal groups. Each communal group was shown to be comprised of a single female and two or more males. Consequently, the overall sex ratio amongst breeding birds was biased, with almost twice the number of males as females. In contrast the sex ratio amongst fledgling chicks did not differ significantly from 1:1. The patterns of reproductive success in breeding pairs and communal groups of the brown skua from the Chatham Islands population were determined using multilocus DNA fingerprinting. Sixteen breeding groups were examined, the parentage of 45 chicks produced over three breeding seasons was established using the probes 33.15 and 33.6. No evidence was found of either extra-pair paternity or extra-group fertilisation and there was no evidence of egg dumping by females in any breeding group. These results suggest that long-term banding records for breeding pairs and communal groups accurately reflect the overall reproductive success of these individual groups. In addition, preliminary band sharing analysis indicated that adult members of communal groups were not closely related. These findings are also supported by banding records and are in contrast to the findings of the majority of communally breeding species studied. In the 10 communally breeding groups examined, multiple paternity within a clutch was recorded on two of the 12 occasions in which two chicks were reared. Furthermore, analysis of parentage of the chicks belonging to communal groups in which the adults had remained unchanged for two or more seasons showed that some males had variable reproductive success in different seasons. These records suggest that estimates of reproductive success of individuals based on a single season's data can be misleading. Should temporal changes in paternity (and/or maternity) be shown to be common phenomena in other species, this would have major implications for the interpretation of many parentage studies. The explanation of altruistic behaviour is one of the central issues in contemporary evolutionary theory and behavioural ecology. One of the best known examples of apparent altruism is the helping behaviour which occurs in communal breeding groups such as those found in the brown skua. Within these groups individuals often help to raise offspring which are not their own. This behaviour is an apparent enigma in a world in which organisms are assumed to act in a selfish manner. Consequently, this behaviour has become a focal example at the centre of much evolutionary debate. A variety of theories have been suggested to explain helping behaviour, the most recent is that helping is an unselected consequence of the evolution of communal breeding. This hypothesis is discussed in relation to the recent literature and it is concluded that it does little to advance the current debate. An alternative theoretical approach to helping behaviour is outlined. In conclusion the general findings from the investigation of communal breeding in the brown skua are summarised and these findings are discussed. Finally, possible areas of future research are outlined.
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11

MacDiarmid, Robin M. "Tamarillo mosaic potyvirus: characterization and resistance." Thesis, University of Auckland, 1994. http://hdl.handle.net/2292/2320.

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The export of tamarillo is an important component of the New Zealand, exotic fruit industry. However, the quality of tamarillo fruit is severely decreased by tamarillo mosaic potyvirus (TaMV) which detrimentally affects fruit colour resulting in the fruit being unacceptable on the international marketplace. Virus incidence was surveyed in four tamarillo growing regions. Viruses were detected by indicator plant symptomology, and the incidence confirmed by dot blot analysis or ELISA. TaMV was present in 100% of tamarillo trees analyzed in ten of the twelve orchards surveyed. Incidence of potato aucuba mosaic potexvirus, cucumber mosaic cucumovirus, alfalfa mosaic virus and arabis mosaic nepovirus, which had all been previously reported in tamarillo was also determined. Tomato spotted wilt tospovirus was identified for the first time in tamarillo plants in New Zealand. TaMV RNA was purified from infected tamarillo leaves and a 1600 base pair cDNA clone generated to the 3'-terminus. The clone was sequenced and the location of the gene for the coat protein was identified by direct amino-terminal sequencing of purified TaMV coat protein. Comparison with other potyvirus coat protein sequences established that TaMV represents a new member of the potyvirus group. Seven chimeric transgenes containing TaMV sequences were constructed in three different binary vectors suitable for Agrobacterium-mediated transformation of plants. Of the twenty-six independent Nicotiana benthamiana plant lines generated expressing either TaMV coat protein or TaMV RNA sequences modified to block translation, eight plant lines demonstrated resistance to virus infection (more than 10% of resistant plants/line). All plants of one line, PT#25, were resistant to TaMV infection using dilute inocula; 40% were also resistant after inoculation with more concentrated inocula. The level of accumulation of CP in transgenic plant lines did not correspond with the degree of resistance to TaMV infection. In eight of the twenty-six transgenic lines a proportion of plants demonstrated recovery from systemic infection. Recovery was manifested as an absence or significant reduction of virus symptoms in newly developed leaves of plants that had previously shown symptoms of systemic TaMV infection. The mechanism of recovery from systemic infection is discussed. The protocol for the regeneration of transgenic tamarillo by Agrobacterium-mediated transformation was improved. The efficiency of transformation was optimized and the rate of transgenic shoot elongation was increased. Two tamarillo plants transformed with a transgene designed to express the TaMV coat protein were produced and were demonstrated to express the neomycin phosphotransferase gene and TaMV coat protein gene. However, following challenge with a low concentration of inoculum, micropropagants of these tamarillo plants failed to demonstrate resistance to TaMV infection. Three in vitro enzymatic activities of the virus encoded cytoplasmic inclusion protein were studied. The cytoplasmic inclusion protein was purified to near homogeneity using differential centrifugation and sucrose gradient purification. RNA-stimulated ATPase activity was demonstrated and the Km and Vmax determined. RNA binding and energy-dependent dissociation were characterized. RNA helicase activity of the cytoplasmic inclusion protein was demonstrated in the presence of NTP using RNA duplexes with single-stranded overhangs. These results have confirmed and extended the previous findings of the likely RNA helicase function of the potyvirus cytoplasmic inclusion protein, and suggest possible future mechanisms for obtaining resistance to potyviruses.

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12

Stevens, Peter M. (Peter Michael). "Host races and cryptic species in marine symbionts." Thesis, University of Auckland, 1990. http://hdl.handle.net/2292/2321.

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The Pinnotheridae is a family of decapod crustaceans comprising more than 120 mostly microphagous and commensal species. As symbionts of a variety of aquatic invertebrates, pinnotherids typically live in an intimate association with their host depending on it for an almost lifelong source of nourishment and shelter, together with a site for mating. The New Zealand pinnotherid fauna was thought to comprise only one species, Pinnotheres novaezelandiae Filhol, associated with a multitude of hosts. Recently, however, a separate species, P. atrinicola Page, has been described which is regarded as being host specific to the horse mussel Atrina zelandica Gray. In this context, the relationship between pea crabs and their hosts is of special interest, and is the focus of this thesis. An investigation into the population dynamics of the symbiotic relationship between P novaezelandiae and its host, the green-lip mussel Perna canaliculus, at Westmere Reef, Auckland between May 1986 and July 1988 is reported. Ovigerous females and Stage I males and females were found throughout the sampling period, indicating that reproduction is continuous in this species. The developmental composition of the pea crab population reveals that soft-shelled males, usually regarded as an anomalous instar, formed a significant component of the pea crab population at all times. It is suggested that these individuals represent a distinct facies, analogous to the Stage II female instar. The presence of a pea crab was found to have a highly significant detrimental effect on mussel condition. Analysis of the distribution of pea crabs among the mussel population indicates mature crabs display a repulsed distribution favouring to live a solitary existence, whereas younger (pre-hard and Stage I) crabs showed a random distribution in broad agreement with a theoretical Poisson distribution. The biological status of the two described taxa was investigated by a survey of electrophoretically detectable genetic variation of populations from throughout the North Island of New Zealand. Pea crabs from 18 host populations from nine geographically disparate localities were subjected to cellulose acetate and poly-acrylamide electrophoresis. Forty-one enzyme systems were screened for polymorphism. Clearly resolved enzyme phenotypes were obtained at 23 presumptive loci, of which l5 exhibited polymorphism. An analysis of electromorph frequency data revealed that both taxa are highly genetically structured and typified by high levels of polymorphism and heterozygosity; results atypical of brachyuran crabs. P- atrinicola was found to exhibit strong patterns of geographic differentiation and clinal variation in electromorph frequency. Of particular significance is the pattern of genetic differentiation observed among populations of p. novaezelandiae. Hierarchical F-statistics indicated that the preponderance of inter-population differentiation can be attributed to differences in electromorph frequency among host-associated populations of P. novaezelandiae within a sampling locality. Geographic differentiation was a comparatively insignificant factor in the structuring of the sampled P. novaezelandiae populations. Individuals belonging to two genetically very distinct units were found within a newly recorded host species, Mactra ovata ovata Gray at Green and Wood Bays, Manukau Harbour. Hardy-Weinberg analyses indicate the host-associated populations of P. novaezelandiae exhibit such a pronounced pattern of homozygote excess and disturbance from genetic equilibrium in sympatry that it is unreasonable to consider them as a single panmictic population. It is concluded that significant biological discontinuities based on host origin exist within the currently recognised taxon. Such a conclusion is supported by data presented on qualitative differences in host recognition observed between different host-associated populations of P. novaezelandiae. Conservatively these discontinuities indicate host race development, although a viable alternate hypothesis would be the presence of cryptic, host-specific biological species within P. novaezelandiae. Hostrace development as found here is a well recognised phenomenon in insect-host and parasitoid-host relationships, although little studied in marine symbiotic relationships. Such a phenomenon has important implications for ecological, behavioural and physiological studies on marine symbionts in general.

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13

Stevens, Michelle Lisa Glogau. "Aspects of photoadaptation in the intertidal red alga Gracilaria chilensis." Thesis, University of Auckland, 1992. http://hdl.handle.net/2292/2322.

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The intertidal red alga, Gracilaria chilensis Bird, McLachlan et Oliveira (Rhodophyta, Gracilariales), lives in an environment in which light is highly variable in terms of both amplitude and duration. A laboratory investigation of the photophysiology of G. chilensis was conducted to assess the response of the photosynthetic apparatus to light variability characteristic of the natural environment. Freshly collected Gracilaria chilensis was found to exhibit an endogenous rhythm of photosynthesis in conditions of constant limiting light and temperature. However, such a phenomenon was not observed in saturating light. Rhythms of phycobiliprotein concentration and dark respiration were also observed but were not as well-defined and could nor account for the photosynthetic rhythm. The photosynthetic response of G. chilensis to light fluctuations of various durations (0.25 to 900 seconds) and light levels was compared to that in static light. G. chilensis was able to utilise rapidly fluctuating light (< 1 second) more efficiently than fluctuations of longer duration (60-900 second). Mean photosynthetic rates were enhanced by up to l50% in fluctuating light of less than 60 sec duration over that predicted from steady-state. The photosynthetic apparatus of freshly collected G. chilensis was found to have many low-light "shade" acclimation characteristics. These included a low compensation point (5 µmolm-2 s-l) and onset of saturation (80 µmolm-2 s-l) suggesting sensitivity to photoinhibition. However, (laboratory) low-light acclimated G. chilensis was able to tolerate periods of constant high light (2000 µmolm-2 s-l)for periods of six hours without detectable detrimental effect on photosynthetic capacity, although photosynthetic efficiency was significantly inhibited after two hours of this treatment. The time course and characteristics of photoacclimation were determined by culturing G. chilensis in low- (15 µmol m-2 s-l) and high- (180 µmol m-2 s-l) light and high- and low-nitrogen regimes. The observed change in photosynthetic characteristics and pigment concentration indicated that acclimation began after a time lag of l-2 days, was complete after approximately a week and was reversible. Acclimation to growth light included changes in growth rate, P-I response curves, pigment concentration and composition and other biochemical components (e.g. carbon/nitrogen ratio). The nitrogen regime significantly affected pigment concentration in the high-light grown plants and the response suggests pigments play a role in nitrogen storage as well as light harvesting. These various physiological characteristics described above were interpreted as important mechanisms that enable G. chilensis to optimise photosynthetic response in the highly dynamic and stressful zone of the intertidal environment.

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14

Palmer, Jonathan Gray. "A dendroclimatic study of Phyllocladus trichomanoides D. Don (tanekaha)." Thesis, University of Auckland, 1989. http://hdl.handle.net/2292/2473.

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This thesis demonstrates some of the potential of Phyllocladus trichomanoides D. Don (tanekaha) for dendrochronological research, especially dendroclimatology. The type of vegetation associated with sites producing a chronology showed no obvious pattern. The resulting non-specific range of suitable sites for dendrochronological sampling was thought to be a favorable species characteristic. Standardization of tree-ring series using a 50-year Gaussian filter resulted in 28% improvement of retained common variance above that obtained from using conventional polynomial filters. The problem of autocorrelation was investigated. Tanekaha had a consistent autocorrelation pattern through both space (i.e. latitudinal and altitudinal ranges) and time (preserved forest and contemporary stands). P. glaucus (toatoa) also showed a spatially consistent yet distinctive pattern. These different diagnostic patterns implied a non-climatic cause (i.e. physiological), illustrating the need to ensure their removal before species chronologies are compared or used for climate modelling. Low order autoregressive models (ARMA(p,0)) were used to filter out the significant levels of autocorrelation. Subsequent comparison of the chronologies showed a consistent and highly significantly correlated pattern between both sites and species. The paucity of information about the physiology of Phyllocladus spp. led to the testing of several climatic variables in response function analyses. The "best" variables (based on only statistical evidence) were minimum monthly temperature, monthly total raindays and an optimal 12 month span of August (t) to July (t+1). Preliminary attempts with response functions using different combinations of predictor (climate variables) and predictand (chronologies) data sets generally failed to verify. Neither the extent of time from which the response functions were based (no analogue situation) nor a supposed "physiological shock" period (poor growth period) were the causes for non-verification. Further investigation showed that, for both the individual and combined chronologies, the temperature response was similar but rainfall varied. The instability through time of the rainfall response was particularly disconcerting since it broke one of the fundamental assumptions used in dendroclimatology. It was therefore concluded that climatic reconstruction could be attempted only on the temperature data. The temperature data of three seasons (spring, summer and autumn) were tried with transfer function models. Of these the summer season explained the most variance and had the highest reduction of error(RE) values. The selected model used the period 1918 to 1982 for calibration and 1853 to 1917 for verification. Summer temperatures from 1982 to 1750 were reconstructed. The reconstruction modeled only c.30% of the variance but was highly correlated to the summer temperature series developed by Norton (1983). This was interpreted as further independent verification of the reconstructed series. The summer temperatures reconstructed from 1982 to 1750 were concluded to have been similar to the recorded pattern from 1853. The same regressors were applied to the buried forest chronology and the summer temperatures reconstructed from 105 BC to AD 175. Because of the non-continuous tree-ring record no comment can be made of actual ambient conditions in comparison to those of today. However, the reconstructed pre-Taupo summer series did show increased variation towards the time of the eruption and more pronounced cool summers than hot ones. Another applied use of a tanekaha chronology was demonstrated with 14C dating. A statistically significant fit of 14C ages was obtained between twelve contiguous decades from the buried forest tanekaha chronology with that from a Northern hemisphere decadal calibration curve based on Sequioadendron giganteum(Stuiver & Becker 1986). This "wiggle-matching" to the calibration curve places the year of the Taupo eruption as AD 177±18 10. An Independent check of the "new" date was conducted using the most reliable data of Healy et al. (1964) with the same calibration series as described above. A date of AD177±4 44 was obtained which supports the wiggle-matched date. The confidence interval of the wiggle-matched date also coincides with the date proposed by Wilson et al. (1980) of AD186 (based purely on ancient written records).

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15

Sorrell, Brian. "Gas transport and storage processes in the lacunar system of Egeria densa Planch." Thesis, University of Auckland, 1987. http://hdl.handle.net/2292/2475.

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Aquatic macrophytes possess an internal lacunar system of proliferated intercellular airspaces. Lacunar gas exchange processes were investigated in Egeria densa Planch., a submerged freshwater angiosperm. Investigations of oxygen exchange between Egeria shoot segments and the water revealed that up to 17% of the photosynthetically-produced oxygen is retained within the lacunae. A consequence of this partitioning, which results from the relatively low solubility of oxygen in water, is the development of internal lacunar pressures up to 20 kPa above atmospheric pressure. This storage of oxygen in Egeria casts doubts on oxygen-based measurements of productivity in aquatic macrophytes, unless both internal and external sinks are monitored. Pressurisation also revealed that storage is greater in static water than in flowing water, suggesting that boundary layer limitations to oxygen transfer can also affect partitioning. Pressures fall to sub-atmospheric values in the dark, due to respiratory consumption of the internal oxygen. The Egeria respiratory gas exchanges in the dark demonstrated a steady concentration gradient between plant and water within an hour of darkening. However, the material steadily consumes approximately 30% of its respired oxygen from the lacunae, rather than the water. This oxygen supply is again due to the low oxygen solubility. The lacunae also assist the radial oxygen supply into the respiring tissue; it was found that the Michaelis-Menten constant for the respiratory response to oxygen tension in Egeria was some two to three times greater in material with infiltrated lacunae than in uninfiltrated material. Oxygen storage in the stem lacunae resulted in a longitudinal (shoot to root) movement of this gas, which was monitored using a bicompartment apparatus. The root oxygen release rate varied with light intensity and water flow rate in a similar manner to the internal pressure changes. Further experiments, involving measurements of the oxygen flux rates in the Egeria rhizosphere, demonstrated that this root oxygen loss is capable of effecting substantial diurnal oxygen fluctuations in the surrounding sediment. These processes may be interrupted by natural infiltration of the airspaces, but the factors involved here remain uncertain. The mean internal oxygen transport rate in Egeria (6.28 μ102 h-1) was consistent with estimates of lacunar oxygen concentration gradients calculated from Fick's Law, suggesting that diffusion is the oxygen transport mechanism in Egeria. However, by connecting shoots into manometers, internal pressure gradients of some 0.9 kPa m-1 were detected. These gradients were 103 -fold greater than the pressure gradient required to account for oxygen transport in Egeria, but were transient features, as the pressure equilibrated throughout the lacunar system 20 - 30 minutes after a dark/light change. Mass flow was therefore proposed as a transitory, but potentially significant, contribution to oxygen transport. Root to shoot carbon dioxide transport was measured using 14CO2 tracing. The CO2 uptake (mean internal transport = 4.96 μ1CO2 h-1) represented <10% of the total carbon fixed; the concentration of root-derived carbon in shoot tissue declined rapidly from the root insertion point. These results are compared with those of previous studies, and the significance of the Egeria lacunar system assessed.

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16

Francis, Malcolm 1954. "Population dynamics of juvenile snapper (Pagrus auratus) in the Hauraki Gulf." Thesis, University of Auckland, 1992. http://hdl.handle.net/2292/1976.

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The population dynamics of juvenile snapper, Pagrus auratus, were investigated in the Hauraki Gulf, north-eastern New Zealand, between 1982 and 1990. Attention focused on age and growth, temporal and spatial variation in abundance, and recruitment. Daily increment formation was validated in the sagittae of snapper up to about 160 days old. Increment width varied with time of year, and snapper age, and increments were not resolvable with a light microscope during winter. Increment counts inside a prominent metamorphic mark showed that larval duration was 18-32 days, and was inversely related to water temperature. Spawning dates were back-calculated from increment counts in settled juveniles, and ranged from September to March with a peak in November-January. The onset of spawning was temperature dependent. Fast-growing snapper had smaller sagittae than slow-growing snapper, indicating an uncoupling of otolith and somatic growth. Snapper gonads differentiated first as ovaries during the second year of life, and then some juveniles changed sex to become males during their third year. Sex change occurred before maturity, so snapper are functionally gonochoristic. Growth was slow during the larval phase, but increased rapidly after metamorphosis to about 0.6-0.9 mm.day-1. From the first winter, growth followed a well-defined annual cycle, with little or no growth during winter, and linear growth of 0.16-0.43 mm.day-1 during spring-autumn for 0+/1+ and 1+/2+ snapper. Snapper grew faster at higher temperatures. Trawl catch rates were affected by numerous gear and environmental factors, but probably provided reasonable estimates of snapper relative abundance. Recommendations are made for improving snapper trawl survey procedures. There was a strong annual abundance cycle in the Kawau region, peaking in spring, and declining to a minimum in winter. Snapper were patchily distributed at a spatial scale of 1-2 km, probably because of preference for specific micro-habitats. Year class strength of 1+ snapper varied 17-fold over seven years, and was strongly positively correlated with autumn sea surface temperature during the 0+ year. The strengths of the 1991 and 1992 year classes are predicted to be below average, and extremely weak, respectively.

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17

Page, Roderic D. M. (Roderic Dugald Morton). "Panbiogeography: a cladistic approach." Thesis, University of Auckland, 1990. http://hdl.handle.net/2292/1999.

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This thesis develops a quantitative cladistic approach to panbiogeography. Algorithms for constructing and comparing area cladograms are developed and implemented in a computer program. Examples of the use of this software are described. The principle results of this thesis are: (1) The description of algorithms for implementing Nelson and Platnick's (1981) methods for constructing area cladograms. These algorithms have been incorporated into a computer program. (2) Zandee and Roos' (1987) methods based on "component-compatibility" are shown to be flawed. (3) Recent criticisms of Nelson and Platnick's methods by E. O. Wiley are rebutted. (4) A quantitative reanalysis of Hafner and Nadler's (1988) allozyme data for gophers and their parasitic lice illustrates the utility of information on timing of speciation events in interpreting apparent incongruence between host and parasite cladograms. In addition the thesis contains a survey of some current themes in biogeography, a reply to criticisms of my earlier work on track analysis, and an application of bootstrap and consensus methods to place confidence limits on estimates of cladograms.

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18

Pankhurst, Patricia Melva. "Growth, development and visual ontogeny of two temperate reef teleosts Pagrus auratus, (Sparidae) and Forsterygion varium, (Tripterygiidae)." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/2000.

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Growth, development and behaviour were examined in artificially reared larval Pagrus auratus and Forsterygion varium, from the time of hatching. Yolk-sac larval P.auratus hatched at a small size (2.00mm SL), without functional eyes, mouth or digestive tract, and for three days spent long periods at rest. Growth was initially rapid but slowed by 3 days as yolk reserves neared depletion. By days 4-5, the mouth had opened, eyes were functional, yolk was depleted, and a rudimentary gut had formed. Larvae were now able to maintain a horizontal swimming mode and were actively searching for and attacking prey. First feeding was observed in some larvae. Growth was retarded during the transition from endogenous to exogenous nutrition and then increased as feeding proficiency improved. Yolk-sac F.varium hatched at a larger size (4.78mm SL), with functional eyes and jaws. Larvae were able to maintain a horizontal swimming mode from hatching. First feeding was observed from the first day after hatching. F.varium larvae grew steadily from the time of hatching. Ocular morphology was examined in larval, juvenile and adult P.auratus and F.varium. There was a 96 fold increase in eye size, from 0.23mm diameter in a 4 day old larval P.auratus (3.4mm SL) to a maximum diameter of 22mm in an adult of 333mm body length. F.varium displayed a 26 fold increase in eye size, from 0.28mm diameter in the smallest larva (5.00mm SL) to a maximum eye diameter of 7.2mm in a 11gmm long adult. Larval fish had pure cone retinae, however putative rod precursor cells were present from hatching in F.varium and from 18 days in P.auratus. Juvenile and adult fish had duplex retinae with cones arranged in a square mosaic in which 4 twin cones surround a central single cone. Hypertrophy of cone ellipsoids with increasing eye size, resulted in maintenance of a closely packed array in fishes of all sizes. The appearance of retinomotor movements was coincident with the development of a duplex retina in both species. Theoretical spatial acuity (calculated as a function of cone spacing and focal length of the lens) was poor in the smallest larval fish (2° 1' and 1° 8' minimum separable angle in 4 and 1 day old P.auratus and F.varium respectively) but improved to asymptotic values in adults (3'- 4', and 9' in P.auratus and F.varium respectively). Behavioural acuity (determined using the optokinetic response) of 4 day old larval P.auratus (37° 30') and 1 day old F.varium (29°) was very much lower than histological estimates. Behavioural acuity improved to 8° 8' in 16 day old P.auratus and 4° 18' in 14 day old F.varium, but did not attain theoretical estimates for fish of that size (55' and 54'). A rudimentary retractor lentis muscle was first apparent in larval fish 1 week after hatching, and was coincident with the formation of a posterior lental space. Presumably larval fish eyes were incapable of accomodative lens movements until this time. A relative measure of Matthiessen's ratio (distance from lens centre to boundary of the pigmented retinal epithelium/lens radius) measured histologically, decreased from 4.2 and 2.7 in 3 day old P.auratus and newly hatched F.varium, to 2.2 and 2.3 in larvae 22 and 16 days of age respectively. This suggests that growth of the retina and lens were not symmetrical in the eyes of very small larval fish. If Matthiessen's ratio holds for little eyes, then they will initially be strongly myopic. This may account in part for the mismatch between behavioural and theoretical acuity. Perceptive distances of first feeding larval P.auratus and F.varium, estimated for prey items equal in dimensions to maximum jaw widths, were very small (0.2mm and 0.4mm for prey 0.15mm and 0.2mm in size respectively), but increased with increasing body size to 2.1mm and 4.0mm for prey 0.3mm in size, at 16 and 14 days of age respectively. These data have implications for larval feeding in the wild.

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19

Lee, Shao Chin. "Isolation and characterisation of two amylin responsive proteins from rat skeletal muscle." Thesis, University of Auckland, 1997. http://hdl.handle.net/2292/1901.

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Two amylin responsive proteins, here designated ARP1 and ARP2, were discovered from rat skeletal muscle through two dimensional gel electrophoresis analysis. ARP1 was detected only in amylin-stimulated muscles where the insulin-stimulated glucose incorporation into glycogen was inhibited. This protein incorporated 32Pi but not [35S]-methionine in the metabolic labeling experiments. Subsequent molecular characterisation revealed that ARP1 was a novel monomeric form (designated form 1) of protein p20, and two other monomeric forms (designated forms 2 and 3 respectively) of protein p20 were also characterised. The production of ARP1 was not affected by the presence of insulin, but calcitonin gene-related peptide (CGRP) was found to evoke the production of ARP1 in the presence or absence of insulin. In contrast, ARP2 was detected in both control and amylin-stimulated muscles. Amylin stimulation evoked incorporation of [35S]-methionine but not 32Pi into the protein and increased its concentration significantly. It is concluded that amylin elicits the production of ARP1 through phosphorylation and increases the protein biosynthesis of ARP2; the amylin-evoked production of ARP1 is insulin independent; amylin and CGRP share, at least in part, an intracellular signal transduction pathway; and ARP1 and 2 may be involved in the development of insulin resistance. It is suggested that ARP1 and 2 could potentially be used as molecular markers for the analysis of amylin action.

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20

Rho, Jung-hyun. "A novel mucin-desulfating sulfate-6-N-acetylglucosaminidase (sulfoglycosidase) from the anaerobic colonic bacterium Prevotella strain RS2." Thesis, University of Auckland, 2004. http://hdl.handle.net/2292/2275.

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Sulfate removal from sulfomucin is believed to be a rate-limiting step in sulfomucin degradation by bacteria from the digestive tract. A novel sulfomucin-desulfating enzyme has been discovered in the anaerobic bacterium, Prevotella strain RS2, which can grow on colonic mucin as its sole energy source. The enzyme, located in the periplasm, was assayed by measuring p-nitrophenol removal from the model substrate sulfate-6-N-acetylglucosamine-1-p-nitrophenol, sulfate-6-N-acetylglucosamine being the other product. This activity differs from that of sulfatases which remove the sulfate ester group from sulfate-6-N-acetylglucosamine and its analogues substituted at the Cl position. The enzyme has been termed a sulfate-6-N-acetylglucosaminidase or sulfoglycosidase (SGL). The SGL was purified to a single protein band of 100 kDa as analyzed by SDS-PAGE. The purified SGL protein was trypsin-digested and peptide fragments were sequenced. PCR and inverse PCR were then used to amplify the entire sg/ gene from Prevotella strain RS2 genomic DNA. After inserting the gene into a suitable plasmid, active recombinant SGL was expressed using an Escherichia coli expression system. The SGL was characterized using a selection of model substrates, and shown to be an exo-enzyme that removes non-reducing terminal sulfate-6-N-acetylglucosamine residues by glycosidic bond cleavage. When tested against its putative physiological substrate, sulfomucin, the only small molecular size product detected corresponded to a sulfate-6-N-acetylglucosamine residue. Thus the SGL can catalyze a reaction, formerly thought to be performed in bacteria by the combined actions of a N-acetylglucosamine-6-sulfatase and a N-acetylglucosaminidase. Inhibition studies on the SGL were carried out. Inhibitors of the SGL and those of the sulfatases were used to confirm the presence or absence of SGL-like activity in other bacteria that inhabit environments containing sulfomucin. Four isolates, including Prevotella strain RS2, of the thirteen strains tested, appeared to have SGL-like activity. This research on the SGL with its novel catalytic activity, suggests a new mechanism by which sulfomucin desulfation can occur. The physiological importance of the enzyme is postulated to be (i) to provide energy in the form of sulfate-6-N-acetylglucosamine, for the bacterium, (ii) to remove sulfate-6-N-acetylglucosamine groups present on mucin chains, thus creating or removing sites for different adhesins, and (iii) removal of inhibitory sulfate-6-N-acetylglucosamine groups from mucin chains that limit degradation of the chain by exoglycosidases and neuraminidases.

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21

Albertson, Gregory David. "PulA, a thermostable pullulanase from an extreme thermophile Caldocellum saccharolyticum." Thesis, University of Auckland, 1992. http://hdl.handle.net/2292/2436.

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The pullulanase gene from Caldocellum saccharolyticum, an obligate thermophilic anaerobe, was sequenced and expressed in E. coli. Expression and substrate induction studies in E. coli showed that while gene expression was substrate inducible and the enzyme was exported into the growth medium in C. saccharolyticum, expression was non-inducible in E. coli and the enzyme remained in the cytoplasm. The nucleotide sequence of the pulA gene was shown to be 2478 basepairs (bp) in length, coding for a protein of 96 kDa. The proposed promoter sequences showed homology to both the standard E. coli sequences and the consensus sequences obtained from other C. saccharolyticum genes. The enzyme from the native organism was purified from the growth medium and shown to have a molecular mass of approximately 120 kDa. Periodic acid-Schiffs staining showed that this enzyme was glycosylated and substrate characterisation revealed that the enzyme debranched pullulan to produce only maltotriose, but hydrolysed amylopectin, amylose and β-limit dextran to produce a number of smaller oligosaccharides. The enzyme was expressed in E. coli from its own promoters and was purified from the cytoplasmic fraction. Substrate characterisation revealed that the enzyme debranched pullulan to produce only maltotriose, but had only limited activity on β-limit dextran and amylopectin, and no activity on amylose. The pullulanase gene was also expressed under the control of a heat-inducible overexpression system in E. coli and a copper-inducible expression system in yeast. Amino acid homology comparisons of the pullulanase to other pullulanase sequences and related enzymes revealed a high degree of homology, particularly around three highly conserved regions. In α-amylases amino acids in these regions are involved in catalytic activity, substrate binding and metal ion binding.

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22

Campbell, Dana L. M. "Species recognition in zebra finches: testing the effects of sex, sensory modalities, and social ontogeny." Thesis, University of Auckland, 2009. http://hdl.handle.net/2292/4442.

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Species recognition is an integral component of mate selection and must occur in all sexually reproducing organisms to avoid costly hybridisation. Species recognition abilities may be comprised of both innate components and experience during ontogeny through the learning of visual, acoustic, and other sensory species-specific cues. But how greatly is the ability to recognise one‟s own species (conspecifics) over others (heterospecifics) dependent on the phylogeographic relationship of the array of potential species as social partners and to what extent is the discriminatory behaviour modulated by subject ontogeny versus species identity? Using a model system, which is widely studied in all disciplines of avian research, the zebra finch (Taeniopygia guttata castanotis), I aimed to investigate the visual and acoustic cues involved in conspecific recognition by both female and male individuals of this species. I used an array of previously untested phylogeographically relevant estrildid heterospecifics as my stimuli and tested subjects of diverse experimental ontogenetic treatments. By scoring a wide-selection of measured behavioural responses my research indicates that female and male zebra finches prefer live conspecifics over live phylogeographically relevant heterospecific stimuli and this preference is more consistent by females than males. Female zebra finches rely on both visual and acoustic features of potential social partners for accurate species discrimination; in this regard video playbacks or the diverse colour morphs of domesticated zebra finches may be useful tools for further experimentation. Additionally, females display significant individuality in their behavioural responses which may be relevant for pair bonding decisions made by both sexes. I further documented that normally-reared zebra finches will prefer song playbacks of their own species but that both rearing in an indoor restricted acoustic environment of conspecifics or cross-fostering to another species will reduce discrimination preferences, although the results may depend on the behavioural metrics analysed. This dissertation is presented as a general overview with details of my specific contributions towards the work included in this thesis, followed by discrete review and data chapters, and a final general discussion.

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23

Brown, Samuel David James. "Molecular systematics and colour variation of Carpophilus species (Coleoptera: Nitidulidae) of the South Pacific." Diss., Lincoln University, 2009. http://hdl.handle.net/10182/1430.

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The sap beetle genus Carpophilus Stephens (Coleoptera: Nitidulidae) is a large genus consisting of over 200 species and are found worldwide. Several species are important pests of crops and stored products, and are frequently intercepted as part of biosecurity operations. The genus is poorly known taxonomically, and there are several species groups that are challenging to identify by morphological methods. In particular, two species found across the Pacific, C. maculatus Murray and C. oculatus Murray are frequently confused with each other. These two species are similar in size and colour, but differ primarily by the shape of the colour pattern on their elytra. However, this colour pattern is highly variable within both species, leading to ambiguity in the indentification of these species. Within C. oculatus, three subspecies have been described based on differences in the male genitalia and pronotal punctation: C. o. oculatus and C. o. gilloglyi Dobson are distributed widely across the Pacific, while C. o. cheesmani Dobson is known only from Vanuatu. A search of literature records and specimen collections revealed 32 species of Carpophilus recorded from the Pacific region. In addition there remain several unidentified specimens representing at least four species, two of which will be described subsequent to this research. A number of species recorded in the literature may have been misidentified, and these require further field collections and inspection of museum specimens to confirm their presence in the Pacific. To test the validity of the subspecies of C. oculatus, and its distinctiveness from C. maculatus, a phylogeny of available specimens of Carpophilus was inferred from one mitochondrial gene (cytochrome c oxidase subunit I (COI)), and two nuclear genes (28S ribsomal RNA (28S) and the internal transcribed spacer 2 (ITS2)). These data show large genetic distances between the three subspecies of C. oculatus of 7-12%. Given these distances are similar to those between other species in the genus, this indicates these subspecies may be elevated to full species. The data also consistently support a monophyletic relationship between C. o. oculatus and C. o. gilloglyi. Nuclear genes also support C. o. cheesmani as part of a clade with the other subspecies, but these relationships are unresolved in COI. Carpophilus maculatus was not supported as being the sister taxon of the C. o. oculatus and C. o. gilloglyi clade. Other relationships within Carpophilus were unresolved, possibly due to a combination of incomplete taxon sampling, and saturation of substitutions within the COI gene. Phylogeographic analysis of specimens collected from several localities within the range of C. oculatus showed that, with only one exception, there were no shared haplotypes between archipelagoes. This result suggests it may be possible to determine the provenence of intercepted specimens, providing further information regarding potential invasion pathways. A degree of geographic structuring was also present within C. o. gilloglyi, being separated into a western clade found in Fiji and Rotuma and an eastern clade distributed from the Kermadec Islands and Tonga to French Polynesia. This separation was most profound in COI data, with a mean pairwise distance between the clades of 7%. ITS2 data also demonstrates a degree of differentiation between the two clades, based on differences in the insertions and deletions between the clades. The variability in the shape and colour of the elytral pattern of C. oculatus was also investigated. Colour was quantified using a method based on Red-Green-Blue (RGB) colour values derived from digital photographs, while an outline analysis of the elytral pattern was conducted using elliptic Fourier analysis (EFA). Principal Components Analysis of the RGB values and EFA coefficients showed no clear separation between subspecies, nor were any trends correlated with host fruit or collection localities. Variation at all levels and all measures studied in this thesis show that this geographic region and this genus of beetles offer intruiging insights into speciation, biogeography and biological invasions. There is much scope for further research on the causes and consequences of this variation and the lives of these interesting insects.

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24

Zhang, Liangtao. "Identification of Hordeum vulgare-H bulbosum recombinants using cytological and molecular methods." Thesis, University of Auckland, 2000. http://hdl.handle.net/2292/2355.

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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.

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25

Eaton, Carla Jane. "Investigation of signalling involved in maintaining the mutually beneficial association between Epichloe festucae and perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand." Massey University, 2009. http://hdl.handle.net/10179/1179.

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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.

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26

Yee, Thomas William. "The Analysis of binary data in quantitative plant ecology." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/1973.

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The analysis of presence/absence data of plant species by regression analysis is the subject of this thesis. A nonparametric approach is emphasized, and methods which take into account correlations between species are also considered. In particular, generalized additive models (GAMs) are used, and these are applied to species’ responses to greenhouse scenarios and to examine multispecies interactions. Parametric models are used to estimate optimal conditions for the presence of species and to test several niche theory hypotheses. An extension of GAMs called vector GAMs is proposed, and they provide a means for proposing nonparametric versions of the following models: multivariate regression, the proportional and nonproportional odds model, the multiple logistic regression model, and bivariate binary regression models such as bivariate probit model and the bivariate logistic model. Some theoretical properties of vector GAMs are deduced from those pertaining to ordinary GAMs, and its relationship with the generalized estimating equations (GEE) approach elucidated.
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27

Snowden, Kimberley Cathryn. "The molecular response of wheat roots to aluminium stress." Thesis, University of Auckland, 1994. http://hdl.handle.net/2292/1967.

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Aluminium (Al) toxicity to plants is a significant problem, limiting agricultural production in up to 40% of the world's arable soils. In spite of a large amount of research, there is still no consensus on the physiological mechanisms of Al toxicity in plants. In addition, very little is known about the molecular response of plants to Al stress. This body of research was aimed at identifying the changes in gene expression that occurred in the root tips of plants that had been stressed with Al. A cDNA library made from the root tips of Al-treated wheat (Triticum aestivum L., cultivar Warigal) plants was differentially screened to identify clones whose expression was induced by Al stress. Seven cDNA clones, representing five different genes were identified as being induced in the presence of Al. Initial sequencing and northern analysis revealed that none of the clones isolated were full-length, and that some contained multiple cloning adaptors at their 5' ends. A new cDNA library was then constructed from the root tips of Al-treated Warigal plants, and homologues to each of the original five genes were isolated. These five clones were named wali1 to wali5 (for wheat aluminium induced). Northern analysis showed that wali1, -3 and -5 were induced 24 to 96 h after Al treatment, and their expression declined when the Al was removed. wali4 had a similar pattern of expression with a transient increase in expression also observed after 0.5 h of Al stress. Each of these four genes was induced by inhibitory concentrations of Al in two wheat cultivars - Warigal, an Al-sensitive cultivar, and Waalt, an Al-tolerant cultivar, - and also in two inbred lines of wheat, RR (Al-tolerant) and SS (Al-sensitive). The fifth gene (wali2) had a bimodal pattern of induction, and was induced by Al only in the Al-sensitive Warigal and the Al-tolerant RR. The nucleotide sequence of each of the wali clones was determined, and the databases were searched for homologous sequences. Wali1 was found to be homologous to a group of metallothionein-like proteins (MLPs) from plants, and wali4 was homologous to phenylalanine ammonia-lyase (PAL). wali3 and wali5 encode related, cysteine-rich proteins with homology to Bowman-Birk proteinase inhibitors, and wali2 encodes a novel protein with a repeating motif of cysteine amino acids. The induction of the wali genes was investigated in response to a number of other stresses through northern analysis. The expression of wali1, -3, -4 and -5 was induced in root tips of wheat after 2 d treatments with toxic levels of all other metals tested (Cd, Fe, Zn, Cu, Ga, In and La). The expression levels of wali1, -3, -4, and -5 also increased in the root tips of plants grown in the presence of low levels of Ca (10μM). The transcript levels of wali1, -3 and -5 increased in wounded leaf and root tissue, whereas the transcript levels of wali4 increased only in wounded leaves. The expression of wali2 was greatly reduced by low concentrations of Ca, and showed no induction, or a variable response with most of the other treatments. The site of expression of wali1, -2, -3 and -5 in root tips (and wali1 also in leaf tissue) was identified using in situ hybridisation. Wali1 was expressed predominantly in the meristematic tissue of the root tip, while wali3 and wali5 were expressed predominantly in the cortical tissue of the root. wali2 expression was detected primarily in the epidermis and root cap. Some changes in the site of expression of these genes were evident in the roots of Al-treated plants. In leaf tissue, wali1 expression was found in the mesophyl1 layer of cells. The coding sequences for wali1,-2,-3 and -5 were each cloned into the bacterial expression vector pGEX-2T. The resultant fusion proteins between glutathione S-transferase (GST) and the walis were then successfully purified from E coli. Antibodies were made to the wali1-GST fusion protein and purified by immunoaffinity chromatography. However, when used in western analysis, no specific bands corresponding to the native wali1 protein were identified. The wali2-GST protein was used in a south-western procedure to determine if the protein was capable of binding DNA, but no DNA binding to this protein was detected under the conditions tested. The wali3 and wali5 fusion proteins were tested in proteinase inhibitor assays, where no inhibition of either trypsin or chymotrypsin was detected. It is possible that the native wali3 and wali5 proteins may not function as proteinase inhibitors, or that the lack of activity detected for the fusion proteins may be due to incorrect folding or processing in the bacterial system. This research constitutes the first identification of plant genes whose expression is increased by Al stress. The genes identified are also induced in response to other environmental and nutrient stresses, indicating that they form part of the plant's general response to stress.
Appendix 4 restricted.

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28

Henry, Stephen Michael. "Further insight into the Lewis histo-blood-group system as revealed from study of Polynesian and Caucasian plasma and erythrocyte glycosphingolipids." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/1975.

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This project involved the study of Lewis and related blood group glycosphingolipid isolated from individuals with normal and aberrant Lewis/secretor phenotypes. The objective was to find a biochemical basis for the unusual expression of Lewis and secretor phenotypes in Polynesians and to use this information to shed light on the "normal" expression of Lewis antigens. By using purified glycolipids, presenting them in the cell free environment of thin layer chromatography to Lewis antibodies and by determining structures by mass spectrometry it has been shown that: l. The Lec epitope is a terminal Galβ1-3Gal sequence, and not an internal branch as proposed by Hanfland (Hanfland et a1.,1986). 2. Lec or H-5-1 are present in Lewis negative phenotypes and their consequent consumption by the Le and ,Se transferases resulting in the known Lea and Leb antigens can be seen in the Lewis positives. 3. Phenotypically Le(a-b-) individuals have small amounts of Lewis antigens. This clearly demonstrates that although the Lewis negative phenotype exists at the crude serological level, this phenotype is not an "all-or-nothing" phenomenon at the chemical level. This also allows it to be postulated that the le gene is probably partially active. 4. Le(a+b+) individuals have both Lea and Leb glycolipids in the erythrocyte membrane and in plasma. Observed phenotyping anomalies appear to be related to there being quantitatively less Leb-6 in the Polynesian Le(a+b+) erythrocyte membrane than in the Le(a-b+) membrane. 5. The Le(a+b-) phenotype of Polynesians is actually the Le(a+b+) phenotype but with serologically undetectable Leb. This allows it to be postulated that the nonsecretor gene (se) is absent in Polynesians. 6. Extended structures are present in most of the Polynesian samples which is in support of a postulated weak secretor gene (Sew). It now appears that the difference between the extended Lewis glycolipids of Caucasians and Polynesians is quantitative. The postulated, Sew transferase appears to be inefficient and allows for increased formation of elongated glycoconjugates (polyglycosylceramides) to result. 7. Reduced fucosyltransferase activity allows increased elongation of the precursor chain to occur, which allows it to be postulated that fucosylation of the precursor prevents, or at least markedly reduces, chain elongation. It is speculated that, as almost everyone is either Lewis and/or secretor positive, perhaps the prevention of chain elongation is a biological reason as to why the Lewis and Secretor polymorphisms exist. 8. Differences in ceramide patterns of Lewis active glycolipids suggests that the small intestinal tract is not the only origin of plasma glycolipids, or there is differential absorption. 9. There is no plasma glycolipid-based reason for there being increased H type 2 antigen reactivity in the Polynesian erythrocyte membrane, nor a reason for the H antigen association with the Le(a+b+) phenotype.

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29

Hieber, A. David (Andrew David) 1966. "Characterisation of glycoprotein II from bovine adrenal medulla." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/1988.

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Glycoprotein II (GpII) is a glycoprotein isolated from the membranes of chromaffin granules in the adrenal medulla. The chromaffin granules of the adrenal medulla are responsible for the biosynthesis, storage and secretion of catecholamines, neuropeptides and various proteins. The abundance of chromaffin granules makes them an excellent model to further study the organelles specialized in the synthesis and secretion of hormones and neurotransmitters, from both endocrine and synaptic vesicles. When viewed by two-dimensional electrophoresis GpII is a heterogeneoug glycoprotein (80000-100000 daltons) consisting of two components, upper (GpIIa) and lower (GpIIb). Protein and immunological characterisation revealed that the two components of GpII are distinct from each other. Further molecular characterisation of GpIIa showed that it consisted of a 1224 base pair cDNA, encoding a 383 amino acid polypeptide, with a calculated molecular mass of 40500 daltons. This characterisation also revealed 19 potential glycosylation sites, with deglycosylation studies further demonstrating that an estimated 55% of the molecular mass of GpII is asparagine-linked carbohydrate. The presence of poly-N-acetyllactosamine carbohydrate groups on GpII was also demonstrated. The predicted amino acid sequence of GpIIa shares a 72% amino acid identity with the human lamp-l type protein, which belongs to a highly conserved group of lysosomal associated membrane glycoproteins (lamp proteins). As the C-terminal region of GpIIa was identical to the C-terminal region of lamp proteins, a synthetic peptide to the C-terminal region of GpII was used to raise antisera to this region. This antisera was then used to demonstrate that the C-terminal region of GpII wag present on chromaffin granules and facing the cytoplasm. This same C-terminal region on lamp proteins is known to be a cytoplasmic tail that is essential for the intracellular targeting of lamp proteins to the lysosome. A common feature to the pathways of both GpII and lamp proteins is their appearance on the cell surface followed by internalisation via endocytosis. As endocytosis of the chromaffin granule membrane is known to occur, the possibility that GpII was a marker for endocytosis was investigated. The presence of GpII within clathrin coated vesicles was shown, and evidence was presented to demonstrate that the C-terminal region of GpII interacts with adaptor proteins of clathrin coated vesicles. Adaptor proteins are known to mediate between the cytoplasmic domain of proteins internalised by endocytosis, and the outside coat of clathrin. The evidence presented would suggest that the cytoplasmic tail of GpII is a potential marker for endocytosis within the chromaffin granule membrane. The results presented in this study indicate that GpII is the secretory granule and species counterpart to the lamp proteins. This finding however raises some interesting questions regarding intracellular targeting between the lysosomes and secretory granules within the chromaffin cell.

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30

Hiyama, Jun. "Isolation and characterisation of N-glycans of ovine and human luteinizing hormones." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/1989.

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Gonadotrophic hormones are heterodimeric glycoproteins and their N-glycans attached to specific amino acid residues are currently thought to play important roles in hormonal biosynthesis, secretion and function. The studies reported in this thesis aimed at isolation and characterisation of structural properties of the N-glycans on ovine and human luteinizing hormones. Initially, chromatographic methods were developed using reverse-phase HPLC for the analytical separation of the three human pituitary glycoprotein hormones and their subunits. Separation of intact oLH and its subunits was also effected by a single HPLC step. A preparative procedure was developed for the efficient purification of hLH and hTSH from crude human pituitary extracts using hydrophobic chromatography which gave highly purified hormones in good yields and with high biological activities. This method did not significantly influence the hormones' extensive charge heterogeneity and it offered potential advantages in the characterisation of their carbohydrate structures. A preparative scheme was developed for the isolation of the N-linked oligosaccharides from each glycosylation site of o- and hLH. Charge heterogeneity of oligosaccharides, which were released by hydrazinolysis from subunits and glycopeptides, was characterised by anion-exchange HPLC. 1H-NMR analysis showed that the structures of all three N-glycans on hLH were highly heterogeneous but mainly diantennary complex-type, with site-specific patterns of terminal sialylation and sulphation as well as core-fucosylation. Sulphated/sialylated and/or disialylated oligosaccharides were the major components at each site. A set of new mono- and disialylated oligosaccharides with the terminal sequence NeuAcα2-6GalNAcβ1-4G1-cNAcβ1-2Manα1-3 was identified. This finding suggested unique site-specific terminal sialylation of oligosaccharides at Asn 78 (hLHα) by an unknown α2-6 sialyltransferase(s) in the human pituitary gonadotroph cell. Each glycosylation site in oLH had a distinct set of oligosaccharides ranging from mainly monosulphated hybrid-type at the two sites of oLHα to predominantly disulphated diantennary complex-type on oLHβ. Core-fucosylation also differed at each site. These results suggested that processing of the oligosaccharides of the α- and β-subunits by α-mannosidase II and α1-6 fucosyltransferase was differently regulated by protein structure in oLH. Whereas hCG, hLH and oLH share similar biological activities, no apparent relationship between their N-glycan structures was found, which suggested that specific branching and peripheral structures of N-glycans on LH and hCG may not be essential for biological function, although the N-glycan nearer the N-terminus of the α-subunit of hCG has been implicated in hormonal activity.

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31

Podivinsky, Ellen. "Molecular studies on actinidin, a cysteine protease from kiwifruit." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/2001.

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Research in this thesis describes the characterisation of mRNA sequences coding for actinidin, a cysteine protease found in abundance in the fruit of kiwifruit (Actinidia deliciosa). The first step in the characterisation required the isolation of mRNA from ripe kiwifruit tissue. The suitability of a number of RNA extraction procedures was investigated. The method finally adopted differed from that used for unripe fruit tissue, and was chosen as a result of the nature of the polysaccharide that contaminated nucleic acids prepared from extracts of kiwifruit fruit tissue. RNA extracted from ripe fruit was used to synthesis a partial cDNA library and clones for actinidin were isolated. A number of these cDNA clones were sequenced; three clones were almost full-length. The actinidin cDNA clones obtained fall into two broad sequence classes. The majority of them encode acidic proteins (pI˜4.7), with 97% homology to the published amino acid sequence of actinidin. The second class encode basic proteins (pI˜8.1), with 83% homology to the published amino acid sequence of actinidin. Both classes of actinidin cDNA sequence encode zymogens, which contain N- and C-terminal extensions not present in the mature form of the enzyme. The N-terminal extension of both sequence classes includes a putative signal peptide. Northern hybridization analysis was used to investigate the tissue specificity of actinidin mRNA expression, and the expression of mRNA for the two actinidin sequence classes during fruit ripening. Both actinidin sequence classes were expressed differentially during the latter stages of kiwifruit fruit development and through post-harvest fruit ripening. The expression of both sequence classes increased from just prior to fruit maturity through ripening and reached a maximum as fruit attained the stage of 'eating' ripeness. The level of expression of the sequences encoding acidic actinidin reached a plateau at this point, while the expression of the sequence encoding basic actinidin appeared to decrease slightly as fruit continued to ripen. The sequences encoding acidic actinidin were expressed during ripening at a much higher level than those encoding basic actinidin. No actinidin mRNA was detected in other tissues except for very low levels of the acidic form in kiwifruit leaf, and low levels of the basic form in senescing petals. A full-length, acidic, actinidin cDNA sequence was introduced into tobacco (Nicotiana tabacum) plants via Agrobacterium tumefaciens-mediated transformation. Using the binary vector pGA643, the sequence was introduced in both the sense and antisense orientation relative to the cauliflower mosaic virus 35S promoter and transgenic plants were obtained for both sequence orientations. The presence of the T-DNA cassette (containing the actinidin sequence) in the plant genomes was determined using PCR analysis, and confirmed by Southern hybridization. A number of the transgenic plants contained multiple insertions of the actinidin sequence, and most plants contained at least one intact copy of the T-DNA cassette. The transcription of the introduced actinidin sequence was investigated by Northern hybridization analysis. All of the plants containing actinidin in the sense orientation, and some of those incorporating the antisense construct, transcribed the actinidin sequence. Attempts to detect actinidin protein in the transgenic plants were unsuccessful. Acidic actinidin was identified as one of the most abundant bands in the total protein profile from ripe kiwifruit fruit tissue. The identity of the protein was confirmed by N-terminal sequence analysis. The electrophoretic mobility of actinidin, both in the total cell homogenate and when partially purified, suggested that the first step in post-translational processing of the zymogen may be the removal of the N-terminal extension. Actinidin was also partially purified and used to raise antibodies. Poor specificity of the antibody for actinidin led to preliminary evidence for the glycosylation of actinidin.

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Spiers, Andrew J. (Andrew Julian). "Molecular and genetic analysis of RepA from the P307 RepFIB replicon." Thesis, University of Auckland, 1992. http://hdl.handle.net/2292/2044.

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The work in this Thesis concerns the replication control system of the P307 plasmid RepFIB replicon. The basic replicon occupies ≈ 1.6kb of DNA and contains a single large open reading frame (repA) flanked on either side by a series DNA repeat elements. The organisational structure of fie replicon has placed RepFIB into the Step function class of replicons. The placement of RepFIB within this group, as well as a strong homology between RepFIB and mini-P1, has resulted in a series of predictions concerning the control elements of RepFIB replication. The aim of this work was to test some of these predictions and to characterise the fundamental control elements utilised by the replicon. This Thesis describes three different active promoter elements found embedded within the repeat elements flanking repA. Although the functional significance of two of the promoter is unknown (orip and EFp), the third is responsible for the expression of RepA and has been designated 'repAp'. All three promoters are sensitive to RepA in trans, demonstrating that repA is autoregulated and that RepA is a DNA-binding protein capable of recognising copies of the repeat elements. RepA DNA-binding has also been demonstrated in vitro using a modification of the Western analysis technique (referred to a 'Western-DNA') in order to complement the in vivo experimental results. Although the coding region of repA had been determined in earlier work, the identification of the translational start codon was uncertain. This uncertainty has been resolved by limited N-terminal sequence analysis of a RepA:β-galactosidase fusion protein which has demonstrated that translation begins from a CTG codon located upstream of the predicted start sites. Finally, a series of genetic experiments have been used to determine the functional significance of RepA binding to the repeat elements. The repeat group upstream of repA are involved in autoregulation and also form part of the origin of replication, whilst the downstream repeats appear to be involved in the sensing and setting of plasmid copy number. Although the work presented in this Thesis does not directly test the applicability for RepFIB of various control models proposed to explain the behaviour of Step function replicons, the nature and type of control elements identified in RepFIB support the placement of RepFIB within the Step function class. As a result of this work, it is clear that RepFIB is confronted by the same kind of control paradox faced by replicons such as mini-P1 and mini-F. All three replicons use autoregulation and titration to control the supply of initiator protein required for replication. However, concurrent autoregulation and titration appear to be incompatible in current control models, and the identification of both mechanisms in these replicons has lead to a control paradox. Some of the results presented here suggest potentially valuable avenues of future research which may help resolve the paradox faced by RepFIB and other Step function replicons.

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33

Daja, Mirella Maria. "Enzyme activities associated with gonadotropic hormones." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/2311.

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A structural relationship between gonadotropic hormones and certain types of enzymes has been suggested in previous studies and an investigation into the possibility of enzymatic activity associated with the gonadotropic hormones has been the primary focus of the research presented in this thesis. Partial sequence homology between human chorionic gonadotropin (hCG) and α-chymotrypsin prompted the recent proposal of a tertiary structure of hCG using α-chymotrypsin as a folding template, which suggested the possibility of intrinsic peptidase activity associated with hCG. Highly purified hCG (CR127) was assayed for enzymatic activity against a range of synthetic peptide substrates and was found to exhibit Arg-specific peptidase activity. This activity was almost completely inhibited by diisopropylfluorophosphate (DFP), soybean trypsin inhibitor (STI), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and to a lesser extent by N-α-p-tosyl-L-lysine chloromethyl ketone (TLCK), which indicated that the observed protease activity was serine protease-like. To establish whether this activity was intrinsic to the hormone or due to contaminants, extensive purification procedures were carried out. Hydrophobic interaction chromatography (HIC) and soybean trypsin inhibitor-affinity chromatography were found to effectively separate the protease activity from the hormone, indicating the presence of exogenous protease contaminants in the highly purified preparation of hCG. Further analysis by [3H]-DFP labelling of hCG and SDS-PAGE of the isolated contaminants revealed the presence of possible serine proteases with apparent molecular masses of 60 and 20 kD. Because serine proteases are known to stimulate cAMP production in the same target cells, it was necessary to determine the effects of the contaminating proteases on the receptor binding of hCG and cAMP production. The presence of these contaminants was found to have no apparent effect on the receptor binding capability of hCG, however the in vitro biological activity of hCG as determined by maximal cAMP production was decreased after HIC-HPLC purification of the hormone. These observations suggested that the serine protease-like contaminants contributed to the total cAMP production, thereby introducing significant error in biological assays that use hCG (CR127). The possible intrinsic enzymatic activity of hCG against its receptor as a natural substrate was further investigated. A membrane-bound receptor preparation was isolated from porcine ovaries and a receptor binding assay successfully established. The effects of hCG binding upon the membrane-bound receptor were studied and receptor proteolysis was observed. However, this proteolysis could not be definitively attributed to the actions of hCG. A purified receptor was subsequently prepared by hCG-affinity chromatography and analysed by SDS-PAGE with detection by autoradiography and silver staining. The purified receptor was found to have undergone proteolysis during the purification procedure, presumably following incubation with the hCG affinity matrix. Recent reports of the presence of homologous amino acid sequences in the active site of thioredoxin and the β-subunit of the gonadotropic hormones luteinizing hormone (LH) and follicle stimulating hormone (FSH), and subsequent demonstration of thioredoxin-like activity associated with these hormones, prompted an investigation into the possibility of thioredoxin-like activity associated with hCG. LH, FSH and hCG were all assayed for their ability to promote reactivation of reduced and denatured RNase. Although LH was shown to be capable of reactivating reduced RNase, the level of activity detected was significantly lower than that previously reported, whereas FSH and hCG were not found to be capable of this thioredoxin-like activity. These results suggested that the previously reported thioredoxin-like activity may be due to contamination of the hormone preparation, by the ubiquitous enzyme thioredoxin. The possibility of LH possessing intrinsic dithiol-disulphide interchange activity was investigated further using [3H]-iodoacetic acid. RNase/LH were incubated in an attempt to quench a dithiol intermediate. Preliminary results suggested that the presence of LH in this reaction increased the amount of protein radiolabelled, however, the isolation of a radiolabelled dithiol intermediate which could be conclusively identified as LH was not forthcoming. Furthermore the lack of RNase reactivation activity in hCG, suggests that the putative thioredoxin-like activity of LH, if intrinsic, may not be involved in receptor activation and/or signal transduction, as hCG and LH share the same receptor and should therefore have a similar mechanism of activation.

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34

Janssen, Bart-Jan. "Agrobacterium-mediated gene transfer into kiwifruit." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/2313.

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A system has been developed to aid in the establishment of Agrobacterium-mediated transformation for new plant species. A series of binary vectors have been constructed that express a chimaeric β-D-glucuronidase (GUS) gene in plants cells but not in bacterial cells. This feature allows GUS activity from transformed plant cells to be assayed in the presence of Agrobacterium. Preliminary experiments examined the expression of these chimaeric GUS genes in transformed petunia leaf discs. GUS expression was detectable 2 days after inoculation, peaked at 3 – 4 days and then declined; if selection was imposed expression increased again after 10 - 14 days. The amount of expression observed 4 days after inoculation correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3 - 4 days after inoculation. Surprisingly, GUS expression was concentrated in localized zones on the circumference of the disc; within these zones essentially all the cells appeared to be expressing GUS. These results suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of the petunia leaf explants, but that the frequency of stable integration is several orders of magnitude lower. A reliable Agrobacterium-mediated transformation system has been established for kiwifruit (Actinidia deliciosa var. deliciosa cv. Hayward) by using transient expression of GUS to monitor gene transfer frequencies. In vitro culture of kiwifruit plants and conditions for regeneration of plants from leaf discs have been established. Several factors were found to improve gene transfer frequencies in kiwifruit: (i) healthy actively growing source tissue; (ii) the use of Agrobacterium strain A281; (iii) the presence of a layer of moistened filter paper between the leaf explants and the cocultivation media; and (iv) the presence of 20 μM acetosyringone in both the bacterial culture media and in the cocultivation media. Pre-culture of leaf explants significantly inhibited gene transfer, particularly at the cut edge of the explants. Using the optimized transformation system, at least one transgenic plant can be regenerated from each leaf inoculated. Stable transformation frequencies have been shown to vary significantly between different binary vectors. Phenotypic, PCR, and Southern analysis has confirmed the presence of stably integrated T-DNA in several transgenic kiwifruit plants.

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35

Merriman-Smith, B. Rachelle. "Glucose Transporters in Diabetic Complications of the Lens." Thesis, University of Auckland, 2001. http://hdl.handle.net/2292/2338.

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Lens transparency is primarily maintained by the anaerobic metabolism of glucose. Glucose is transported from the aqueous humuor to the lens epithelial cells however, it has not yet been established how glucose penetrates to the inner part of the lens. The core of the lens is acidic, approximately pH 6.5, as an effect of the accumulation of lactate, the end-product of glycolysis. This confirms that glucose is drawn deep into the core of the lens. Until recently it was assumed that glucose was transported to the core via a gap junction-mediated route by cell-cell diffusion. However, passive diffusion is limited in capacity and is unlikely to be sufficient for nutrient transport especially for larger lenses. Instead, an active circulation system has been proposed that has the potential to transport glucose deep into the lens via an extracellular route. This would imply that fibre cells may have evolved their own glucose uptake system, yet no direct evidence to this effect has been available. My thesis describes new molecular evidence that both epithelial and fibre cells have evolved their own glucose uptake system. The rat lens expresses the facilitative glucose transporters GLUTI and GLUT3 differentially. GLUTI is predominantly expressed in the epithelium while GLUT3 is predominantly expressed in the fibre cells of the lens. In the normal lens, this makes good physiological sense. GLUTI has a high Km suitable for situations of high glucose concentrations, as is the case for the epithelium where the aqueous humour mirrors glucose concentrations found in blood. GLUT3 has a lower Km and is particularly suitable for the fibre cells, where the supply of glucose from the tortuous extracellular space is limited. The discovery of GLUT3 in the fibre cells lends strong support for the existence of an active circulation system in the lens and makes it seem unlikely that glucose is transported into the core via gap junction-mediated diffusion. In the diabetic state, sorbitol - a product of glucose metabolism, occurs at elevated levels of about 30 times more than that of the normal, suggesting a significant increase in glucose uptake. This imposes an osmotic stress on the lens, which can be countered by regulated cell volume decrease only in the outer but not in the inner cortex. As a consequence inner cortex tissue breaks down and opacities result. My studies of the diabetic rat lens shows that the situation is made worse by an apparent up-regulation of GLUT3 in the fibre cells. Quantitative RT-PCR shows that the GLUT3 transcript is up-regulated six-fold during the initial weeks of diabetic insult in the streptozotocin rat model. The up-regulated GLUT3 protein is detected in the region where maximum tissue damage occurs. These results suggest a new mechanism for the initial tissue damage in the diabetic lens, whereby increased uptake of glucose leads to an over-production of sorbitol which causes osmotic stress on the fibre cells that is beyond their defense capability of regulated cell volume decrease. While my results described above have revolutionized our view of nutrient transport in the lens and its potential role in the early stages of diabetic cataract, the picture is only complete when the functionality of GLUT3 can be demonstrated. For this purpose, vesicles were prepared from isolated lens fibre cells and subjected to quantitative uptake studies using a fluorescent glucose derivative. Uptake was greatly reduced using the GLUT-specific inhibitor phloretin demonstrating that GLUT3 of the rat lens fibre cells is indeed fully functional. In summary, my results add a new dimension to our understanding of how the lens maintains homeostasis and tissue transparency. The lens has evolved an ingenious system to supply, nutrients to the core region to compensate for the absence of a vasculature. However, by achieving this, it has rendered itself unable to defend itself against the adverse effects of high glucose. My results also contribute towards developing new strategies of rational drug design to prevent or delay cataract.

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36

Simpson, Robert Malcolm. "The biosynthesis and control of indoleacetic acid." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/2344.

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Attempts were made to form indoleacetic acid in cellfree extracts of mung bean (Vigna radiata) shoots. The extracts were incubated with radiolabelled tryptophan and other substrates and cofactors thought to be involved in indoleacetic acid biosynthesis. After incubation indolepyruvate and indoleacetic acid were separated and quantified by HPLC. There was no significant difference in the conversion of tryptophan to indolepyruvate and indoleacetic acid between the incubations and control incubations using boiled extract. The concentrations of indolepyruvate and indoleacetic acid in mung bean hypocotyl suspension cultures were measured using GC-MS SIM over the growth of the culture, a period of 29 days. Indoleacetic acid concentrations, although scattered, mostly remained at constant low levels in the range of 6 to 9ng/g fwt of culture. The indolepyruvate levels steadily increased to a maximum level after 14 days, then remained at this level, 10 to 12 ng/g fwt, for the remainder of the culture period. This plateau in indolepyruvate concentration matched the period that the suspension culture was in the logarithmic phase of growth. An aromatic amino acid aminotransferase was purified over 33,000 fold from the shoots and primary leaves of mung beans, as determined using a tryptophan aminotransferase activity assay. The enzyme was a monomer, with a molecular weight of about 58kDa. The pH optimum was broad, with a maximum at about 8.6. The relative activities of the aromatic amino acids were: tryptophan 100, tyrosine 83 and phenylalanine 75, and the Kms were 0.095, 0.08 and 0.07mM respectively. The enzyme was able to use 2-oxoglutarate, oxaloacetate and pyruvate as the oxo acid substrate at relative activities 100, 128 and 116 and Kms 0.65, 0.25 and 0.24mM respectively In addition to the aromatic amino acids the enzyme was able to transaminate alanine, arginine, leucine and lysine to a lesser extent, and showed slight activity with asparagine, aspartate, histidine, valine and D-tryptophan and tyrosine. Inhibition studies showed that the alanine, aspartate and histidine activities were part of the aromatic amino acid aminotransferase activity. The enzyme was not inhibited by indoleacetic acid, although naphthaleneacetic acid did inhibit slightly. There was evidence of substrate inhibition by hydroxyphenylpyruvate at high concentrations. Addition of the cofactor pyridoxal phosphate only slightly increased the activity of the enzyme. The enzyme was blotted onto a PVDF membrane cleaved by in situ trypsin digest. Three of the tryptic fragments were sequenced. These fragments had approximately 60% sequence similarity with plant aspartate aminotransferases and tyrosine aminotransferases.

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37

Taylor, Jacqueline Ann, and Jackie (name change) O'Flaherty. "Factors affecting the metabolic control of cytosolic and lysosomal glycogen levels in the liver." Thesis, University of Auckland, 1985. http://hdl.handle.net/2292/2380.

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Although glycogen is a chemically homogeneous material it is polydisperse, exhibiting a broad molecular weight spectrum and a metabolic lability that is molecular weight dependent. The lower molecular weight (β-particle) glycogen was found to be extremely labile, while the higher molecular weight (α-particle) exhibited a far lower metabolic activity, indicating that it may act as a glycogen store for mobilisation in stress situations. These observations, coupled to the existence of Pompe’s Disease, a glycogen storage disease involving the lysosomal system, supports the hypothesis that α - and β -particulate glycogen may be partially separated from one another within the cell i.e. compartmentalised. By the use of a rapid differential centrifugation technique it was possible to show, both physiochemically and ultrastructurally, the existence of glycogen of a very large molecular size associated with the lysosomal fraction. This glycogen exhibited a different molecular weight distribution from that isolate from the liver as a whole i.e. cytosol + lysosomal. It is suggested that appreciably more than 10% of cellular glycogen is located within the lysosomes and that this is of predominantly high molecular weight. The size-distribution of liver glycogen was shown to be distinctly affected by the anti-inflammatory drugs, salicylate and Indomethacin. By measurement of the incorporation of radioactive glucose into glycogen, salicylate was shown to have a depressing effect on overall liver glycogen metabolism. These effects appear to arise from a stabilisation of the lysosomal membrane by the drugs. The incorporation, via liposomes, of purified anti-1,4-α-glucosidase antibodies into the liver lysosomes of normal Wistar rats and rats with a genetic deficiency of phosphorylase kinase, caused a distinct decrease in 1,4-α-glucosidase activity and in the content of high molecular weight glycogen. These changes were enhanced by prolonged liposomal-antibody treatment and suggested that a possible feedback control mechanism operates in the incorporation of glycogen into lysosomes. The 1-4- α -glucosidase inhibitor, Acarbose, when injected intraperitoneally into normal and phosphorylase kinase-deficient rats similarly disturbed liver lysosomal metabolism, causing distinct and persistent inhibition of enzymes and acute disturbances of lysosomal glycogen metabolism. Again a feedback control mechanism appears to operate, which effects cytosolic carbohydrate metabolism. The biochemical effects closely resembled those occurring in Pompe’s disease and were confirmed by electron microscopy. A model for the adult form of the lysosomal storage disease has been suggested.

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38

Powell, Kevin F. H. (Kevin Frederick Herbert). "Gene sequencing and in vitro synthesis of the rotavirus non-structural glycoprotein." Thesis, University of Auckland, 1986. http://hdl.handle.net/2292/2385.

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1. Recombinant DNA techniques have been applied to the dsRNA genome of the bovine rotavirus Nebraska Calf Scours Diarrhoea virus (NCDV). The sequence of a full - length cloned copy of genomic segment 10 of NCDV has been determined using the Sanger dideoxynucleoside sequencing technique by subcloning cDNA into M13 vectors. 2. Genomic segment 10 codes for the non-structural protein NCVP5, a protein which appears to be involved in virus maturation (Estes et al., 1983). Determination of the nucleic acid sequence of the gene has enabled the amino acid sequence o f the bovine NCVPS protein to be inferred. Comparison of the inferred amino acid sequence with homologous sequences derived from other virus strains (Both et al., 1983c; Baybutt and McCrae, 1984; Okada et al., 1984; Ward et al., 1985) has enabled conserved regions of the molecule to be identified. A small region of the NCVPS protein has been identified (residues 131-161) which exhibits considerable variability between rotavirus strains. 3. A computer-based algorithm has been utilised to predict the folding pattern of NCDV gene 10 mRNA. This reveals a 'panhandle ' structure which differs from that proposed for the related gene of strain Wa rotavirus (Okada et al., 1984) but both molecules possess a common feature in that the initiation codon falls within a potentially-stable duplex formed with a portion of the 3 untranslated region. 4. A series of four site-directed deletion mutants of the cloned gene were constructed in order to investigate the functional significance of the three N-terminal hydrophobic regions of the NCVP5 protein. Two mutants were constructed using conveniently-located HindIII and BamHI restriction enzyme sites. The other two mutants were generated using M13 vectors and synthetic oligonucleotides. These modifications yielded genes coding for proteins in which portions of the first and second hydrophobic regions had been deleted. 5. DNA corresponding to the 'wild-type' coding region was inserted into an SP6 transcription vector to enable mRNA to be produced in vitro. This mRNA, when incubated in a reticulocyte lysate, directed the synthesis of a protein of the correct size (20 K). The addition of dog pancreatic microsomes to the reaction yielded a protein product (29 K) of a size consistent with the glycosylated ('wild-type') form of the NCVP5 protein. 6. The four variant forms of the NCVP5 gene were also inserted into SP6 transcription vectors and the protein products synthesised by the resulting mRNAs studied. All four mRNAs directed synthesis of variant protein products of the anticipated size. 7. The ability of the four variant proteins to become glycosylated and to associate with membranes was investigated. The topology of the proteins in the membrane was examined by digestion with proteolytic enzymes. Variant proteins altered in the first or second hydrophobic regions retained their ability to associate with membranes, suggesting that the third hydrophobic region, which was not altered, might play a role in membrane association. 8. A model for the disposition of NCVPS in the endoplasmic reticulum is proposed in which the first hydrophobic region is located within the lumen of the endoplasmic reticulum, the second hydrophobic region spans the membrane and the third hydrophobic sequence associates independently with the membrane from the cytoplasmic side leaving the C-terminus of the molecule exposed to the cytoplasm. The model proposed accounts for the experimental observations but is in conflict with current mechanisms proposed for the insertion of proteins into membranes. (Wickner and Lodish, 1985).
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39

Beuning, Lesley L. "Cytokinins and the division or expansion of plant cells." Thesis, University of Auckland, 1988. http://hdl.handle.net/2292/2406.

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The effect of cytokinins was studied in three systems: the alga Chlorella, callus cultures and etiolated cucumber cotyledons. In Chlorella cultures: 1) A range of concentrations of 6BA and IP had no effect on growth; 2) Low concentrations of an anticytokinin had no effect on growth, whereas higher concentrations appeared to be inhibitory. 3) Characterisation of the Chlorella species suggested that it was surrounded by an impermeable sporopollenin layer which hindered the uptake of cytokinin. 4) The uptake of radioactive adenine occurred readily, whereas the uptake of radioactive 6BA was very slow in both growing and saturated cultures of Chlorella. 5) Extracts isolated from Chlorella and the medium in which Chlorella was growing contained cytokinin-like activity in two bioassays. 6) HPLC analyses of these extracts showed that there were fractions which eluted at the positions of IP and IPA. In callus cultures: A.1) A carrot callus was grown from the secondary phloem of the storage root of carrot. 2) This callus, which was grown on 2,4-D and kinetin, produced roots and shoots when subcultured onto IAA and kinetin. 3) Growth on 2,4-D alone was independent of the presence of kinetin. 4) Growth was inhibited in the presence of an anticytokinin, suggesting that the callus produced a cytokinin. B.1) A tobacco callus was grown from a young leaf of tobacco. 2) This callus habituated to cytokinin independence following subculture onto lower concentrations of kinetin. 3) Subculture of the habituated callus onto a higher concentration of kinetin resulted in the production of roots and shoots. C.1) Cytokinin-dependent soybean and tobacco callus cultures were obtained from the Botany Department, University of Otago. 2) Analysis of the total proteins from suspension and callus cultures of soybean by 1-D polyacrylamide gel electrophoresis showed one small 6BA-induced change in the proteins from the suspension cultures. In etiolated cucumber cotyledons: 1) 6BA caused the expansion of excised etiolated cucumber cotyledons after a 24h-hour incubation in the dark in a solution containing 6BA, in comparison to cotyledons incubated in water only. 2) The cotyledons curved upwards and in the light microscope the cells of the vascular bundles and the lower epidermis exhibited greater expansion than the upper epidermis. 3) Electron microscopic examination showed that the central vacuole of palisade cells from cotyledons treated with 6BA had expanded and that the cytoplasm had probably lost water and was compressed by the vacuole against the cell wall. 4) In contrast to other research, there was no apparent increase in polysome formation in 6BA-treated cotyledons in comparison to untreated cotyledons examined in the electron microscope. 5) A number of protein extraction methods were tried before a method was found which produced a protein extract suitable for both 1-D and 2-D polyacrylamide gel electrophoresis analyses. 6) 1-D and 2-D polyacrylamide gel electrophoresis showed that a number of proteins either increased or decreased following the treatment of cotyledons with 6BA. 7) A number of RNA extraction methods were tried, to obtain RNA suitable for translation in vitro. Only one method produced RNA which appeared to be free of contaminating substances. Weak translation of this RNA was obtained in vitro and it might be possible to develop conditions for optimal translation of the RNA given an adequate supply of an in vitro translation system.

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40

Kennedy, Martin A. "Transcriptional promoters in a replication region of F plasmid." Thesis, University of Auckland, 1986. http://hdl.handle.net/2292/2482.

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This thesis describes aspects of genetic regulation within and near a replication origin (ori-1) of the F plasmid. A number of transcriptional promoters were isolated, precisely mapped, and characterized with respect to their strengths and modes of regulation. The principal techniques employed in these investigations were: "shotgun" molecular cloning of restriction fragments into a galactokinase-based promoter selection vector, assays for galactokinase activities, DNA sequencing and S1 nuclease mapping of transcripts. Major findings from this study can be summarized as follows: 1). Promoters for the essential replication genes pifC and E were cloned and shown to be autoregulated at the transcriptional level. 2). An E.coli protein, integration host factor (IHF), was found to modulate the activity of the pif operon promoter. 13). Two promoters which direct transcription in opposite directions from within the minimal ori-1 region were discovered. 4). Transcription from both ori-1 promoters was shown to be repressed by the mini-F encoded D protein. 5). Precise transcriptional startsites of the pifC gene and the two ori-1 promoters were determined. 6). A mini-F protein (D) was shown to resolve dimers of a plasmid which contains a site-specific recombination locus from near ori-1, and a facile assay system for this function was developed.

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41

Simpson, Wayne Roydon. "A high frequency change, which is both inducible and reversible, results in altered colony morphology of a fungal symbiont (Neotyphodium lolii) and dwarfing of its grass host (Lolium perenne) : this thesis is presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Microbiology at Massey University, Palmerston North, New Zealand." Massey University, 2009. http://hdl.handle.net/10179/1139.

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Fungal endophytes of the genus Neotyphodium form stable symbiotic associations, with grasses, that are symptomless and generally considered to be mutualistic. The benefits that these fungi confer to their grass hosts are exploited in pastoral agriculture systems. The production of a range of secondary metabolites, specifically alkaloids including peramine and ergovaline can give their host plants an ecological advantage in certain environments. Neotyphodium endophytes are asexual and have lost the ability to transfer horizontally between hosts making seed transmission a vital feature of the association. This thesis reports the occurrence of phenotypically different perennial ryegrass plants (Lolium perenne) in a population infected with Neotyphodium lolii. Here we show that the change in the plants is directly attributable to a variant endophyte that they host. Isolation of the variant endophyte reveals a change in colony growth compared to the wild-type resident endophyte in the population, which has a white and cottony phenotype. Colonies of the variant endophyte are smaller than wild-type colonies and mucoid, with hyphal filaments forming aggregates. Evidence shows that the switch between colony morphologies occurs at a very high frequency, is reversible, and appears to be environmentally induced. This suggests that the switching phenomenon involves gene regulation rather than mutation. When endophyte-free plants are infected, with either white and cottony (wild-type) or mucoid (variant) fungal colonies, they assume a morphology consistent with the state of the fungus at the time of inoculation, that is normal or dwarfed, respectively. In addition, re-isolation of endophyte from either normal or dwarfed plants always yields white and cottony or mucoid colonies, respectively, suggesting that the host environment stabilizes the state of the fungus. Proteomic profiling revealed differences in protein expression between plants infected with either the wild-type or mucoid fungus. Furthermore, host plants containing the mucoid fungus have never flowered or produced seed. Thus, if this change in the fungal symbiont occurs in a competitive natural environment the mucoid fungus and its host plant may not persist beyond the first generation. This thesis provides insights into the plastic nature of fungal endophyte/grass symbiota and discusses possible mechanisms for the observed morphological switching in culture and host dwarfing.

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42

Pontin, David R. "Factors influencing the occurrence of stinging jellyfish (Physalia spp.) at New Zealand beaches." Lincoln University, 2009. http://hdl.handle.net/10182/1580.

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Individuals of the cnidarian genus Physalia are a common sight at New Zealand beaches and are the primary cause of jellyfish stings to beachgoers each year. The identity of the species and the environmental factors that determine its presence are unknown. Lack of knowledge of many marine species is not unusual, as pelagic invertebrates often lack detailed taxonomic descriptions as well as information about their dispersal mechanisms such that meaningful patterns of distribution and dispersal are almost impossible to determine. Molecular systematics has proven to be a powerful tool for species identification and for determining geographical distributions. However, other techniques are needed to indicate the causal mechanisms that may result in a particular species distribution. The aim of this study was to apply molecular techniques to the cnidarian genus Physalia to establish which species occur in coastal New Zealand, and to apply models to attempt to forecast its occurrence and infer some mechanisms of dispersal. Physalia specimens were collected from New Zealand, Australia and Hawaii and sequenced for Cytochrome c oxidase I (COI) and the Internal transcribed spacer 1 (ITS1). Three clans were found: a Pacific-wide clan, an Australasian clan and New Zealand endemic clan with a distribution confined to the Bay of Plenty and the East Coast of the North Island. Forecasting Physalia occurrence directly from presence data using artificial neural networks (ANN) proved unsuccessful and it was necessary to pre-process the presence data using a variable sliding window to reduce noise and improve accuracy. This modelling approach outperformed the time lagged based networks giving improved forecasts in both regions that were assessed. The ANN models were able to indicated significant trends in the data but would require more data at higher resolution to give more accurate forecasts of Physalia occurrence suitable for decision making on New Zealand beaches. To determine possible causal mechanisms of recorded occurrences and to identify possible origins of Physalia the presence and absence of Physalia on swimming beaches throughout the summer season was modelled using ANN and Naϊve Bayesian Classifier (NBC). Both models were trained on the same data consisting of oceanographic variables. The modelling carried out in this study detected two dynamic systems, which matched the distribution of the molecular clans. One system was centralised in the Bay of Plenty matching the New Zealand endemic clan. The other involved a dynamic system that encompassed four other regions on both coasts of the country that matched the distribution of the other clans. By combining the results it was possible to propose a framework for Physalia distribution including a mechanism that has driven clan divergence. Moreover, potential blooming areas that are notoriously hard to establish for jellyfish were hypothesised for further study and/or validation.

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43

Jankovic, Dragana. "Direct selection and phage display of the Lactobacillus rhamnosus HN001 secretome : a thesis presented to Massey University in partial fulfillment of the requirements for the degree of Doctor of Philosophy." Massey University, 2008. http://hdl.handle.net/10179/869.

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Bacteria communicate with their hosts in part via surface, secreted and transmembrane proteins (collectively the secretome) resulting in probiotic (beneficial) or pathogenic (harmful) outcomes to the host. Therapeutic benefits of probiotic bacteria have been shown previously, but the molecular mechanisms and the health-promoting effector components involved are still being elucidated. Some evidence suggests that probiotic bacteria can competitively adhere to intestinal mucus and displace pathogens. The adherence of probiotic bacteria to human intestinal mucus and cells appears to be mediated, at least in part, by secretome proteins. Secretome proteins-encoding open reading frames can be identified in bacterial genome sequences using bioinformatics. However, functional analysis of the translated secretome is possible only if many secretome proteins are expressed and purified individually. Phage display technology offers a very efficient way to purify and functionally characterise proteins by displaying them on the surface of the bacteriophage. While a phage display system for cloning secretome proteins has been previously reported it is not efficient for enrichment and display of Gram-positive secretome proteins. In this study a new phage display system has been developed and applied in direct selection, identification, expression and purification of Gram-positive Lactobacillus rhamnosus strain HN001 secretome proteins. The new phage display system is based on the requirement of a signal sequence for assembly of sarcosyl-resistant filamentous phage virions. Using this system 89 secretome open reading frames were identified from a library of only 106 clones, performing at least 20-fold more efficiently than the previously reported enrichment method. Seven of the identified secretome proteins are unique for L. rhamnosus HN001. A L. rhamnosus HN001 shot-gun phage display library was also constructed to capture proteins that mediate adhesion or aggregation, initial steps in establishing host-microbe contact or forming multicellular aggregates, both of which may lead to beneficial effects – colonisation of the gastro-intestinal tract and exclusion of pathogens. In search for proteins involved in adhesion, a L. rhamnosus HN001 shot-gun phage display library was screened against the human extracellular matrix component fibronectin commonly used as binding target by bacteria that colonise diverse tissues. This screen selected, instead of a fibronectin-binding protein, a protein that binds to avidin, used to immobilise biotinylated fibronectin. Affinity screening of the shot-gun library for binding to L. rhamnosus HN001 cells identified a secretome protein, Lrh33, as an HN001-cell surface binding protein. This protein contains two bacterial immunoglobulin-like domains type 3. Analysis of phage-displayed nested deletions of Lrh33 determined that the proximal (N-terminal) immunoglobulin-like domain is not sufficient for binding; only the constructs displaying both domains demonstrated binding to HN001. Lrh33 does not have any similarity to previously identified Lactobacillus-binding proteins and no match in the NCBI database (at a cutoff value of > e-13), hence it represents potentially a new type of bacterial auto-aggregation protein.

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44

Cooney, Terrence Patrick. "Studies on the biosynthesis of indole-3-acetic acid in tomato shoots." Thesis, University of Auckland, 1989. http://hdl.handle.net/2292/2071.

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The relative contributions of the three main intermediates of indole-3-acetic acid (IAA) biosynthesis from L-tryptophan (L-Trp); indole-3-pyruvate (IPyA), tryptamine (TNH2) and indole-3-acetaldoxime (IAOX), were investigated in vivo in tomato shoots. Initially, L-Trp, D-Trp, IPyA, TNH2 and IAA were purified from shoots, identified by full-scan mass spectrometry and their concentrations measured using gas chromatography with an electron capture detector. High specific activity [5-3H]IAOX and [5-3H]IPyA were synthesized from L-[5-3H]Trp and used as internal standards. Purification of endogenous IPyA was enabled by forming a stable pentafluorobenzyl oxime derivative in the crude plant extract. The respective endogenous concentrations of L-Trp, D-Trp, TNH2, IPyA and IAA were found to be 2,520, 103, 146.3, 5.9 and 8.5 ng g-1 f. wt. However, IAOX could not be identified as a natural constituent of tomato shoots by full-scan GC-MS. Secondly, incubation of tomato shoots for 6, 10 and 21 h in 30% 2H2O was used as a means of labelling IAA and its putative precursors in vivo. L-Trp, D-Trp, TNH2, IPyA and IAA were then extracted and purified and the 2H content measured by combined gas chromatography-mass spectrometry. These indole compounds were labelled rapidly with up to four 2H atoms. Direct comparison of the number and the amount of 2H atoms incorporated (pattern) was obtained from the mass spectral data on the common m/z 130 ion and its isotope peaks. IAA and L-Trp demonstrated an increase in 2H label with up to 17% and 21% of their molecules labelled at 10 h respectively. This was followed by a significant decrease in 2H label at 21 h to 12% for both L-Trp and IAA. This decrease in 2H label was attributed to an increase in protein catabolism, following shoot excision, resulting in the dilution of free L-Trp pool(s) with unlabelled L-Trp from which IAA is biosynthesized. This is reflected in the observed 1.6 to 1.8 fold increase of free L-Trp from 10 to 21 h. In contrast, tryptamine demonstrated a continual increase in 2H label with an average of 8, 20 and 28% of the molecules labelled at 6, 10 and 21 h respectively, suggesting that TNH2 and IAA were synthesized from separate Trp pools. In addition, the relatively slow rate at which 2H is incorporated into tryptamine would not be sufficient to account for the rate at which IAA becomes labelled. However, IPyA demonstrated a rapid increase in 2H with 22% and 37% of its molecules labelled at 6 and 10 h respectively. From the rate at which IPyA was labelled with 2H and the concentration of IPyA in tomato shoots a rate of synthesis for IPyA in tomato shoots was estimated which was sufficient to provide most of the shoot IAA requirements. Furthermore, the extent to which IAA and IPyA were labelled relative to that of total L-Trp would imply that a smaller more rapidly metabolised pool(s) of L-Trp was the precursor of these compounds. The rate and extent that D-Trp was labelled was consistently less than that of IAA precluding it as a possible precursor of IAA. These results indicate that in tomato shoots IAA is biosynthesized from a rapidly metabolized sub-pool(s) of L-trptophan predominantly via IPyA.

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45

Gallie, Jenna. "Evolutionary and molecular origins of a phenotypic switch in Pseudomonas fluorescens SBW25 : a thesis submitted in partial fulfilment of the requirements for the degree of Ph.D. in Evolutionary Genetics at Massey University, Auckland, New Zealand." Massey University, 2010. http://hdl.handle.net/10179/1215.

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Survival in the face of unpredictable environments is a challenge faced by all organisms. One solution is the evolution of mechanisms that cause stochastic switching between phenotypic states. Despite the wide range of switching strategies found in nature, their evolutionary origins and adaptive significance remain poorly understood. Recently in the Rainey laboratory, a long-term evolution experiment performed with populations of the bacterium Pseudomonas fluorescens SBW25 saw the de novo evolution of a phenotypic switching strategy. This provided an unprecedented opportunity to gain insight into the evolution and maintenance of switching strategies. The derived ‘switcher’ genotype was detected through colony level phenotypic dimorphism. Further microscopic examination revealed the cellular basis of phenotypic switching as the bistable (ON/OFF) expression of a capsule. Transposon mutagenesis demonstrated that the structural basis of the capsule was a colanic acid-like polymer encoded by the Pflu3656-wzb locus. Subsequently, whole genome re-sequencing enabled elucidation of the series of mutational events underlying the evolution of capsule bistability: nine mutations were identified in the switcher. Present in both forms of the switcher, the final mutation – a point mutation in a central metabolic pathway – was shown to be the sole mechanistic cause of capsule switching; it ‘set the stage’ for a series of molecular events directly responsible for bistability. Two models were proposed to explain capsule switching at the molecular level: the genetic amplification-reduction model, and the epigenetic feedback model. Collective results of biochemical and genetic assays proved consistent with the epigenetic model, whereby a decrease in flux through the pyrimidine biosynthetic pathway activates an already-present feedback loop. Subsequent analysis of a second switcher (evolved independently of and in parallel with the first) revealed a radically different genetic route leading to phenotypically and mechanistically similar capsule switching. In addition to providing the first empirical insight into the evolutionary bases of switching strategies, the work presented in this thesis demonstrates the power of natural selection – operating on even the simplest of organisms – to forge adaptive solutions to evolutionary challenges; in a single evolutionary step, selection took advantage of inherent intracellular stochasticity to generate an extraordinarily flexible phenotype.

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46

Liu, Yunhao. "Structural and biochemical analysis of HutD from Pseudomonas fluorescens SBW25 : a thesis submitted in fulfilment of the requirements for the degree of Master of Science in Molecular Biosciences at Massey University, Auckland, New Zealand." Massey University, 2009. http://hdl.handle.net/10179/1074.

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Pseudomonas fluorescens SBW25 is a gram-negative soil bacterium capable of growing on histidine as the sole source of carbon and nitrogen. Expression of histidine utilization (hut) genes is controlled by the HutC repressor with urocanate, the first intermediate of the histidine degradation pathway, as the direct inducer. Recent genome sequencing of P. fluorescens SBW25 revealed the presence of hutD in the hut locus, which encodes a highly conserved hypothetical protein. Previous genetic analysis showed that hutD is involved in hut regulation, in such a way that it prevents overproduction of the hut enzymes. Deletion of hutD resulted in a slow growth phenotype in minimal medium with histidine as the sole carbon and nitrogen source. While the genetic evidence supporting a role of hutD in hut regulation is strong, nothing is known of the mechanism of HutD action. Here I have cloned and expressed the P. fluorescens SBW25 hutD in E. coli. Purified HutD was subjected to chemical and structural analysis. Analytic size-exclusion chromatography indicated that HutD forms a dimer in the elution buffer. The crystal structure of HutD was solved at 1.80 Å (R = 19.3% and Rfree = 22.3%) by using molecular replacement based on HutD from P. aeruginosa PAO1. P. fluorescens SBW25 HutD has two molecules in an asymmetric unit and each monomer consists of one subdomain and two ß-barrel domains. Comparative structural analysis revealed a conserved binding pocket. The interaction of formate with a highly conserved residue Arg61 via salt-bridges in the pocket suggests HutD binds to small molecules with carboxylic group(s) such as histidine, urocanate or formyl-glutamate. The hypothesis that HutD functions via binding to urocanate, the hut inducer, was tested. Experiments using a thermal shift assay and isothermal titration calorimetry (ITC) analysis suggested that HutD binds to urocanate but not to histidine. However, the signal of HutD-urocanate binding was very weak and detected only at high urocanate concentration (53.23 mM), which is not physiologically relevant. The current data thus does not support the hypothesis of HutD-urocanate binding in vivo. Although the HutD-urocanate binding was not confirmed, this work has laid a solid foundation for further testing of the many alternative hypotheses regarding HutD function.

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47

Gregory, Samuel James. "Investigation into the relationship between aluminium treatment and the superoxide dismutase (SOD) enzyme system in Lolium perenne (L. perenne cv. Nui) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (with Honours) in Plant Biology at Massey University." Massey University, 2009. http://hdl.handle.net/10179/1216.

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Lolium perenne cv. Nui is a cultivar of ryegrass grown throughout New Zealand in pastures due to favourable traits such as high palatability for livestock and its ability to withstand intensive grazing. However, the productivity of pastures is reduced when levels of aluminium and other metals accumulate in soils to toxic levels, a phenomenon referred to as the ‘acid soil syndrome’. In response to this toxicity, plants activate a series of antioxidant reactions, with one catalysed by the superoxide dismutase (SOD) enzymatic system. The enzyme system comprises three isoenzymes, a Cu/ZnSOD, FeSOD and a MnSOD which catalyse the same reaction but differ in amino acid sequence, molecular mass and the metal ion co-factor (hence Cu/ZnSOD, FeSOD and MnSOD). Together these isoenzymes combat the damaging effect of superoxide radicals which accumulate due to metal toxicity. In this thesis, the isolation of genes encoding isoenzymes of the SOD enzyme from L. perenne cv. Nui is described. As well, the growth of L. perenne cv. Nui and changes in expression of the SOD genes encoding each isoenzyme in response to aluminium treatment (0.2mM AlCl3) is investigated. A 1072 bp FeSOD gene sequence and a 705 bp MnSOD gene sequence were isolated from shoot tissue of L. perenne cv. Nui using a combination of RT-PCR with degenerate primers and 3'-RACE. The FeSOD gene comprised 572 bp of the coding sequence and 500 bp of 3'-UTR while the MnSOD gene comprised 508 bp of coding sequence and a 197 bp 3'-UTR. By alignment of each sequence with the gene from the database with highest identity it was predicted that the translation start codon (ATG) is located a further 196 bp upstream for the FeSOD gene (aligned with an Oryza sativa FeSOD sequence as a reference) and a further 152 bp upstream for the MnSOD sequence (aligned with a Triticum aestivum MnSOD sequence as a reference). Using RT-PCR with degenerate primers, a 313 bp CuSOD sequence was predominantly cloned from shoot tissue of L. perenne cv. Nui, but it was not possible to generate the 3'-UTR using 3'-RACE. For growth analysis, seedlings of L. perenne cv. Nui were germinated and acclimatised in Hoagland’s solution, and then subjected to either aluminium treatment (0.2mM AlCl3) or no treatment to act as a control over a designated time course of 0, 4, 8, or 24 hours. Two growth trials were conducted that differed in the age of seedlings used and plant tissues were separated into root and shoot tissues. Similar growth trends were observed in both trials, but the sampling regime in the second growth trial meant that statistical analysis could be carried out. In this trial, analysis revealed that over a time course of 24 hours exposure to 0.2mM aluminium, both root and shoot tissue fresh weight did not significantly differ when compared to the control (no aluminium). A general trend of an increase in root and shoot fresh weight was observed in plants treated with aluminium, but this trend was not significant at P=0.05. No significant change in fresh weight partitioning from shoot to root, or root to shoot in response to aluminium was also observed. Using semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqRTPCR) and primers based around the 3'-UTR with RNA isolated from plants grown in the second hydroponic trial, it was determined that under the conditions used, expression of the FeSOD and MnSOD genes isolated in this study were neither up-regulated or downregulated in response to aluminium treatment in both shoot and root tissue. Further, using degenerate primers to detect expression of one or more genes encoding the Cu/ZnSOD isoenzyme, total expression of the Cu/ZnSOD isoenzyme was also unresponsive to aluminium treatment.

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48

Campbell, Kirsten L. "A study of home ranges, movements, diet and habitat use of kereru (Hemiphaga novaeseelandiae) in the southeastern sector of Banks Peninsula, New Zealand." Master's thesis, Lincoln University. Bio-Protection and Ecology Division, 2006. http://theses.lincoln.ac.nz/public/adt-NZLIU20080317.131118/.

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The present study is part of the Kaupapa Kereru Programme. The main aim of the programme is to increase the numbers and range of kereru (Hemiphaga novaeseelandiae) on Banks Peninsula. Home ranges, movements, diet and habitat use of 15 kereru captured in Hinewai Reserve, Banks Peninsula, were investigated from February 2005 to February 2006. Hinewai Reserve is the largest tract of regenerating native forest in a highly modified urban-rural landscape. Phenology of 11 plant species predicted to be key kereru foods, was studied to determine the pattern of food availability in Hinewai Reserve. Twelve radio-tagged kereru resided in the Hinewai Reserve study site (Otanerito Valley and Sleepy Bay) and three resided in Akaroa. Ripe fruit was available from January to August; the height of the fruiting season was in autumn. The bulk of new leaf growth occurred in spring and early summer although new leaves were available on broom and tree lucerne year round. Peak flowering occurred in spring. Kereru in Akaroa ate a total of 21 plant species; six of these species were native and 15 introduced. Kereru in the Hinewai Reserve study site ate a total of 26 plant species; 20 of these species were native and six introduced. Fruit was preferred when readily available. Native fruit appeared to be preferred over fruit of introduced species in Akaroa, where both types were available. New foliage of introduced legumes and deciduous species appeared to be preferred over new foliage of native species at both sites during winter and spring. These species were important food sources prior to the breeding season and may be selected specifically for their nitrogen and protein content. Food is currently not a limiting factor for kereru survival or reproductive success. Considerable variation in the use and preference of vegetation types of individual kereru made it difficult to identify trends in habitat selection. Use and preference for many vegetation types was seasonal; this was certainly because of the availability of food species included in or close to these vegetation types. Overall, native vegetation communities were used more than communities dominated by introduced species and forest communities were used more than non-forest communities. Kanuka (Kunzea ericoides) was used most often for non-feeding activities and 67% of observed nests were built in kanuka. Annual home ranges and core areas in the Hinewai Reserve study site (mean of 15.9 and 2 ha respectively) were significantly larger than those found in Lyttelton Harbour, Banks Peninsula in previous research (mean of 8 and 0.08 ha respectively). Home ranges were larger when fruit was eaten, than when no fruit was eaten indicating that kereru are more sedentary when feeding on foliage. Kereru from the Hinewai Reserve study site made no excursions >5 km and no daily movements >2 km. Kereru from Akaroa and Sleepy Bay travelled into Otanerito Valley to feed on horopito in autumn, indicating that there may have been a lack of fruit in their local areas during autumn. No kereru in Otanerito Valley travelled outside of the valley. The distribution of high quality food sources is likely to have caused the observed differences in home range and core area size between localities. Kereru in Lyttelton Harbour may have been restricted to small patches of high quality resources in a study area consisting largely of unsuitable habitat. In Hinewai Reserve, high quality resources were spread over larger areas and were more uniformly distributed. The density of kereru was unknown at both study sites, and this confounded assessment of habitat quality. However, it is likely that the Hinewai Reserve study site would support a higher number of kereru. The main factor limiting population growth in the present study was failure of nests at the egg and chick stage. The fledge rate was 17%. Two of fifteen adult kereru died. Control of predators should be the first aspect of management that is focused on, and will almost certainly increase reproductive success of kereru and loss of breeding adults. As the population of kereru on Banks Peninsula increases due to predator control in existing kereru habitat, food may become a limiting factor. Habitat can be improved for kereru by planting a diverse range of plant species that provide food year-round. Native fruiting species are greatly recommended for habitat enhancement and should be selected so that fruit is available for as much of the year as possible. Native and introduced legumes should also be made available as foods for winter and spring. As most land on Banks Peninsula is privately owned, co-operation and enthusiasm of the community is critical for successful management. Information and support needs to be given to landowners wishing to enhance their properties for kereru.

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49

Roldan, Marissa B. "Expression of ACC oxidase genes in white clover (Trifolium repens L.) roots in response to phosphate supply : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Molecular Biology at Massey University, Palmerston North, New Zealand." Massey University, 2008. http://hdl.handle.net/10179/1133.

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The differential expression of members of the Trifolium repens ACC oxidase (TR-ACO) gene family and accumulation of TR-ACO proteins in white clover roots, and the temporal TR-ACO gene expression and TR-ACO protein accumulation in response to phosphate (Pi) stress has been investigated. Four-node stolon cuttings of wild type and transgenic white clover (designated TR-ACOp::GUS and TR-ACO1p::mGFP5-ER) plants were rooted and acclimatised in Hoagland’s solution, and then subjected to either a Pi sufficiency (1 mM Pi) treatment or a Pi depletion (10 µM Pi) treatment over a designated time course. Using semi quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqRT-PCR) and gene-specific primers it has been determined that the TR-ACO genes are differentially expressed in the roots of white clover. The TR-ACO1 transcript abundance was greater in the lateral roots when compared to the main roots. By immunodetection analysis using antibodies raised against TR-ACO1, recognition of a protein of expected size (ca. 36 kDa) was also greater in the lateral roots. The tissue-specific localisation of TR-ACO1 promoter activity was investigated first by light microscopy using a single genetic line of white clover transformed with a TR-ACO1p::GUS gene construct, and results then confirmed by confocal microscopy using several genetically independent lines of transgenic plants transformed with a TR-ACO1p::mGFP5 ER gene construct. In these lines, the TR-ACO1 promoter activity was primarily located in the meristem of the main and lateral roots, lateral root primordia as well as in the pericycle of the root with nodes of expression in the emerging lateral roots, suggesting a role for ethylene in the development of young tissues where cells are actively dividing. In terms of TR-ACO2, greater transcript abundance and protein accumulation of TR-ACO2 were also observed in the lateral roots when compared to the main roots. Histochemical GUS staining of roots of a single genetically-independent line transformed with a TR-ACO2p::GUS construct showed predominant promoter activity in the mature tissues of both the main and lateral roots but not in the meristematic tissues. In contrast, TR-ACO3 showed greater transcript abundance in the main roots relative to the lateral roots, and the promoter activity, as determined using a single genetically- independent line of TR-ACO3p::GUS transformed plants was predominantly in the mature tissues of the main roots In response to Pi depletion, the members of TR-ACO gene family were temporally expressed in the white clover roots. Using sqRT-PCR, the TR-ACO1 transcript abundance was greater in Pi depleted roots at 12 h and 24 h after Pi depletion in both wild type plants and in the one genetically-independent line of white clover transformed with the TR-ACO1p::mGFP5-ER construct examined. Similarly, by western analysis using both a-TR-ACO1 and commercially available a-GFP antibodies (for the transformed line), a greater accumulation of proteins was consistently observed in Pi depleted roots from the first up to the seventh day after Pi depletion. By confocal microscopy, it was determined for several genetically-independent line of white clover transformed with TR-ACO1p::mGFP5-ER that under Pi depletion more intense GFP fluorescence over a time course of 1 d, 4 d, and 7 d was observed, when compared to plants grown under Pi sufficiency. For TR-ACO2, there was no significant difference in transcript accumulation and protein accumulation in response to short term Pi depletion of up to seven days. However, at 15 d and 21 d after Pi depletion there was a greater protein accumulation in the roots of Pi depleted plants relative to the Pi sufficient roots. Further, when main and lateral roots were compared, a greater protein accumulation occurred in the lateral roots. For TR-ACO3, there was no consistent trend of transcript accumulation in response to Pi depletion over a 24 h period. While a marked reduction in transcript accumulation was noted in Pi depleted roots at 1h, 12 h, 24 h, there was an increase in transcript accumulation at 6 h and 18 h after Pi depletion, indicating that factors other than Pi supply may be affecting gene regulation. Root morphological studies revealed an increase in the main root length and lateral root production in white clover in response to Pi depletion with a greatest growth rate noted between the sixth and ninth day after Pi depletion, and this period overlapped with accumulation of TR-ACO1 protein suggesting a role for ethylene in the Pi stress induced lateral root production in white clover. The differential regulation of the three TR-ACO genes in white clover roots in response to Pi depletion further suggests the divergence in terms of regulation of the ethylene biosynthetic pathway, which may play an important role in fine tuning the responses of plants to particular environmental cues.

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50

Albert, Nick William. "Two novel MYB transcriptional activators regulate floral and vegetative anthocyanin pigmentation patterns in Petunia : [a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Molecular Biology at Massey University, Palmerston North, New Zealand] EMBARGOED UNTIL 12 MARCH 2012." Massey University, 2009. http://hdl.handle.net/10179/1347.

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Mr Albert investigated the genetic mechanisms controlling complex floral and vegetative pigmentation patterns in Petunia. He discovered two new MYB transcription factors that control the timing and spatial location of anthocyanin pigment production in flowers and leaves, giving rise to specific colour patterns. He showed that complex pigmentation patterns are formed by tightly controlling the expression of genes required to synthesise anthocyanin pigments and involves proteins that both activate genes and repress them from being expressed. The interactions between distinct classes of transcription factors form an intricate network and hierarchy, allowing fine control of gene expression and strict control of pigment production. These findings will aid in the development of ornamental plants with new pigmentation patterns and also this research serves as a model for how plants control the expression of genes to produce health-promoting plant compounds.

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Dissertations / Theses on the topic 'Fields of Research – 270000 Biological Sciences'

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