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Item Regulation of paxilline biosynthesis in Penicillium paxilli : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2000) Telfer, Emily JaneProduction of the indole-diterpenoid paxilline was examined in the filamentous ascomycete Penicillium paxilli. Paxilline is a secondary metabolite, that is synthesised via a specific secondary metabolite biosynthetic pathway. The primary precursors of paxilline biosynthesis, mevalonate and isopentenyl pyrophosphate, are synthesised via the isoprenoid pathway and the paxilline biosynthetic pathway branches from isoprenoid biosynthesis after the synthesis of farnesyl pyrophosphate. The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co) reductase is the rate limiting step of the isoprenoid biosynthesis. Genes for (hmg) and β-tubulin (tub-2), were isolated from a genomic DNA libray and characterised by DNA sequencing and RT-PCR. The steady state mRNA levels of hmg and tub-2 were compared with genes isolated from the paxilline biosynthetic gene cluster, using a semi-quantitative RT-PCR gene expression assay. A distinct pattern of expression was identified for genes involved in the biosynthesis of paxilline. Increased expression of these genes occurs 36 h prior to the detection of paxilline in liquid culture. P. paxilli physiology and paxilline production was analysed in liquid culture after the development of reproducible growth conditions that results in the formation of homogeneous loose hyphal fragments and detectable paxilline after 72 h. The morphology of P. paxilli in paxilline-inducing media was examined microscopically and key physiological markers, culture pH and biomass accumulation, were also analysed. Paxilline levels in both mycelia and culture supernatant were analysed with HPLC and TLC. This confirmed that paxilline is not released into the media until 144 h when large scale autolysis is observed. Initial experiments to examine paxilline production in cultures supplemented with a biological buffer suggest that phase switching between primary growth and secondary growth may be triggered by changes in ambient pH. The presence of alternative carbon sources also affected the rate of paxilline production and preliminary results indicate that biosynthesis of paxilline may be under carbon catabolite repression by glucose. Levels of HMG CoA reductase are known to be regulated at many levels, including mRNA transcription, protein inactivation and protein degradation, in response to excess sterols. A number of putative sterol response elements (SRE), which control transcription of hmg in higher eukaryotes, where identified in the 5' UTR of hmg from P. paxilli. In higher eukaryotes, the extremely complex 5' UTR of hmg has been proposed as the site of regulation for biosynthesis of non-sterol end-products. This complexity appears to be conserved in the 5' UTR of hmg from P. paxilli and another filamentous fungus Neotyphodium lolii Lpl9. Intronic sequences are spliced from the 5' UTR of both genes and there are additional intronic sequences present that could produce alternative transcripts. At least two different hmg transcripts were identified from P. paxilli with 5' RACE. The mechanism by which these alternative transcripts arise is unclear at present, but could involve alternative splicing of the 5' UTR intron or initiation of transcription from alternative start sites.Item Linkages between hyporheic and benthic invertebrate communities in New Zealand gravel bed rivers : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology at Massey University(Massey University, 2000) Fowler, Reece TrevorSurber samples from the benthos and T-bar samples (14 mm PVC pipe diameter) from the hyporheos were collected from 8 Southern Hawkes Bay rivers, differing in environmental conditions and geological age, and 8 rivers of the Cass/Craigieburn region of Canterbury that had differing physical stability and hyporheic physicochemical characteristics. Gravel bedded rivers of the Southern Hawkes Bay (North Island) and Cass/Craigieburn region (South Island) were sampled to examine the spatial distribution of the hyporheos in relation to physicochemical factors, bed stability and the significance of the hyporheic zone as a refuge and/or source of invertebrate colonists to the benthos. Physical bed movement, the duration of channel dewatering and flow variance were used to measure disturbance to the hyporheos and benthos, respectively. Benthic invertebrate communities of many streams are subject to constant disturbance from fluctuations in flow, temperature, and bed movement. In contrast, the hyporheos residing in the hyporheic zone may not experience the same disturbance regimes because bed movement occurs less often in the hyporheic zone and there is less diel temperature, dissolved oxygen and flow fluctuations. In the rivers investigated, broad (between river) and finer (within river) scale environmental factors influenced the composition of the hyporheos. Local geomorphology appeared to have a significant effect on the hyporheos by markedly increasing the proportion of epigean animals (benthic animals resident in the hyporheos) at sites of young geomorphology (less that 0.05 mya). In contrast, hypogean animals (animals seldom encountered from the benthos) dominated geologically older sites (greater than 0.4 mya). Species diversity in the hyporheic zone was highest with intermediate levels of bed movement where both hypogean and epigean animals were present. The greater abundance of epigean fauna with increasing bed movement may reflect differences in disturbance regime between sites and in turn interstitial flow rates. However, it is unlikely that the geological age and stability of bed substrates determined community composition alone. Water chemistry was also an important factor determining community composition; epigean animals were more abundant in the hyporheic zone when hyporheic water chemistry was similar to the surface river water (e.g., higher dissolved oxygen and lower conductivity). In contrast, hypogean animals dominated the hyporheos when water chemistry was least like the surface water (e.g., lower dissolved oxygen and higher conductivity). Dewatering and bed movement may disturb the hyporheos by desiccating or physically disturbing the hyporheic zone, respectively. If the potential colonists of the hyporheic zone are removed by desiccation or physical abrasion, the types of invertebrate taxa colonising will change because some invertebrates may be less capable of colonising from this region. The proportion of epigean animals may increase in the hyporheic zone with greater bed movement, but decline during stable conditions or at stable sites because the interstitial environment becomes less like the surface. Thus, the hyporheic zone may harbour benthic invertebrates during disturbance events and may be an important source of colonists for the benthos in frequently disturbed sites. In contrast, during stable conditions, or at stable sites, factors other than disturbance (e.g., substrate compactness, organic matter supply, competition or surface/groundwater interactions) may be more important in determining community composition. The diverse hyporheos identified in New Zealand rivers may be influenced by substrate composition, which may in turn alter interstitial flow patterns, discharge variance and the probability of bed movement.Item Homogeneous and heterogeneous enzymeimmunoassays for the home detection of fertility : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Biochemistry at Massey University(Massey University, 2000) Cooke, Delwyn GayThe physiology of the menstrual cycle has been reviewed and the suitability of the ovarian steroid urinary metabolites estrone glucuronide and pregnanediol glucuronide as markers of fertility and as utilised by the home Ovarian Monitor fertility assay was discussed. The biomaterials for the homogeneous enzymeimmunoassay which forms the basis of the Ovarian Monitor home fertility assay were prepared. One of the major difficulties in preparing a signal generator for use in home tests is the separation of the unconjugated enzyme material from the desired signal generators. In this thesis a new procedure was developed for the purification and isolation of the complex range of signal generators formed during acylation of hen egg white lysozyme with estrone glucuronide. A cation exchange column in the presence of 7 M urea allowed the separation to be carried out in the absence of the hydrophobic effects which complicate other schemes. Even under these conditions complex behaviour was seen which could be rationalised in terms of the tertiary structures of the conjugates and their electrostatic fields. A second step involving hydrophobic interaction chromatography gave two pure mono conjugated estrone glucuronide lysozyme conjugates in good yield the activities of which were highly inhibited (>90%) by anti-estrone glucuronide antibodies. The availability of the pure conjugates allowed the effect of tertiary structure on the immunoassays to be evaluated. The results showed that both mono acylated hen egg white lysozymes could be used to give good standard curves for use in monitoring menstrual cycles for the naturally occurring periods of fertility and infertility. Since the specific activity of human lysozyme is three times that of hen egg white lysozyme, in an attempt to provide a more rapid test for fertility human lysozyme estrone glucuronide conjugates were synthesised with estrone glucuronide for the first time. However, despite the fact that these two enzymes had very similar tertiary structures they behaved completely differently in the protein chemistry and immunological experiments reported in this thesis. The human enzyme was more easily acylated to give pure mono acylated conjugates in high yield and the conjugates were more easily purified to give highly inhibitable conjugates (>95%). The differences in behaviour could be accounted for in terms of the sequence differences between the two lysozymes and the relative exposure of the lysine residues. A fast assay (1-2 minutes) was developed for urinary estrone glucuronide using the three new signal generators. However, the sensitivity of the assays was less than half that of the hen egg white conjugates making them unsuitable for use in home assays for fertility. The assays could be useful for women using fertility drugs such as clomiphene. The lack of sensitivity of the assays and other binding behaviour indicated a much tighter binding to the antibody than with the hen egg white conjugates. This important difference was accountable on the basis of the extra extension of the lysine residues in the human enzyme. A new method for producing estrone glucuronide conjugates of the active enzyme horse radish peroxidase was evaluated. The mono substituted hemin conjugates reconstituted with the apo protein to give active peroxidases with good specific activities (~50%) and good stability. The procedure is such that any small molecule can be attached to the enzyme using the same procedures and a large range of signal generators can be formed for immunoassays. However, both assay formats examined failed to produce an assay in this thesis. The reconstituted enzymes, although binding to the immobilised anti- estrone glucuronide antibodies as required did not produce the necessary colours. The reasons for this were discussed.Item Breeding systems and rarity in New Zealand Myosotis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology at Massey University, Palmerston North, New Zealand(Massey University, 2001) Brandon, Andrea MaryRarity in New Zealand Myosotis was explored at several different levels for this thesis. Within the genus, there are many rare species, encompassing several different types of rarity, and many of these are considered threatened in some way. In addition, the genus contains species that exhibit a wide array of floral forms, each of which can be associated with a distinct form of rarity. The reproductive biology of selected Nelson taxa was investigated in several species covering a range of floral morphologies, including the previously unstudied brush blossom floral syndrome. Species were found to fall into one of two mating types based on the degree of herkogamy A glossary of technical terms follows the appendices exhibited by the flowers. Species with flowers that are herkogamous throughout their life, require pollinators to set seed while those that are not herkogamous at some stage during anther dehiscence, are self-fertile and able to self-pollinate autonomously. All species studied were self-compatible. Considerable variation in seed production was observed under natural conditions for those species that required pollinators to set seed. For these pollinator-requiring species, local population density had strong effects on seed set, while population size had no effect. These plants were always pollen limited in low-density patches. For a self-fertile species, seed set was always high and unaffected by either local density or population size and pollen was never limiting. These results indicate reproductive success in pollinator-requiring species of Myosotis is subject to Allee effects and these effects occur at a very local scale. These results have implications for the assessment and management of threatened Myosotis species in particular and rare plants generally. It is essential to know the pollination requirements and levels of density dependence for reproduction both for the assessment of threat and for determining management strategies for threatened plants. Self-compatible, but pollinator-requiring species are prone to autogamy and geitonogamy and therefore inbreeding depression. One strategy to avoid this is to increase dichogamy and synchronise inflorescence development. Pollination was studied in the geographically restricted, ultramafic endemic Myosotis monroi, a species that has a relatively large floral display and whose flowers often bear precocious styles prior to buds opening. In general, precocious styles are assumed to be receptive and indicate protogyny. Stigmas were receptive at this stage and some pollination occurs during this precocious phase. Stylar precocity effectively lengthens the female-only phase in this species. This is the first time that precocious styles have been proved to be receptive at this stage. M. monroi also shows far greater phenological synchrony of within plant flowering than five other species of New Zealand Myosotis. It is thought that the impact of a large floral display on levels of geitonogamy in M. monroi is alleviated to some degree by the relatively long, initial female-only phase and phenological synchrony of flowering stages. Rarity is generally considered "the precursor to extinction" (Darwin, 1872). However, there are several different forms of rarity and not all rare species are threatened. I examined the different rarity patterns observed within New Zealand Myosotis. New Zealand Myosotis species are never common, all are rare in different ways and some species are threatened. Some species are known to occur at one locality where they may be locally common, others may occur in two or more widely disjunct geographic areas, while others may be widespread but never common where they occur. A comparative analysis was carried out to determine whether there are morphological correlates of the rarity patterns seen within the genus. Local abundance, population disjunctions and distribution patterns of 33 Myosotis taxa were compared to aspects of their morphologies, including traits related to mating systems, dispersal ability and life-histories. Taxa requiring pollinators to set seed had smaller range sizes and higher local population densities than those that were able to self-pollinate. Apparent adaptations for dispersal and life-history traits were not correlated with range size. The disjunct distributions exhibited by some taxa within the genus were not associated with any of the morphological traits. My results can assist threat assessment and conservation management for New Zealand Myosotis. Locally dense, geographically restricted taxa are pollinator requiring while sparse, widespread taxa are selfing. Cases that break this general rule may be used to identify taxa at risk. In the most recent taxonomic treatment, New Zealand Myosotis is initially split into two major clades depending the degree of anther exertion exhibited by the flowers. This has led to the recognition of species that cannot be distinguished in any other way. Species limits were examined in one such species complex; the vegetatively indistinguishable M. forsteri and M. venosa. Several floral and vegetative characters were measured and compared. Filament length was the only significant character that could be used to distinguish the species, and even this was not 100% reliable. Filament length determines the degree of self-pollination that can occur and whether or not reproduction is assured. The continuous variation observed in filament, style and anther lengths and corolla sizes shows that there is a lot of phenotypic variation within each taxon. Geographic clines are observed in vegetative traits that are independent of mating system. This, in addition to the continuous variation observed in floral characters, lends some strength to the proposition that this species pair may be switching between the two breeding systems. The increased knowledge provided by the studies contained in this thesis on New Zealand Myosotis has provided a much-needed boost to our understanding of the population dynamics of these rare species, which ultimately can be used to guide conservation management for those taxa considered at risk of extinction. It can assist in identifying those populations that are not threatened and it can direct efforts towards the more pressing problem situations. It has also highlighted the necessity for a taxonomic revision for the southern hemisphere section of the genus.Item Evolution of the New Zealand alpine flora : origins, diversification and dispersal : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Biology and Biotechnology at Massey University, Palmerston North, New Zealand(Massey University, 2000) Winkworth, Richard CharlesThis thesis describes molecular systematic studies that test hypotheses concerning the age and origins of the New Zealand alpine flora. Analyses of nuclear and chloroplast DNA markers for two plant groups that have radiated extensively in the alpine zone of New Zealand - the genus Myosotis (Boraginaceae) and the Australasian apioid Umbelliferae - are reported. The molecular results suggest that the diversification of these groups in New Zealand has occurred since the late Tertiary. This finding corroborates recent molecular systematic studies on other New Zealand plant groups. Taken together, these studies suggest that New Zealand's modern floristic diversity was greatly influenced by dramatic global climate change during the late Tertiary and Quaternary. These results are also consistent with those reported for plant groups overseas. In these, recent diversification has occurred with Quaternary climatic fluctuations (Comes & Kadereit, 1998) and colonisation of insular environments (Crawford & Stuessy, 1997) The molecular analyses also suggest that since the late Tertiary, Myosotis and the Australasian Apioideae have been involved in transoceanic dispersal events both to and from New Zealand. However, while most other molecular studies have provided evidence for the importance of circum-polar westerly winds, the present data suggests that, for Myosotis and with less confidence the apioid genera, some dispersal events have been in a westerly direction. Since this finding was made, late Tertiary and Quaternary westward dispersal has also been inferred for other New Zealand alpine plants. These observations suggest that passive eastward wind dispersal can not explain the distributions of all southern Pacific plant groups. The present study, as well as other recent studies, has highlighted the need to develop molecular tools and analytical approaches for describing the potentially complex evolutionary relationships between taxa that have originated since the late Tertiary. Here, the amplified fragment length polymorphism (AFLP) method was investigated as a means of identifying fast evolving genome regions in New Zealand Myosotis. Preliminary analyses suggest that this is a useful approach for locating highly variable molecular markers. However, like other rapidly evolving regions (e.g. Buckler et al., 1997) some of the derived markers were multiple copy and polymorphic at different loci within a single genome. This feature of fast evolving genome regions is problematical since bifurcating evolutionary analysis models will poorly represent such complex data. For this reason split-decomposition was investigated as an alternative method for data representation. This approach was found to have both advantages and limitations for studying late Tertiary or Quaternary radiations.Item An investigation of the factors regulating house mouse (Mus musculus) and ship rat (Rattus rattus) population dynamics in forest ecosystems at Lake Waikaremoana, New Zealand : a thesis presented in fulfilment of the requirements of the degree of Doctor of Philosophy in Ecology at Massey University, Palmerston North, New Zealand(Massey University, 2000) Blackwell, Grant LeonardFactors regulating the eruptive population dynamics of house mice, Mus musculus, and ship rats, Rattus rattus, were investigated over 29 months in mixed forest at Lake Waikaremoana, New Zealand. Mice and rats are generally present at low density, but erupt periodically following synchronous southern beech (Nothofagus spp.) seeding. A range of factors proposed as important in shaping the population dynamics of these species was investigated. These included rodent diet and habitat use, and the roles of food availability and predation pressure. Changes in rodent population dynamics were investigated using three relative density estimates: footprint tracking tunnels; and two kill trapping indices. Tracking tunnels gave reliable density estimates, but were influenced by sampling effort and habitat type. Rats had an opportunistic, omnivorous diet, and had no measurable detrimental effects of stomach parasite infection. Rats were generally more common in forest with the most food, but became equally abundant in all areas following widespread synchronous tree seeding. Rats were more numerous in areas with predators removed. Mice were found in all areas following Nothofagus seeding. Mice became scarce as food levels dropped, suggesting that the forest habitat does not contain enough food to support them in most years. The roles of food limitation and predation in shaping rodent population dynamics were investigated initially by computer modelling. The model showed that predators could not prevent a rodent population eruption, nor limit peak prey-population density. However, predation may be important during the decline and low phases of the eruption. The predictions of the model were tested in a large-scale field experiment. Predators were removed from a 750 ha peninsula in the study area. Rodent population dynamics during an eruption were compared in large areas with and without predators present. Predators did not prevent a prey eruption or limit peak population size as predicted by the computer model. There was evidence that predators limited prey populations during the post-eruption low phase, but the role of predation during the rodent decline remains unclear. Thus, the eruptive population dynamics of mice and rats in forest ecosystems in New Zealand are driven primarily by spatial and temporal variation in food supply, with predation by a single common predator potentially important during the crash and low phases following a population eruption.Item Molecular genetic analysis of plant Mei2-like genes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand(Massey University, 2002) Alvarez, Nena de GuzmanMolecular and genetic methods were used to analyse how a novel class of genes, plant Mei2-like genes may be involved in the regulation of morphogenesis in plants. The study specifically aimed to 1) further characterise maize te1 (the first plant Mei2-like gene to be genetically analysed) and understand the morphological basis of the te1 mutant phenotype and 2) analyse the function of Arabidopsis Terminal Ear Like (TEL) genes using expression analyses and reverse genetics strategy. te1 maize mutants are initially characterised by abnormal phytomer formation and development. A more detailed morphological analysis shows that mutant plants 1) have smaller vegetative shoot apices than the wild type, 2) initiate leaves at a higher, more distal position on the apical dome and 3) have higher plastochron ratio. Molecular analyses of kn1 expression pattern, a marker of leaf founder identity, show that dowregulation of kn1 transcripts occur higher up the dome. Clonal analyses show that fewer number of leaf founder cells are recruited to form the leaf. TEL1 and TEL2 are expressed in distinct overlapping domains in the undifferentiated region of the shoot apical meristems during the embryo, vegetative and reproductive stages of Arabidopsis development suggesting involvement of these genes in regulating meristem development and subsequent maintenance. The distinct expression of TEL1 in both the embryonic SAM and RAM raises the possibility of a unifying regulatory mechanism in the formation of the root and the shoot. The absence of TEL single knockout phenotypes supports the idea of functional redundancy. When the TEL genes were both knocked out, double mutant phenotypes show apical-basal pattern defects, ectopic production of numerous secondary shoots, production of numerous leaves and basic embryonic pattern defects such as deletions of apical and/or basal region of the seedling. Results of this study support the hypothesis that plant Mei2-like genes are important in regulating morphogenesis in plants and that they are required in the early patterning of the basic plant body.Item Regulation of apoptosis in neural cells : two methods for overcoming asynchrony : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 2000) François, FleurProgrammed cell death, or apoptosis, plays a major role in the development of the nervous system and in the pathogenesis of neurodegenerative diseases. Although many proteins that play a key role in apoptosis in other systems also appear to function in neurons, the mechanism that triggers apoptosis in neurons is unknown. Apoptosis occurs asynchronously in neural and differentiated neuronal cells, which makes biochemical studies difficult because a small number of cells are at a particular stage at any one time. Two strategies were devised to overcome asynchrony during neural cell death. The first strategy was to separate rat pheochromocytoma (PC12) cells at different stages of commitment to cell death on the basis of cell density using equilibrium density gradient centrifugation. Three populations were defined. Cells in population 1 were the most dense and committed to cell death. They showed extensive loss of mitochondria1 cytochrome c, DNA fragmentation, and chromatin condensation. Population 3 contained live cells that floated to the top of density gradients. Population 2 displayed some chromatin condensation, yet little DNA fragmentation and loss of cytochrome c. This population showed upregulation of the pro-death factor, c-Jun, and downregulation of pro-survival kinase, Akt. Importantly, these cells could be rescued from death by nerve growth factor (NGF) and thus represent an intermediate stage of apoptosis, upstream of irreversible commitment. The second strategy was to create a cell-free system to reconstitute apoptosis. The addition of cytochrome c to human neuroblastoma (SY5Y) cell extracts activated caspase-9 and -3, and nucleolytic events in PC12 nuclei. Using this system, requirements for ATP and phosphatase activity for caspase activation and nuclear apoptosis were characterised. In addition, pro-survival molecules Akt and Creb were identified as caspase substrates during apoptosis in vitro. To assess whether these events occurred in vivo, the kinase inhibitor staurosporine and the topoisomerase inhibitor camptothecin were used to induce apoptosis in intact SY5Y cells. The pro-survival signalling kinase Raf-1 was downregulated during both staurosporine- and camptothecin-induced apoptosis, but Akt was only downregulated by camptothecin. These studies illustrate the complex interactions of apoptosis and signalling mechanisms in neural cells.Item Nitric oxide production in the mammary gland : a thesis submitted in partial fulfilment for the degree of Doctor of Philosophy at Massey University, New Zealand(Massey University, 2001) Turner, Sally-AnneAlthough the effects of nitric oxide (NO) have been widely studied in many different cell and tissue types, very little is known of the role it plays in the mammary gland. Thus, the production of NO by mammary gland was investigated in a series of experiments. NO is a free radical gas which is produced by a wide variety of cells by the action of the enzyme nitric oxide synthase (NOS) on arginine. This results in the formation of citrulline and NO. The study first examined several methods for their suitability for the detection of NO or NOS. Evaluation of the methods revealed that the indirect measurement of NO production by the detection of nitrate and nitrite (NOx), the spontaneously produced metabolites of NO, was the most valid and reliable. The measurement of NOx was carried out in culture medium using a fluorescent-based assay, which was developed by the modification of published methods, during the course of this study. Comma-D cells (murine mammary epithelial cell line) were used to investigate the production of NOx by the inducible form of NOS (iNOS) following treatment with cytokines and cytotoxins. The cell's response was characterised and showed that mammary epithelial cells produce NOx in a dose dependent manner in response to interferon-γ (IFN-γ). Lipopolysaccharide (LPS), a component of bacterial cell walls, was employed to examine the response of the mammary epithelial cells to cytotoxins and it was found that the treated cells produced more NOx than the untreated ones, however, no dose response was apparent. The specific iNOS inhibitor, aminoguanidine (AG) and general NOS inhibitor Nω-nitro-L-arginine (L-NNA) were both used to confirm that the NOx measured in the medium was produced by NOS. The production of NOx by the mammary gland was also examined in cultured explants of mammary tissue taken from pregnant (D 12-14 of pregnancy) and lactating (D 12-14 postpartum or D 17-18 postpartum) rats. A significant difference was found in the basal production of NOx between the different developmental stages. The method of euthanasia of the rats also affected the amount of NOx produced. The inclusion of prolactin (PRL) also increased the production of NOx from both Comma-D cells and explants of mammary tissue. Xanthine oxidase (XO), an enzyme responsible for the conversion of NOx to NO under anaerobic conditions, does not interfere with the determination of NO production using the NOx assay. The measurement of NOx was carried out in the milk of cows following the intramammary infusion of Streptococcus uberis or interleukin-1β (IL-1β). By comparing the milk NOx concentration with the somatic cell count (SCC) and electrical conductivity (EC) of the milk, it was concluded that the source of the NOx in the milk could not be attributed entirely to the epithelium or the somatic cells. The experiments in this Thesis clearly show that the mammary gland is capable of the production of NO in response to a variety of situations and that the regulation of the production is very complex. The work also identifies some new areas of research, which if completed would further enhance the understanding of the role NO plays in the mammary gland.Item Fine scale spatial behaviour of indigenous riverine fish in a small New Zealand stream : a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Conservation Biology at Massey University, Palmerston North(Massey University, 2009) McEwan, Amber JulieThe substrate and flow characteristics of a 100m reach of a small, North Island, New Zealand stream were mapped and drawn to a 0.25m2 grid scale. One hundred and thirty four individual fish, representing five native and one introduced species were PIT tagged and then monitored with a portable transceiver over 41 occasions during day and night in autumn to winter of 2008, then on 3 occasions in January 2009. Redfin bullies (Gobiomorphus huttoni), shortjaw kokopu (Galaxias postvectis) and koaro (Galaxias brevipinnis) were most commonly represented and redetected in the 100m reach (75%, 73%, and 83% detection rates respectively). Redfin bullies with a lower condition factor than conspecifics were less likely to be redetected and gravid fish were considered more at risk of infection or death associated with PIT tagging. Shortjaw kokopu were less likely to be redetected but more likely to retain tags in the longer term than both redfin bullies and koaro. No difference was found in tag detection rates at a range of flow levels, nor between day and night surveying, although a small decline in detection rates occurred as water temperature decreased. Four hundred and twelve locations of untagged fish were collected during 14 night samples and added to the dataset of 557 locations of PIT tagged fish. A total of 1112 (82% of the reach) 0.25m2 grid squares were inventoried for microhabitat characteristics using 16 physical variables which, together with fish locations, enabled the microhabitat characteristics of the grid squares where fish were found to be compared with those where fish were not found. Redfin bullies and shortjaw kokopu showed strong associations with large substrates and large interstitial refuge spaces and both species showed marked diel differences in microhabitat utilisation. Koaro were more dependent on velocity and surface turbulence and used similar microhabitat types regardless of diel period. No size-based or seasonal differences were found regarding microhabitat use. Potential segregation was observed between shortjaw kokopu and koaro but no other biotic influences on habitat utilisation were apparent. Three floods occurred during the 2008 sampling period which facilitated the collection of fish behavioural data in relation to high flows. A total of 31 individuals were detected during flood conditions and these were found either within 0.5 metres of the base flow stream bed edge or inside the base flow stream bed in areas with large boulder substrates. A subset of the population was found returning to the same locations during multiple floods. Individual fish detected during high flows were significantly less familiar (see pages 68-69 for a detailed description of the term “familiar” in this context) in comparison to the subset of individuals that were commonly resident in the study reach during base flow conditions, showing that tagged fish made larger scale movements during flood conditions. While small changes in community composition occurred that were able to be attributed to flood-induced microhabitat changes, overall a remarkable level of persistence was observed in the tagged community, with over half of all individuals remaining in or returning to the same 100m section of stream following each flood.