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Item Biofilm formation of Vibrio parahaemolyticus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology at Massey University, Campus Manawatū, New Zealand(Massey University, 2024-05-20) Wang, DanVibrio parahaemolyticus in seafood can cause food poisoning. There is increasing concern with the increase in reports of illness globally believed to be due to climate change affecting sea temperatures. Biofilm formation of V. parahaemolyticus is an additional concern as biofilms are more resistant to cleaning and sanitation than planktonic cells. However, little is known about the biofilm formation of V. parahaemolyticus. Strain variation and the factors determining biofilm formation were investigated in this study with the aim to provide information that can be used to design more effective control strategies. This study identified two robust biofilm forming strains (PFR30J09 and PFR34B02) from nine V. parahaemolyticus seafood isolates. Comparative genome analysis unveiled 136 unique accessory genes in robust biofilm formers. Protein-protein-interaction analysis showed interactions between UDP-glucose metabolism (Gene ontology (GO): 0006011), cellulose biosynthesis (GO: 0030244), rhamnose metabolism (GO: 0019299) and O antigen biosynthesis (GO: 0009243). Cellulose contributed to robust biofilm formation. Cellulose biosynthesis was identified as being acquired from within the order Vibrionales. The cellulose synthase operons consisting of genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC were present in 15.94% (22/138) of V. parahaemolyticus. Strong biofilm-forming V. parahaemolyticus showed greater resistance to sanitizers of biofilm cells than the weaker biofilm forming cells. The effective concentrations of sodium hypochlorite for inactivating most V. parahaemolyticus biofilm cells were higher than the recommended concentration. Available chlorine of 1176 mg/L inactivated 1.74-2.28 log10 CFU/cm2 of biofilm on stainless steel surfaces and 4704 mg/L inactivated > 7.00 log10 CFU/cm2 of biofilm (to undetectable levels, < 10 CFU/cm2), except for biofilms formed by the strong biofilm formers. Peracetic acid (PAA) at 200 ppm (89.56 mg/L PAA, 471.64 mg/L hydrogen peroxide) inactivated > 5.00 log10 CFU/cm2 of biofilm from stainless steel surfaces (except for those the strong biofilm formers, see Figure 4.4). RNA sequencing (RNA-seq) identified 74 differentially expressed genes when comparing planktonic and biofilm cells of V. parahaemolyticus. These represented the rearrangement of nucleotide and energy metabolism in biofilm cells. Biosynthesis of secondary metabolites, purine and pyrimidine metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation were deemed essential in the young V. parahaemolyticus biofilms. Genes of purH, purF, pdhA are potential genetic targets for biofilm prevention and control of V. parahaemolyticus. Understanding V. parahaemolyticus biofilm formation will help to design strategies to overcome the limitations of chemical sanitizers, improving product safety and quality in the seafood industry.Item Mass spectrometry-based metabolomics as a tool for biomarker discovery and diagnosing metabolic health : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science, School of Health Science, Massey University, Palmerston North, New Zealand(Massey University, 2021) Zhanxuan, WuAlthough obesity and prediabetes have been long-established risk factors for type 2 diabetes (T2D), excess fat deposition in visceral and ectopic organ sites (i.e. the “risky” fat depot) has been increasingly recognised as key conduits for T2D. However, quantification of these fat depots relies on expensive and time-consuming imaging techniques. There is a need for identification of biomarkers predictive of risky fat depot levels. Metabolomics is a promising tool for discovering novel markers and generating mechanistic speculation. This PhD project aims to identify and understand plasma metabolite markers associated with metabolic risk factors including elevated fasting plasma glucose (FPG), fat deposition in visceral (VAT) and ectopic organ sites (liver and pancreas) in non-T2D human. To achieve this, a workflow to select the optimal injected concentration of different sample types for two complementary LC-MS untargeted analyses (polar metabolites and lipidomics) was established and applied to examine the value of measuring the plasma metabolome as a proxy for metabolite concentrations in various tissue sites including adipose tissue, muscle and the liver. Lastly, plasma metabolomic signatures for elevated FPG and VAT were characterised and metabolite markers predictive of VAT and ectopic fat deposition in the liver and pancreas were identified. This PhD study highlighted the critical importance in optimising injected concentration for LC-MS analysis of different sample types to ensure the maximal number of linear features were obtained, and for the first time showed the plasma metabolomics profile was more reflective of the liver profile than muscle or adipose tissue. Subsequent metabolomics characterisation of clinical plasma samples reported profound associations between FPG or VAT with changes in several glycerolipid species independent of gender, ethnicity, age and body mass index (BMI). VAT was additionally associated with changes in phospholipid, ether-linked phospholipid and sphingolipid species independent of covariates. Liver fat deposition was predicted by a number of glycerolipid, phosphatidylethanolamine and dihydroceramide lipid species whose plasma concentrations were linearly correlated with the liver counterparts. A novel marker, sulfolithocholic acid, for the prediction of pancreatic fat independent of age, BMI and visceral adiposity was also identified. Finally, the study also reported an improved prediction of ectopic fat deposition by utilising a panel of metabolite markers compared to clinical measurements, and demonstrated the usefulness of the metabolomic signature to identify a subset of normoglycaemic individuals with a worse cardiometabolic profile. Findings from this PhD study highlighted the value of metabolomics as a promising tool to capture metabolic risk, and that candidate markers identified by metabolomics may offer opportunities for improved risk prediction and stratification, disease progression monitoring and to develop alternative means for the measurement of the effectiveness of dietary interventions.Item Identification of mechanisms defining resistance and susceptibility of Camellia plants to necrotrophic petal blight disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatū, New Zealand(Massey University, 2019) Kondratev, NikolaiSpecies in the genus Camellia, which includes the tea crops, oil-producers and valuable ornamental plants, have economic and cultural significance for many countries. The fungus Ciborinia camelliae causes petal blight disease of Camellia plants, which has a short initial asymptomatic phase and results in rapid necrosis and fall of blooms. Ciborinia camelliae is a necrotrophic pathogen of the family Sclerotiniaceae, which also includes two broad-host range necrotrophic pathogens, Botrytis cinerea and Sclerotinia sclerotiorum. Previously it was shown that some Camellia plants, such as Camellia lutchuensis, are naturally resistant to petal blight. In order to find molecular mechanisms underpinning this resistance, a genome-wide analysis of gene expression in C. lutchuensis petals was conducted. The analysis revealed a fast modulation of host transcriptional activity in response to C. camelliae ascospores. Interaction network analysis of fungus-responsive genes showed that petal blight resistance includes increased expression of important plant defence pathways, such as WRKY33-MPK3, phenylpropanoid and jasmonate biosynthesis. A much-delayed activation of the same pathways was observed in the susceptible Camellia cultivar, Camellia ‘Nicky Crisp’ (Camellia japonica x Camellia pitardii var. pitardii), suggesting that failure to activate early defence enables C. camelliae to invade and cause tissue necrosis. Early artificial induction of defence pathways using methyl jasmonate reduced the rate of petal blight in susceptible ‘Nicky Crisp’ plants, further verifying the role of a rapid defence activation in petal-blight resistance. Overall, transcriptomic and functional analysis of the Camellia spp.- C. camelliae interaction demonstrated that the same plant defence pathways contribute to both resistance and susceptibility against this necrotrophic pathogen, depending on the timing of their activation. To further understand the molecular mechanisms of petal blight resistance, the role of the phenylpropanoid pathway, identified as a key feature in the transcriptome study above, was investigated in more detail. This pathway produces various metabolites, including phenolic acids, aldehydes, and alcohols, which have numerous physiological functions and also participate in the production of flavonoids and lignin. Resistant C. lutchuensis was shown to rapidly activate the expression of core phenylpropanoid genes after treatment with C. camelliae ascospores. LC-MS-based quantification of phenylpropanoid compounds demonstrated that within the first 6 h of the infection, resistant plants had already accumulated coumaric, ferulic and sinapic acids, while at 24 hpi, concentrations of coumaraldehyde, sinapaldehyde, and caffeyalcohol were significantly increased. Thus, I further hypothesized that the compounds produced by the phenylpropanoid pathway may have fungistatic activity. Indeed, all tested phenylpropanoids inhibited the growth of C. camelliae in agar plates with different efficacy. Moreover, the application of phenylpropanoid compounds, including ferulic and coumaric acids, fully prevented the formation of petal blight lesions on susceptible Camellia ‘Nicky Crisp’ petals. Taken together, it can be concluded that the phenylpropanoid pathway may contribute to the early defence against the petal blight via the rapid production of fungistatic compounds. Ultimately, these compounds could be used to develop natural antifungal sprays to protect susceptible Camellia flowers. The analysis of the C. camelliae secretome using LC-MS/MS detection of proteins showed that the pathogen produces a large number of carbohydrate-active enzymes in liquid culture and plant petals. Injection of these proteins induced necrosis not only in susceptible Camellia petals but also in petals of the resistant species and leaves of non-host Nicotiana benthamiana. It was proposed that these enzymes can contribute to the virulence of the pathogen by inducing cell death and facilitating necrosis propagation. Thus, the early defence responses of resistant Camellia plants may possibly stop the development of C. camelliae before it starts releasing carbohydrate-active enzymes during the necrotrophic step of the infection. Overall, the results of this research further expand our understanding of plant- necrotroph interactions, suggesting that the timing of plant immune responses may be a crucial factor defining the outcome of the necrotrophic infection.Item Biomarker development to assess bone health : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Palmerston North, New Zealand(Massey University, 2019) Cabrera Amaro, Diana LeticiaPostmenopausal women experience an accelerated bone loss with increased fracture risk caused by oestrogen deficiency. Biomarkers of bone turnover assess the changes of bone metabolism in postmenopausal women; however, prediction of bone loss with these common biomarkers cannot be achieved because bone biomarkers might not reflect the bone microenvironment status. Thus, there is a need for discovering new bone biomarkers that can efficiently predict bone loss in postmenopausal women. Previous studies suggest that the ovariectomised sheep in combination with injected glucocorticoids may be a reliable model to evaluate the biological response to oestrogen withdrawal as well as the bone remodelling process. The purpose of this research programme was to test the following hypotheses: 1) ovariectomising sheep in combination with monthly injections of glucocorticoids would result in decreased bone mineral density (BMD) and increased plasma bone remodelling marker concentration over a shorter period of time; 2) the plasma metabolome and lipidome of ovariectomised sheep would be different, and the biochemical changes in plasma and bone remodelling would be associated with bone loss; 3) and finally, there would also be a difference in the plasma metabolome and lipidome of Singaporean–Chinese postmenopausal women according to their bone mineral density status. The hypotheses were evaluated using the OVX sheep in combination with glucocorticoids as a large animal model for postmenopausal osteoporosis, as well as comprehensive LC–MS-based metabolomics as a diagnostic approach to identify lipids and metabolites associated with bone loss in postmenopausal women. The OVX sheep model was successfully validated over five months of this study period, and bone mineral density was decreased and bone biomarkers increased after five months. Then, plasma samples from this animal model were analysed to measure the metabolome and lipidome of the OVX sheep. In the OVX sheep, metabolite and lipid alterations associated with bone loss included methionine, glutaric acid, tryptophan, 5-methoxytryptophan, CL and CerP, and these correlated with OC, CTx-1, femoral BMD and lumbar spine BMDThese studies revealed dynamic changes of the metabolite and lipid profiles from affected sheep, such as perturbation in multiple amino acids, metabolites, and fatty acid β-oxidation. Additionally, the results from the Singaporean–Chinese postmenopausal women showed alterations in proline, threonine, methionine, 4-aminobutyric acid, aminopropionitrile, phosphatidic acid, diacylglycerol, CerP and phosphatidylinositol correlated with low femoral neck BMD. Methionine and CerP were the common compounds altered in OVX sheep and SC women with low BMD when compared with healthy groups. Those compounds, which are known to be involved in bone remodelling, have the potential for studying early bone loss in postmenopausal women, where identifying new bone-specific biomarkers may aid in clarifying novel molecular mechanisms of bone loss.Item Development of reference ELISA assays for urinary oestrone-3 ¯-glucuronide and pregnanediol-3 ¯-glucuronide using timed urine specimens : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1999) Binnie, Janette ElizabethEnzyme-linked immunosorbent assays (ELISA) have been developed which measure oestrone glucuronide (E1-3G) and pregnanediol glucuronide (PdG) in timed, diluted urine samples. Measurement of these urinary metabolites allows information to be collected, non-invasively, on the hormonal interplay between the ovaries and the hypothalamicpituitary axis, which determines or helps to make predictions about the potentially infertile and fertile phases of the human menstrual cycle. Immunoglobulin Class G (IgG) antibodies raised in sheep against the analyte of interest (E1-3G and PdG) were adsorbed onto polystyrene microtitre wells. The enzyme conjugate tracer was horseradish peroxidase (HRP), and was prepared by conjugation with either E1-3G or PdG using the active ester coupling procedure. A direct competitive immunoassay configuration in which both analyte and tracer were added to the wells simultaneously allowed a direct competition between them for the immobilised antibody sites. A chromogenic detection system involving o-phenylenediamine (OPD) was used for the measurement of the amount of bound tracer (HRP conjugate) which could be related to the amount of analyte in a urine sample. The sensitivity of the E1-3G assay was 3.4 nmoles/ 24 h, and for the PdG the sensitivity was 0.5 μmoles/ 24 h. Both assays were reliable, and were successfully validated against World Health Organisation (WHO) assays performed on the same urine samples in a multicentre study of the Ovarian Monitor (project #90905). The E1-3G and PdG reference assays developed in the present study are acceptable for use in the laboratory and can be used to validate new non-instrumental colour tests, or other home fertility kit assays currently being developed.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 Identification and characterization of Dothistromin biosynthetic genes in the peanut pathogen Passalora arachidicola : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 2008) Guo, YananDothistromin (DOTH) is a secondary metabolite produced by the fungal peanut pathogen Passalora arachidicola and pine needle pathogen Dothistroma septosporum. The chemical structure of DOTH is similar to a precursor of aflatoxin (AF) and sterigmatocystin (ST), which are secondary metabolites produced by Aspergillus species. A size fractionated genomic library was made and 11 putative DOTH genes were identified in P. arachidicola. The DOTH genes in P. arachidicola were compared to DOTH genes in D. septosporum as well as to AF and ST genes in Aspergillus species. The DOTH gene products in P. arachidicola showed 73 - 96% amino acid identity to DOTH genes in D. septosporum and 50 - 69% amino acid identity to AF/ST genes in Aspergillus. The DOTH biosynthesis genes in P. arachidicola had similar gene organization and direction of transcription to DOTH biosynthesis genes in D. septosporum and is similar in that 11 putative DOTH genes are separated into three mini-clusters. This differs from the AF/ST clusters in which 25 AF/ST genes are tightly clustered in a 70 kb region. Identification of transcription factor binding sites upstream of DOTH genes in P. arachidicola and D. septosporum suggested similar co-regulation of DOTH gene expression in P. arachidicola and D. septosporum. Tandem and inverted repeat sequences were identified in intergenic regions in the P. arachidicola DOTH gene cluster, but the distribution of those repeats appears to be random. This suggests that the fragmentation of the DOTH biosynthesis gene cluster is not due to retrotransposon activity or recombination between repeat sequences. The DOTH biosynthesis gene clusters in P. arachidicola and D. septosporum could be ancestral to AF/ST biosynthesis clusters in Aspergillus species.