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    The tale of the shear-thickening mamaku polysaccharide, from forest to gut : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Food Technology), Massey University, Palmerston North, New Zealand. EMBARGOED until further notice.
    (Massey University, 2023) Bisht, Akshay
    The New Zealand black tree fern (Cyathea medullaris, ‘mamaku’ in the Māori language) is grown across the Pacific Islands and has a long history of use for therapeutic benefits or as food by Māori people. The water-soluble gum extract from mamaku fern contains a novel glucuronomannan biomacromolecule, called mamaku polysaccharide (MP), which has been shown to exhibit a unique shear-thickening (i.e., increase in viscosity on shearing) behaviour at a similar shear rate as that found in the human stomach. Herein, the objective was to gain a better technical and physiological understanding of MP for designing a novel shear-thickening ingredient for the industry with proven effects in humans. The shear-thickening behaviour of MP was sensitive to the harvesting age of mamaku fronds and industrial operations such as high temperature and shear. With the increase in harvesting age, the molecular weight of MP reduced, which consequently reduced the shear viscosity. The shear-thickening behaviour was lost in MP from old fronds. Furthermore, the temperature treatment disintegrated the backbone of MP into smaller fragments which caused a reduction in viscosity and extent of shear-thickening. Similar rheological trends were observed post-shear treatment, however, there was no evidence of depolymerisation. A combination of in vitro models revealed that mamaku gum extract could improve host gut functioning by reducing the activity of digestive enzymes (α-amylase, pepsin and lipase) and binding bile acids. Mamaku gum can act as a substrate for colonic fermentation, promote the production of short-chain fatty acids and alter the colonic microbial composition. Upon ingesting mamaku gum, the shear-thickening behaviour may develop in the oesophagus causing a possible choking hazard. Therefore, the potential of using the whole pith—natural entrapment of MP in the tissue of pith—as an alternative to gum extract was studied. Freeze-dried pith was ground to powder. The powder particles swelled upon rehydration with water and released the water-soluble MP into the continuous phase in a time-dependent manner. The presence of enough MP in the continuous phase to form polymer-polymer interactions resulted in a shear-thickening behaviour of the pith powder suspension similar to the MP extract solution. Moreover, the co-consumption of 1 h pre-hydrated mamaku pith powder with a carbohydrate-rich meal significantly reduced the postprandial glycaemic response (blood glucose peak height) in human participants. Additionally, the consumption of mamaku pith powder in rats could alter colonic microbiota. Interestingly, more than half of the MP (uronic acid) consumed by rats survived the gut transition and was obtained in faeces, suggesting that MP could potentially be used as a laxative. Thus, mamaku pith could be used as an alternative to gum extract to develop a natural shear-thickening ingredient which may potentially help to manage diabetes and improve colon health.
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    Design and engineering of self-assembling antigens towards particulate vaccines : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Chen, Shuxiong
    Natural and synthetic self-assembling polymers and proteins could be bioengineered to display and/or encapsulate antigens to serve as innovative antigen carrier systems for the induction of desirable immunities. Polyhydroxyalkanoates (PHAs) are naturally occurring polyesters synthesized as cytoplasmic polyester inclusions (polyester particles) by various bacteria. The particles have been used as an antigen delivery platform by translationally fusing antigens to the particle surface-associated protein, PHA synthase. Furthermore, it has been found that protein inclusion bodies contain a large amount of correctly folded and biologically active proteins and could be engineered to perform as an antigen carrier system. Tuberculosis (TB) is a global health issue for both humans and animals. Inaccurate diagnosis and inefficacious vaccination make TB control problematic. The Mantoux tuberculin skin test gives false positive results if humans or animals are vaccinated with the Bacille Calmette-Guérin (BCG) strain or exposed to environmental mycobacteria. BCG cannot provide effective protection against TB. Subunit vaccines have great promise to protect against infectious diseases, but they are often weak immunogenically. A strategy to circumvent this problem is the use of self-assembly particulate vaccines, which could present multiple copies of antigens and serve as a depot for prolonged multivalent antigen display to induce enhanced immunogenicity. In this thesis, four specific TB diagnostic antigens — CFP10, Rv3615c, ESAT6, and Rv3020c — were displayed on polyester particles. The results showed that polyester particles displaying TB antigens specifically distinguished TB-infected from non-infected cattle. Antigen immunogenicity was dramatically enhanced after the display on polyester particles, which lowered the antigen concentration (0.1 to 3 μg dose/inoculum) required for skin tests. Mycobacterial vaccines H4 (Ag85B-TB10.4) or H28 (Ag85B-TB10.4-Rv2660c) were bioengineered to display H4/H28 on polyester particles and/or self-assemble H4/H28 into protein inclusion bodies. The results demonstrated that polyester particle-/protein inclusion body-based particulate TB vaccines increased overall immunogenicity by enhancing humoral (for example, IgG1 and IgG2c) and cellular (for example, IFNγ and IL17A) immune responses when compared to respective soluble antigens.
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    Fate of hydroxyapatite nano particles during in vitro gastrointestinal digestion : a thesis presented in partial fulfilment of the requirements for the degree of Master in Food Technology at Massey University, Riddet Institute and Massey Institute of Food Science and Technology, Palmerston North, New Zealan
    (Massey University, 2018) Choki, Kinley
    There is an increasing change in population demographics towards an aging population in the world, which had led to the availability of various commercial nutritionally supplemented products. Hydroxyapatite (HA), with chemical formula Ca10(PO4)6(OH)2, is an insoluble calcium salt used for calcium supplementation because of its similarity to the minerals found in human bone and teeth. The insoluble calcium salts are preferred over the soluble ones because of their high heat stability during milk processing under high heat treatment. However, the drawback of insoluble calcium salts is the tendency to sediment during storage resulting in unfavourable gritty texture. Thus, reduction in particle sizes into micron to nano-size improves the dispersion of these insoluble salts. However, the application of nano-sized particles in food products have raised concerns from both the regulatory organizations and consumers on the implications related to both the environmental and health safety aspects. Thus, the objective of the study is to determine the digestion behaviour of nano-sized needle/rod shaped HA (nHA) when added into skim milk during in vitro gastrointestinal digestion. Determination of calcium such as soluble and ionic calcium was conducted to determine the dissolution of nHA. The structural changes and the crystallographic changes of nHA were determined using electron microscopy and x-ray diffraction techniques. The results of in vitro gastric digestion showed presence of undissolved nHA particles even after 240 min of gastric and 120 min of intestinal digestion when examined under TEM, while the XRD analysis detected the presence of crystalline nHA in the first 120 min of gastric digestion. Thus, the possible mechanisms leading to the incomplete dissolution of nHA under acidic conditions of the stomach are discussed subsequently.
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    Citric acid production by the yeasts Candida guilliermondii and Yarrowia lipolytica : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology and Bioprocess Engineering at Massey University
    (Massey University, 1993) Thomson, Karen Roberts
    The aim of this thesis was to investigate the relationships, for a citric acid­ producing strain of yeast, among the growth rate, sugar uptake rate and the citric acid production rate, and to investigate the hypothesis that citric acid production occurs when the growth rate slows, but the sugar uptake rate is maintained. As previous experimental work in the Department of Process and Environmental Technology (formerly Biotechnology Department) of Massey University had been performed in shake flask cultures only, it was desired to scale-up the culture into a 21 laboratory scale batch culture, and then into a chemostat culture. The first yeast investigated, Yarrowia lipolytica IMK2, failed to successfully scale-up, so further investigations were performed using the yeast Candida guil!iermondii IMK1. Experiments were performed in shake flask culture to investigate the effect of using mixed carbon sources to adjust the carbon uptake rate, and hence the citric acid production rate, but no effect was noticed with the mixtures tested. Batch fermenter experiments were performed to investigate the effect of the culture pH, and the aeration rate, on citric acid production. The aeration rate was not observed to have an effect on the culture in the range tested (0.06 - 0.333 vvm), but the culture pH was observed to have an effect, with the maximum production occurring at pH 4.3, and no citric acid production occurring below pH 3.5. Chemostat culture experiments were performed to investigate the effect of culture pH and the specific growth rate on citric acid production. The specific growth rate was observed to have a significant effect, with the specific citric acid production rate increasing as the growth rate decreased. The effect of the culture pH was found to vary with the growth rate, with the maximum production rate and yield occurring at pH 3.8, and a growth rate of 0.02 h-1 • From cultures where the glucose was exhausted from the medium, and therefore glucose was a limiting nutrient, the specific citric acid production rate was observed to decrease as the glucose uptake rate decreased. Thus, it could be concluded that the specific citric acid production rate increased as the growth rate decreased, provided that the sugar uptake rate remained high.
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    Characterisation of malate-dependent mutants of the yeast Schizosaccharomyces malidevorans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University
    (Massey University, 1991) Hansen, Nicolette Olivia
    The phenotypes of several UV induced malate-dependent mutants of Schizosaccharomyces malidevorans were characterised and compared to the previously characterised malate-dependent strain, S. malidevorans #11. This strain uses less glucose than the wild type, has an extended life in liquid media, does not sporulate readily and turns indicator medium blue. The malate-dependent mutant strains were analysed for their fermentation characteristics, their complementation groups and their chromosomal patterns on a transverse alternating field electrophoresis apparatus (TAFE). The fermentation patterns of all of the strains were similar to #11. There appear to be three complementation groups involved in the malate-dependent phenotype. The TAFE patterns and subsequent Southern Hybridisation showed that the malate­ dependent mutants had a decreased mobility of chromosome II, while chromosomes I and III were not altered. The chromosome II alteration varied between the different malate-dependent mutants but fell into a small number of discrete patterns.
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    Novel polyhydroxyalkanoate beads for use as a vaccine against tuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatū, New Zealand
    (Massey University, 2017) Rubio Reyes, Patricia
    Tuberculosis was in 1993 declared as a re-emerging disease by the World Health Organization. The only vaccine currently available, BCG, an attenuated strain of Mycobacterium bovis, does not protect adults against the pulmonary disease, which is the form of transmission. New vaccine candidates are being developed to provide protection against tuberculosis. Subunit vaccines offer a safer alternative than whole cell preparations and provide the possibility of utilizing only the components that mediate protective immune responses. This thesis describes the production of bacterially derived polyhydroxyalkanoate (PHA) beads for use as a delivery system for Mycobacterium tuberculosis reverse vaccinology antigens and immune modulators. In the first study, the immunogenicity of beads derived from an endotoxin-free host, Clear coli, displaying M. tuberculosis antigens Rv1626, Rv2032 and Rv1789 was evaluated in mice. Beads displaying Rv1626 were selected for further studies based on the magnitude and specificity of the immune response elicited. In a final study, the immune modulators Cpe30, CS.T3378-395 and Flagellin were co-displayed with Rv1626 antigen on beads and the immunogenicity of these functionalised beads evaluated in mice. Vaccinations with Rv1626 beads and the immune modulators Cpe30 and CS. T3378-395 induced a Th1/Th17 skewed immune response. These beads were then assessed for their ability to protect mice against aerosol challenge with Mycobacterium bovis. Rv1626 beads reduced the bacterial loads in 0.48 log10 compared with the negative control group but the inclusion of immune modulators did not enhance the immunogenicity or protection induced by Rv1626 beads. This study has demonstrated the potential of PHA beads delivering a single reverse vaccinology antigen for protection against tuberculosis infection in mice. While the co-display of immune modulators did not improve the protection induced by the antigen, further studies are needed to determine optimal doses for delivery of immune modulators to enhance protective immunity.
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    Purification of TrkA intracellular domain and the characterization of novel intracellular proteins : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Biology at Massey University
    (Massey University, 1999) Walker, Scott David
    Nerve growth factor (NGF) binds to its receptor, TrkA, at the tips of nerve cell axons to inhibit apoptosis, causing survival and differentiation. Some factors within this process are largely unknown, such as the role of the p75 receptor and the molecular mechanisms that occur within the cell. NGF binding causes dimerization of TrkA, which activates the intracellular kinase domain. Autophosphorylation on tyrosine residues stimulates binding to the receptor of several intracellular proteins that mediate the NGF response. This receptor complex has been demonstrated to be retrogradely transported to the cell body. Retrograde transport is hypothesized to occur in small vesicles that have been isolated in our lab using a cell fractionation protocol using in vitro reactions with an ATP regenerating system. Discovering the initial molecular interactions that occur upon NGF binding could further our knowledge of NGF's inhibition of apoptosis, providing us with a possible tool for treatment of diseases that occur when the regulation of apoptosis no longer exists. Novel proteins that were not previously identified were associated with TrkA in small vesicles after NGF activation. To isolate these proteins for further characterization, TrkA's intracellular domain (TrkAID) was expressed in E. Coli. This protein was found to be constitutively tyrosine-phosphorylated and therefore presumably active. In E.Coli, TrkAID protein was localized to the soluble fraction but smaller amounts were detected in the insoluble fraction. TrkAID was partially purified from the soluble fraction using a combination of salt disruption and denaturing techniques. The unpurified TrkAID was immunoprecipitated from the bacterial soluble fraction with an antibody to the C-terminus of TrkA, and some results suggest that immunoprecipitated TrkAID was able to stimulate ERK activation in untreated PC12 cells, but unfortunately this was not reproducible. If the protein could be purified with a combination of techniques, then it would provide a useful tool for studying the initial events in NGF stimulation, that is, the recruitment of several intracellular proteins to the tyrosine-phosphorylated intracellular domain of TrkA.
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    Polyhydroxyalkanoate beads as a particulate vaccine against Streptococcus pneumoniae and Neisseria meningitidis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand
    (Massey University, 2017) González Miró, Majela
    Streptococcus pneumoniae and Neisseria meningitidis are the major causes of pneumonia and meningitis, respectively, worldwide. Capsular polysaccharide-protein vaccines (conjugate vaccines) provide protection against these diseases but not protection against infections caused by serotypes and serogroups not included in these vaccines. Proteins have been increasingly considered as antigens for vaccine development due to their more structurally conserved composition when compared to capsular polysaccharides. Proteins subunit vaccines are safe and protective; however, they have limitations such as serotype-dependent immunity, and low immunogenicity of the proteins, requiring adjuvant to be included in these formulations or delivery systems that enhance the desired immune response. In addition, complex production procedures are required, increasing production costs and therefore market prices making these vaccines inaccessible for many people affected by these diseases. Recently, bacterial storage polymer inclusions have been developed as protein antigen carriers. Polyhydroxyalkanoate, in particular 3-polyhydroxybutyrate (PHB) inclusions have been successfully bioengineered to display antigens from pathogens like Mycobacterium tuberculosis and Hepatitis C virus. These particulate vaccine candidates elicited both a Th1 and Th2 immunity patterns combined with a protective immune response against Mycobacterium bovis in mice. This thesis focuses on the study of polyhydroxybutyrate (PHB) beads properties as a carrier/delivery system engineered to display antigens from extracellular bacteria. The antigens Pneumococcal adhesin A, Pneumolysin (proteins) and 19F capsular polysaccharide (CPS) from Streptococcus pneumoniae, and Neisserial adhesin A, factor H binding protein (proteins) and serogroup C CPS from Neisseria meningitidis were displayed on the PHB bead surface. These antigenic proteins were produced as fusion proteins on the PHB bead surface, while the CPS was covalently attached by chemical conjugation. Mice vaccinated with these PHB beads produced strong and antigen-specific antibody levels. In addition, splenocytes from the same mice generated both IL-17A and IFN-ɣ production. The antibodies elicited against antigenic pneumococcal proteins were able to recognise the same protein in the context of an Streptococcus pneumoniae whole cell lysate from more than six different strains, while antibodies produced after vaccination with 19F CPS conjugate to PHB showed high opsonophagocytic titers against the homologous strain. In the case of Neisseria meningitidis, bactericidal antibodies were elicited in mice vaccinated with PHB beads displaying proteinaceous and CPS antigens. Overall, this thesis shows that PHB as particulate vaccine candidate holds the promise of a broadly protective vaccine that can be produced cost-effectively for widespread application to prevent diseases caused by Neisseria meningitidis and Streptococcus pneumoniae.
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    Understanding aspects of alginate biosynthesis and regulation by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2017) Wang, Yajie
    Alginate is a medically and industrially important polymer produced by seaweeds and certain bacteria. The bacterium Pseudomonas aeruginosa over-produces alginate during cystic fibrosis lung infections, forming biofilms, making the infection difficult to treat. Bacteria make alginate using membrane spanning multi-protein complexes. Although alginate biosynthesis and regulation have been studied in detail, there are still major gaps in knowledge. In particular, the requirement of AlgL (a periplasmic alginate degrading enzyme) and role played by MucR (an inner membrane c-di-GMP modulator) are not well understood. Here I show that AlgL and MucR are not essential for alginate production during biofilm growth. My findings suggest that while catalytically active AlgL negatively affects alginate production, expressing catalytically inactive AlgL enhances alginate yields. Furthermore, preliminary data show AlgL is not required for the stability or functionality of the alginate biosynthesis complex, suggesting that it is a free periplasmic protein dispensable for alginate production. These findings support the prediction that the primary function of AlgL is to degrade misguided alginate from the periplasm. For MucR, I show for the first time that its sensor domain mediates nitrate-induced suppression of alginate biosynthesis. This appears to occur at multiple levels in a manner only partially dependent on c-di-GMP signaling. These results indicate that MucR is associated with the negative effect of nitrate (and possibly denitrification) on alginate production. On the basis of these results, I propose a combination of nitrate (or denitrification intermediates), exogenous lyases and antimicrobial agents could be used to eliminate established chronic biofilm infections. Furthermore, catalytically inactive AlgL and/or homologs of MucR with disabled sensor motifs could be harnessed in non-pathogenic bacteria for producing tailor-made alginates.
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    An investigation of the extractable insulin levels and pancreas weights of New Zealand sheep : a thesis presented in partial fulfilment of the requirements for the degree Master of Technology in Biotechnology at Massey University, Palmerston North, New Zealand
    (Massey University, 1971) Swan, Janis Elizabeth
    In 1921 Banting and Best were the first workers to successfully obtain the active substance insulin from the pancreas. The dramatic clinical benefits of the hormone were immediately apparent and insulin has become the most important commercially produced protein in the medical field. It also has a unique place in protein chemistry investigations as it was the first protein to be crystallised, the amino acid sequence determined and a biologically active molecule synthesized chemically. Methods for producing insulin were developed by Scott and Best at the Connaught Laboratories, Toronto, in the early 1920's. The Insulin Committee set up by the Connaught Laboratories selected Eli Lilly and Company as the first pharmaceutical company to manufacture insulin. Scott and Best developed methods for the preparation of insulin, including large-scale production methods and these methods were sent to interested manufacturers throughout the world at regular intervals (Best 1960). Active work on insulin has continued both at Connaught Laboratories and at the Banting and Best Institute in Toronto. The more recent work from these groups has been on the physiological effects of insulin, modes of insulin action, and factors affecting secretion of insulin by the pancreas. Many other laboratories throughout the world are also investigating similar aspects of insulin. [From Introduction]