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    An investigation into the interaction of the microbiome-gut-brain axis with stress : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Bear, Tracey
    This thesis aimed to investigate whether changes in the gut microbiota and associated biomarkers were associated with stress-induced anxiety-like and depressive-like behaviour. Two studies used the unpredictable chronic mild stress (UCMS) over 4 or 6 weeks (vs no UCMS, control) in Sprague-Dawley rats. Depressive-like behaviour was measured in female rats using the sucrose preference test, and the Porsolt swim test. Anxiety-like behaviour was measured with the light-dark box test. Faecal corticosterone, caecal microbiota (composition and organic acids), serum gut permeability (lipopolysaccharide-binding protein, LBP) and plasma inflammation (12 cytokines) markers were measured. Atypical behaviours were observed in female rats following UCMS and no depressive-like behaviours were observed. The circulating concentration of cytokines, but not plasma LBP or caecal organic acids, was higher in UCMS-exposed female rats. Relative abundance of taxa from the Clostridiales order and Desulfovibrionaceae family correlated with anxiety-like behaviours and plasma cytokine concentrations, regardless of UCMS. Studies of these atypical behaviours in female rats confirmed expected patterns of sucrose intake in the sucrose preference test and no decreased depressive-like behaviours in the Porsolt swim test with antidepressant citalopram and imipramine drugs. A further study also showed differences in baseline behaviour in male versus female rats, leading the second UCMS study to be in male rats. Increased faecal corticosterone and anxiety-like behaviours were observed in male UCMS-exposed and control rats at week 4 of UCMS compared to baseline. Plasma cytokine concentrations were higher in the UCMS group but higher faecal corticosterone concentrations and anxiety behaviours in control rats suggest that they were more stressed than treated rats. Caecal neurotransmitter concentrations did not differ between treatments nor correlate with serum neurotransmitter, cytokines or LBP concentrations or behaviour. The findings showed an association between the gut microbiota and anxiety-like behaviours, which was not stress dependent. No measured biomarkers explained the observed anxiety-like behaviours. Caecal digesta neurotransmitter profiles were dissimilar to serum profiles indicating it may not be an important influence on serum levels. Despite the atypical behavioural results following the interventions, the results still provided useful and unique information which contributes to the body of Microbiome Gut Brain Axis research.
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    The preventive effect of greenshell mussel meat against osteoarthritis in vivo : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Health Science At Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Siriarchavatana, Parkpoom
    Osteoarthritis (OA) is identified by progressive cartilage erosion of synovial joints. One of the most prevalent OA phenotypes, metabolic OA (MetOA), is linked to metabolic syndrome (MetS). MetS is a combination of obesity, type II diabetes, hypertension, and hyperlipidemia; the effects of these disorders can lead to the development of MetOA. Osteoporosis is characterised by loss of bone mineral density and is causally linked with a decrease in systemic estrogen levels. As MetS, OA and osteoporosis are all prevalent in postmenopausal women, it is possible they may be causally linked. For example, systemic low-grade inflammation in MetS may trigger inflammation in both joints and bone, which could be further aggravated by high fat/high sugar diet (HFHS)-induced obesity and gut dysbiosis. We hypothesized that chronic inflammation would be correlated with MetOA development and therefore decreasing inflammation would be protective. New Zealand greenshell mussel (GSM) contains anti-inflammatory properties shown to reduce OA symptoms and omega-3 fatty acids shown to reduce the development of post-menopausal osteoporosis. We hypothesized GSM could protect against both MetOA and osteoporosis reducing bone resorption, inhibiting inflammation and/or modulating beneficial gut microbes. In vitro, non-polar GSM lipids demonstrated bone-protective properties and significantly reduced osteoclast differentiation, tartrate-resistant acid phosphatase activity, actin ring formation and gene expression of matrix metalloproteinase, cathepsin K, carbonic anhydrase and nuclear factor of activating T cells 1. In vivo, aging, HFHS and OVX produced a rat model mimicking human MetS. Dietary whole GSM powder provided protection by significantly reducing a biomarker of collagen degradation and subsequent joint damage, as well as improving short-term bone mineral density and lean mass accrual. GSM-induced changes in gut microbiota were not correlated with dysbiosis. No changes in inflammatory markers were found, disproving our initial hypothesis and suggesting that chronic inflammation may not be a critical factor in MetOA. In conclusion, GSM as a dietary intervention may reduce the incidence or progression of MetOA but not via altering systemic inflammation or gut dysbiosis.
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    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 Leticia
    Postmenopausal 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.
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    The assessment of dietary iron bioavailability using the piglet as an animal model for the human infant : a thesis presented in partial fulfilment of the requirements for the degree of Master of Master of Science in Nutritional Science at Massey University
    (Massey University, 1998) Gray, Caroline Elizabeth
    The bioavailability of five different iron sources added to a bovine milk based formula was investigated in the anaemic, suckled piglet. These iron sources were ferrous pyrophosphate (FP), ferrous sulphate (FS), milk protein-iron complex (MPIC), ferrous lactate (FL), and haemin (Hm). Forty eight male piglets were removed from the sow at five days of age and randomly assigned to one of six treatment groups (n=8). Iron depletion was achieved in two ways: firstly, by withholding the iron injection usually given to these piglets at birth, and secondly, by feeding the piglets an iron-deficient formula throughout an eleven day adjustment/iron-depletion period. The five formulas containing the supplementary iron and one formula containing no iron (control diet) were bottle-fed to the piglets (336g of liquid formula/kg bodyweight/day) seven times daily for a 25 day repletion period. Analysis of the formulas revealed that iron levels in each of the five iron treated formulas varied over a range of 6.0-8.3 mg/l00g of powdered formula. Iron bioavailibility was assessed in the piglets by a haemoglobin repletion assay. Blood was collected from the anterior vena cava of the piglets on days 0, 11, 24, and 36 of the trial, and the piglets were weighed every three to four days throughout the 25 day repletion period. Blood haemoglobin concentration, haematocrit, unsaturated iron binding capacity, and piglet liveweights were used as indicators of iron status in the piglets. Haemoglobin Repletion Efficiency (HRE%), a measure of the proportion of ingested iron that is incorporated into haemoglobin in the body, was calculated to correct for differences in the iron content of the formula and differences in iron intake by the piglets. The HRE% for the FP, FS, MPIC, FL, and Hm fortified formulas were 23.5, 35.8, 38.0, 32.9, and 3.2%, respectively. There were no significant differences, however, in the mean blood haemoglobin concentrations, haematocrits, HRE% and UIBC for piglets fed the FS, MPIC, or the FL fortified formulas half way through, and at the end, of the repletion. This implied that there were no differences in the bioavailability of these iron sources. In contrast, these parameters were all significantly lower (P>0.05), for the piglets fed either the FP or the Hm formulas. Based on relative biological value, the bioavailability of each iron source, when ranked in order from highest to lowest, was MPIC
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    Airway hypersensitivity and remodelling induced by repeated exposure to ascaris suum antigen : an ovine model of human asthma : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Physiology at Massey University
    (Massey University, 2001) Lamahewa, H Nalaka
    This study was an attempt to develop a model of asthma which shows all the structural changes of airways that occur in human disease by repeatedly exposing sheep to an aerosol of Ascaris suum antigen. Twenty two sheep were tested for cutaneous reactivity to a commercial preparation of the antigen. For each of three experiments three sheep were selected, two skin reactive and one non-reactive. One week prior to the experiment a tracheostomy was performed according to the method described by Dueck et al., (1985). In each group, the respiratory response of the two reactive sheep to the Ascaris antigen was augmented using 2-3 fortnightly respiratory exposures to the antigen (82,000 protein nitrogen units/ml) for twenty minutes delivered via an endotracheal tube passed through the tracheostomy. One of the reactive sheep (experimental) was then further exposed to antigen for twenty minutes daily for two weeks. The other reactive sheep (sensitized control) and the non-reactive sheep (non-sensitized control) were exposed to the saline vehicle alone daily for two weeks. Airway resistance (Raw) and dynamic lung compliance (Cdyn) were measured before the antigen/saline exposures and at five minutes intervals during the exposure. On the last day the experiment was carried out under general anesthesia. In addition to respiratory measurements, cardiac output, pulmonary arterial pressure, pulmonary wedge pressure, systemic arterial pressure and central venous pressure were obtained to calculate cardiac power output. At the end of the last exposure sheep were killed, necropsied and samples of lung and airway fixed for morphological studies. Sixty four percent of the sheep tested showed an immediate skin reaction to the antigen. The antigen exposure caused significant changes in respiratory parameters and increased the cardiac work load of the right side of the heart in the experimental sheep. Morphological studies revealed that antigen exposure caused an increase in number of eosinophils and goblet cells in the airways and an increase in the thickness of the 'pseudo-basement membrane' at some levels of the respiratory tract. Antigen exposure also caused an increase in the percentage smooth muscle area in the airway wall cross-sectional area in the membranous bronchioles. Based on these observations it can be concluded that repeated daily exposure of sheep airway to Ascaris suum antigen can be used to reproduced morphological changes observed in human asthmatic airways.
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    Effects of dietary sheep, cow and goat milk solids on colitis in the interleukin-10 gene deficient mouse model of inflammatory bowel disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2013) Russ, Anna-Lynne Elizabeth
    Inflammatory Bowel Disease (IBD) is a group of chronic, immunologically-mediated gastrointestinal disorders resulting from interactions between environmental influences, host genetic susceptibility, and the intestinal microbiota. Dietary factors can ameliorate symptoms, providing a rationale for using targeted nutrition to alleviate symptoms. Food components, including milk-derived oligosaccharides and conjugated linoleic acid, have shown anti-inflammatory effects in IBD patients or animal models of IBD. Additionally, some ruminant milks are perceived by some IBD patients to have more beneficial effects on their symptoms (goat, sheep) than others (cow). Soy-based milk substitutes are perceived to be more beneficial than milk. No reports describe the effects of milk solids from different species on molecular pathways in the intestine that might explain differential effects in IBD. This thesis aimed to investigate the effects of dietary intervention with milk solids on the severity of colitis (histology) and molecular pathways (microarrays and qPCR) in the interleukin-10 gene deficient (Il10-/-) mouse model of IBD. First, laser microdissection (LMD) combined with microarrays was used to analyse colon epithelium gene expression in 6 and 12 week old Il10-/- mice fed a control diet. This indicated that intact colon was an appropriate tissue in which to study global changes in gene expression when colitis is established. It also showed that studying colon epithelium during the early stages of inflammation (6 weeks old) may identify molecular changes not seen in intact colon. Secondly, analysis of DNA methylation changes (both globally, and in specific inflammation-associated genes (Ppara, Stat1 and Tap2)) in Il10-/- mouse colon showed that changes in total DNA methylation were correlated with changes in global gene expression, and changes in Stat1 methylation during inflammation correlated with Stat1 gene expression. However, these techniques had limitations for obtaining a global overview of molecular changes (DNA methylation) in response to dietary intervention in established inflammation (LMD) and therefore were not applied in the dietary intervention study. Finally, diets containing goat and cow whole milk solids (40% w/w) fed for 6 weeks had anti-inflammatory effects in the colon of 11-12 week old Il10-/- mice, shown by a reduction in colitis severity and immune-related gene expression. Further research is required to elucidate the physiological and molecular mechanisms of these anti-inflammatory effects.
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    Experimental airway hypersensitivity in sheep : a model for asthma : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Pathology at Massey University
    (Massey University, 1990) Chen, Wangxue
    This study aimed to establish an animal model for human bronchial asthma using locally bred Romney sheep. It was then planned to determine whether or not morphological and inflammatory factors in the ovine respiratory tract are associated with a predisposition to allergic bronchial hypersensitivity induced by inhaled Ascaris suum antigen. The skin and airway responses to a commercial A. suum antigen were tested in adult Romney sheep from two local farms with and without previous exposure to pigs. Ninety percent of 101 adult sheep tested showed an immediate skin reaction, and about 70% of 43 adult sheep with positive skin reactions showed an immediate airway response, reflected as a significant increase in airway resistance and/or decreased dynamic lung compliance. Among these 43 sheep, 21 showed changes in both airway resistance and dynamic lung compliance (Group A); ten only in dynamic lung compliance (Group B) and 12 were non-responders (Group C). No significant changes were recorded when the same animals were given an aerosol of phosphate buffered saline. Although the sheep with previous exposure to pigs showed significantly greater skin reactions than those without exposure to pigs, they showed no significant differences in airway response to antigen inhalation. In addition, there was no correlation between the degree of skin reaction and the magnitude of bronchoconstriction. Since no information was available on the respiratory tract-associated lymphoid tissue and cells in healthy sheep, study of this tissue and its associated epithelium was a prerequisite for studies of the morphological and inflammatory mechanisms involved in the development of allergic airway hypersensitivity. The ovine respiratory tract has five forms of lymphoid tissue; intra-luminal, intraepithelial, scattered forms, and dense and nodular aggregations; the dense and nodular aggregations being confined to the pharyngeal tonsil and bronchioles. Morphologically well-developed lymphoepithelium (M cells) is present only in the pharyngeal tonsil region, and absent in the lower respiratory tract. The M cell of the ovine pharyngeal tonsil is ultrastructurally and functionally similar to that in other mucosal tissues of this and other species, but its development and maturation takes place earlier than the bronchus-associated lymphoid tissue. Mast cells in the lower respiratory tract of normal sheep are morphologically heterogeneous, and both formalin-sensitive and formalin-resistant types can be identified. The morphological and histochemical features of formalin-sensitive mast cells are similar to those from the human respiratory tract in several respects which enhances the use of the sheep model in the study of human allergic respiratory disease. A morphometric comparison of airway structure and inflammatory components was conducted between the three groups of sheep with varying airway hypersensitivity. The epithelium of the small airways was significantly thinner and contained fewer goblet cells in the hypersensitive sheep (Groups A and B; than in non-reacting sheep (Group C). Mast cells from the hypersensitive sheep had a significantly greater volume density of secretory granules than those from non-reacting sheep. However, no morphological difference was found in the epithelial integrity of airways between hypersensitive and non-reacting sheep, and the permeability of tracheobronchial epithelium to horseradish peroxidase was of the same order in all groups. Similarly, the airway wall was not significantly thicker in hypersensitive sheep than in non-reacting sheep, and the shortening of smooth muscle required to cause complete airway closure was similar. The numerical density of mast cells, eosinophils, neutrophils and lymphocytes in the airways and lung was not significantly different between the groups. These observations indicate that the Ascaris-induced airway response seen in Romney sheep is similar in several respects to that seen in human asthmatics and these sheep can therefore be used as an animal model to study human asthma. The current findings suggest that the presence of relatively low goblet cell density, thin epithelium, and high volume density of mast cell secretory granules in the small airways and lung may be important inherent factors responsible for the development of airway hypersensitivity in these sheep. It is concluded that most of the other morphological features observed in asthmatics and animal models are likely to be the result of allergic airway reactions rather than a fundamental difference between potentially allergic and non-allergic subjects.
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    Biochemical studies on animal models of ceroid-lipofuscinoses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Veterinary Pathology, Massey University
    (Massey University, 1990) Martinus, Ryan Dennis
    The ceroid-lipofuscinoses are recessively inherited lysosomal storage diseases of children and animals, characterised by brain and retinal atrophy and the accumulation of lipopigment in a variety of cells. A systematic study of isolated lipopigment from an ovine form of the disease had shown the major stored components to be proteinaceous. This thesis presents further characterisation and identification of the stored ovine lipopigment proteins. Separation of the lipopigment proteins by LDS-PAGE showed the presence of the 3.5 kDa and 14.8 kDa proteins noted in earlier studies, and an additional band at 24 kDa. The 14.8 and 24 kDa bands varied between preparations and from different gels of the same isolate. Radioiodination of lipopigment and silver staining of the proteins separated by LDS-PAGE indicated that the 3.5 kDa protein was the dominant protein component. As these proteins were unable to be separated from each other, exploitation of the molar dominance of the 3.5 kDa protein led to its identification by a non traditional sequencing approach. The major stored protein was shown to be the full proteolipid subunit c of the mitochondrial ATP synthase complex. The 14.8 and 24 kDa proteins were shown to be stable oligomers of subunit c. Quantitaion of the sequence data showed that subunit c accounted for at least 50% of the lipopigment mass. No other mitochondrial protein was detected. Analyses of isolated mitochondria showed that they were functionally normal and did not contain excess amounts of subunit c. Subunit c is classified as a proteolipid, due to its lipid-like solubility in chloroform/methanol mixtures. Its storage in lysosome derived lipopigment bodies explained many of the described physical characteristics of lipopigment in the ceroid-lipofuscinoses. Application of the same methodology showed that a bovine, and two distinct canine forms of the ceroid-lipofuscinoses were also subunit c storage diseases. It is postulated that the lesions in the ceroid-lipofuscinoses involve defects in the degradative pathway of subunit c at some point after its incorporation into the inner mitochondrial membrane.