Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Filters

2005 - 200912010 - 20192

Settings

Subject: search.filters.subject.Calcium in the body

Search Results

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Functional consequences of RyR₁ variants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2019) Parker, Remai
    Malignant hyperthermia (MH) is an uncommon pharmacogenetic disorder that is asymptomatic until triggered by volatile anaesthetics or depolarising muscle relaxants. Exposure to such a trigger can result in a potentially fatal hypermetabolic crisis in an MH-susceptible individual. With prior diagnosis, MH episodes can be avoided by using alternative anaesthesia. Diagnostic testing requires a morbidly invasive muscle biopsy for those considered at risk based on family history. Linkage of MH-susceptibility to variants in the skeletal muscle calcium release channel ryanodine receptor 1 (RyR₁) has provided an opportunity for DNA testing as an alternative to the muscle biopsy. DNA-based diagnosis is severely limited by the number of diagnostic mutations identified—only 50 mutations have been established as MH-causative from over 300 genetic variants associated with the disorder. Moreover, DNA testing may only diagnose an individual as MH-susceptible; a negative DNA test is insufficient under current guidelines for a negative MH diagnosis. The purpose of this study was to develop molecular tools to investigate the hypothesis that RyR₁ variants associated with MH-susceptibility cause dysregulation of calcium release from intracellular stores. Two experimental approaches were followed with the objective of expanding the capabilities of DNA-based diagnosis for MH. The first technique was the generation of mammalian cell lines stably expressing recombinant RyR₁ variants by use of the Flp-In™ T-REx™ system from Invitrogen, followed by functional analysis. Four of five genetic variants associated with MH or myopathy had altered sensitivities to an RyR₁ agonist and therefore meet the criteria for use as diagnostic variants for MH-susceptibility. The second molecular technique explored was gene editing, with the aim of showing that a single nucleotide change was both necessary and sufficient to cause MH-susceptibility. This was developed by introducing a well-characterised MH-causative variant into the genome of a human skeletal muscle cell line. Preliminary results indicated that gene editing was successful.
  • Thumbnail Image
    Item
    The role of vegetables in the maintenance of acid-base balance and bone structure : a thesis presented in partial fulfillment of the requirements for the degree of Masters of Science in Nutritional Science at Massey University, Turitea, Palmerston North, New Zealand
    (Massey University, 2005) Singh, Sewakh
    Metabolic acidosis may over time lead to osteoporosis by causing a release of calcium and other mineral phases from bone. The regular consumption of fruits and vegetables is believed to be associated with higher bone mineral density. In the last ten years various population-based studies have found positive effects of fruit and vegetable intakes on bone health. The aim of the present study was to investigate the effects of broccoli, onion, and potato on bone density and strength in male rats. Forty male Spraguc-Dawlcy rats were randomized into four equal groups of ten each. The animals were fed either a base, broccoli, onion, or potato diet for a period of eight weeks. The apparent percentage calcium and phosphorus retained, the serum type 1 collagen C-tclopcptide concentration, bone density and bone strength, and the urinary parameters i.e. ammonia, creatinine, urea, specific gravity and osmolality were determined. The groups on onion and potato diets had significantly higher apparent percentage calcium retained (over the balance period) than the group on control diet (p=0.02l and 0.008 respectively). Apparent percentage calcium retained was also significantly higher in the group on potato diet compared to the group on broccoli diet (p"0.037). There were no significant differences between groups for percentage phosphorus retained on ANOVA. However, the discriminant analysis (multivariate method) showed that the group on the broccoli diet retained significantly more phosphate over the balance period compared to the other groups. The urinary ammonia excretion (over the balance period) was significantly lower in the group on broccoli diet than in the groups on base and potato diets (p=0.040 and 0.055 respectively). As for the urinary urea excretion over the balance period, the group on base diet had significantly higher urea excretion than the groups on onion and potato diets (p=0.002 and p=0.000 respectively). Urinary urea excretion (over the balance period) was also significantly higher in the group on broccoli diet compared to the groups on onion and potato diets (p=0.005 and 0.000 respectively). The differences between groups for the volume of urine produced over the balance period were also significant i.e. the group on broccoli diet produced significantly more urine than the groups on base, onion, and potato diets (p=0.011. p=0.008. and p=0.00l respectively). However, there were no significant differences between groups for urinary specific gravity, osmolality, and creatinine, and bone density, bone breaking strength, and serum type I collagen C-telopeptide concentrations on ANOVA. In conclusion Ig of broccoli per day significantly reduced urinary ammonia excretion and increased apparent percentage phosphorus retained whereas Ig of onion or potato per day significantly increased apparent percentage calcium retained in growing male rats. The decrease in urinary ammonia excretion was most likely due to the buffering of metabolic acids by the bases present in broccoli resulting in decreased ammonia production and secretion. Similarly the increased apparent percentage phosphorus retained (in the group on broccoli diet) may be due to the bases present in broccoli that may have buffered metabolic acids thereby reducing the need for phosphate buffering. This increased apparent percentage phosphorus retained may also be due to a high pH which is known to enhance renal phosphate uptake. The buffering of metabolic acids by the bases present in onion and potato may have reduced the need for calcium buffering resulting in higher apparent percentage calcium retained in the groups on onion and potato diets. Thus broccoli, onion, and potato intake may protect against the bone depleting effects of an acidogenic diet and may also have the potential to increase bone mineral density.
  • Thumbnail Image
    Item
    Characterisation of C-terminal RyR1 variants linked to neuromuscular disorders : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2015) Parker, Remai
    Intracellular calcium influences a large array of cellular processes in skeletal muscle cells and as a result the movement of free calcium is tightly-regulated by a diverse set of calcium channels and accessory proteins. The main store of calcium in skeletal muscle cells is the sarcoplasmic reticulum, from which the ryanodine receptor one calcium channel (RyR1) controls calcium release. Changes in calcium homeostasis often result in the manifestation of neuromuscular disorders, most notably central core disease (CCD) and malignant hyperthermia (MH). While CCD is usually apparent from the presence of certain physical characteristics, MH is typically asymptomatic unless exposed to a trigger, at which point the disease rapidly manifests as a crisis event which is potentially fatal. Currently, the diagnosis of these disorders requires the testing of a muscle biopsy, which is an expensive and invasive procedure, and thus a genetic test would be an ideal diagnostic alternative. For the most part, CCD and MH cases are linked to the inappropriate release of calcium by defective RyR1 channels – located in the calcium storage organelle membrane – but both are complex disorders with variable penetrance and genetic heterogeneity. A hypoactive RyR1 is thought to cause CCD while a hyperactive RyR1 is thought to cause MH, and yet individuals have been observed to be carriers of both diseases. Most of these instances have been linked to variants in the C-terminal region of RyR1, corresponding to the transmembrane portion of the channel. This research described in this thesis focused on the functional analysis of five C-terminal domain RyR1 variants identified in patients with neuromuscular disorders. The ability of the variant RyR1 channels to release calcium in response to a stimulus in a heterologous system was measured and compared with that of the wild type channel. Moreover, one of these variants was also examined in several B-lymphoblastoid cell lines taken from carriers of the variant. Of the five variants tested in the heterologous system, four different phenotypes were observed, reinforcing the theory that these disorders are caused by a variety of factors that combine to produce a complex phenotype.