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Subject: search.filters.subject.310205 Proteomics and metabolomics

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    A proteomic investigation into the bacteriostatic mechanism of glycocin F, secreted by Lactiplantibacillus plantarum KW30 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2021) Stephens, Teiarere Te Horomai
    Antimicrobial resistance to clinical antibiotics has increased significantly over the past decade and continues to pose a serious public health concern worldwide. Due to this rise in antimicrobial resistance, clinical health professionals and scientific researchers alike have been eager to explore alternative options in place of traditional treatments (e.g. antibiotics). Bacteriocins are antimicrobial polypeptides secreted by bacteria. The therapeutic potential of these diverse natural products is largely unexplored and, as such, they constitute a novel source of potentially viable treatment options in the fight against resistant strains of pathogenic bacteria. At the most basic level, bacteriocins can be divided into two groups, those that are modified and those that are not. Glycocins fall into the former group as they are post-translationally modified by one or more monosaccharide moieties. These monosaccharides can be O-linked to the hydroxyl group of serine or threonine residues, or S-linked to the sidechain thiol group of cysteine residues. Glycocin F (GccF) is a doubly glycosylated bacteriocin secreted by the lactic acid bacterium Lactiplantibacillus plantarum (Lb. plantarum) KW30 that inhibits the growth of its bacterial targets. Although it is known that GccF is bacteriostatic, the exact mechanism by which it inhibits cell growth within two minutes, at nanomolar (nM) concentrations, remains unknown. Previous bacterial genomics, nuclear magnetic resonance (NMR) spectroscopy, total chemical synthesis and analysis of synthetic peptides, and transcriptomic studies have provided the framework for this present research. Collectively, these studies led to the proposal that GccF binds to the transmembrane domain of a specific N-acetylglucosamine (GlcNAc) phosphotransferase system (PTS) transporter on the surface of targeted bacterial cells, causing the amplification of a signal that results in the rapid onset of bacteriostasis. This project used proteomic methods to investigate changes that occurred in the proteome of susceptible cells when treated with GccF. Cultures were sampled immediately before, and then at specific timepoints after the addition of GccF. Sampled cells were separated into membrane and cytosol fractions, then analysed for changes in their proteomes. Changes in the abundances of specific target cell proteins were linked to biochemical pathways that may be affected by treatment with GccF, providing clues to its mechanism of action. The results showed clear changes in the abundance of proteins involved in cell wall metabolism and protein translation in GccF-treated Lb. plantarum ATCC 8014 cells.
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    A search for biomarkers of ovine pre-partum vaginal prolapse : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Biochemistry, Massey University, New Zealand
    (Massey University, 2019) Brown, Stuart
    Ovine pre-partum vaginal prolapse (known as bearings in sheep) occurs within a few weeks prior to lambing and unless treated both ewes and unborn lambs will die. Rates of prolapse in New Zealand vary from 0.1 to 5% per annum, varying between season and farms and is a worldwide problem. Much research has been undertaken over many years to determine the cause of this condition but no clear etiology has emerged. In this study plasma samples were collected prior to prolapse occurring in order to determine physiological changes leading to prolapse. 650 ewes were ear tagged and blood sampled on one day prior to lambing, 28 of these ewes subsequently prolapsed. The date of occurrence and tag number of prolapsing ewes was recorded to enable a comparison of the plasma profile of prolapsing ewes and non-prolapsing ewes. An improved method for running sheep plasma on 2D gels was developed resulting in improved protein spot resolution along with a lower coefficient of variation for spot volume. Using this improved method samples were subjected to 2D DIGE (two dimensional differential in gel electrophoresis) to determine if there were differences between the two groups of ewes. One of the differences was in haptoglobin, a major acute phase protein in ruminants, in which some isoforms were upregulated approximately 3 fold prior to prolapse occurring. This may indicate an inflammatory response due to either infection or injury. A good correlation was found between total haptoglobin spot volume data and quantitative haptoglobin assay data from the same samples (r² = 0.91) validating the haptoglobin gel spot data. Another finding was that alpha-1B-glycoprotein was down regulated close to prolapse, however the biological significance of this is unknown. It was also found that there was a negative correlation between cortisol and days to prolapse from sampling (r² = 0.36) i.e. ewes closest to prolapse had higher plasma cortisol concentrations than controls. These findings in conjunction with a literature search, field observations and an argument from logic lead the author to propose that chronic stress or anxiety may raise intra-abdominal pressure and contribute to the development of prolapse.