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Has files: YesSubject: search.filters.subject.310101 Analytical biochemistry

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    Biochemical characterisation of six novel monoamine oxidase inhibitors identified in tobacco smoke : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Health Sciences at Massey University, Wellington, New Zealand
    (Massey University, 2023) Hong, Sa Weon
    Background: Tobacco smoking is supposed to be the most difficult addiction to give up, and nicotine has been observed as the main addictive agent found in tobacco smoke. However, research is showing that nicotine alone does not account for tobacco dependence. One hypothesis is that monoamine oxidase (MAO) inhibition from non-nicotine compounds in tobacco smoke contributes to nicotine addiction. Six new MAO inhibitors in tobacco smoke have been identified before this PhD project. Overall research aim: The overall research aim for this project was to characterize these six novel monoamine oxidase (MAO) inhibitors identified in tobacco smoke and study their interaction with MAO A and B enzymes. Methods: First, non-nicotinic components of tobacco smoke were tested for MAO inhibitory activity, using the kynuramine assay and recombinant human MAO enzymes. Next, a centrifugation-ultrafiltration method and a time-dependent assay were used as the primary tests of reversibility of the phenolic compounds. Then, Lineweaver-Burke (LB) plots were prepared to understand the kinetics and mechanism of inhibition of recombinant human MAO enzymes by the polyunsaturated fatty acids (PUFAs). Finally, molecular docking and in silico studies using SwissADME and PreADMET web tools were performed. Results: Catechols and hydroquinone showed potent irreversible MAO A inhibition. Among these, 4-methylcatechol displayed the highest activity for MAO A with an IC₅₀ value of 0.267 μM after 1h preincubation. Two PUFAs, α-linolenic acid and linoleic acid displayed potent inhibitory effect for MAO A with IC₅₀ values of 15.74 and 23.8 μM, respectively. Kinetic analysis revealed that α-linolenic acid and linoleic acid are competitive inhibitors of MAO A and MAO B. Molecular docking studies suggest that ternary complexes [MAO B-linoleic acid₂ species (EII)] may be formed. Conclusions: This PhD project showed that six novel MAOIs in tobacco smoke inhibited human MAO A and MAO B isoenzymes. The catechols and hydroquinone are irreversible MAO inhibitors, suggesting they may play a role in contribution to the addictive effects of nicotine and the low incidence of Parkinson’s disease in smokers. In addition, α-linolenic acid and linoleic acid are found to be reversible MAO inhibitors, suggesting these PUFAs may play a role in the lower MAO levels or activity in smokers. Overall, these findings suggest that MAO inhibitors from tobacco smoke may have pharmaceutical possibilities, perhaps in smoking cessation, or in relief of anxiety or depression or in Parkinson's and Alzheimer disease.
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    A novel and environmentally friendly method for preserving and depilating sheepskin : comprehensive physical, biochemical and molecular analyses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2021) Tu, Yi-Hsuan
    The first step of leather processing, depilation – in other words, removing hair from skins – accounts for one-third of the leather-making industrial waste due to the production of sulfide and alkaline water waste from the process. This study describes a method that preserves and depilates unwashed sheepskins using milk or milk by-products, including whey and permeate. It doesn’t require the use of harsh chemicals or temperature control, and results in skin that is ready to be tanned. In order to evaluate the products of this process, the depilated skin surface was evaluated not only by eye, but with scanning and transmission electron microscopy (SEM and TEM) which showed there was no apparent damage to the grain or fraying of the collagen bundles. The depilated skin was also processed to leather which was subjected to tear, tensile and shrinkage measurements which were shown to be almost identical to leathers made using the traditional process. Quantitative biochemical analyses, including collagen quantitation, collagen crosslink analysis and glycosaminoglycan (GAG) analysis of sheepskins depilated with this process showed no significant differences in both collagen, and collagen crosslink concentrations in contrast to the 10-fold increase seen in the GAG concentration. A quantitative proteomic analysis showed there was a higher retention of proteins found in the basement membrane of the skin, supporting the observation that permeate depilated skins were smoother than their sulfide depilated counterparts and produced leather with a superior surface. It also showed the loss of specific proteins around the hair follicle, hinting at a possible mechanism for depilation. To further investigate this a microbiological survey of the process was conducted. Traditional culturing methods were used to isolate and identify microorganisms present in the depilation solution after the wool had been removed. Two bacterial species (Lactococcus lactis and Lactobacillus plantarum) and two fungal species (Geotrichum candidum and Yarrowia lipolytica), were routinely identified, all of which are known to secrete a number of hydrolytic enzymes and antimicrobial compounds. This was followed by a metagenomic study of changes in the microbial community over the time course of the depilation. Although there were only 13 dominant bacterial genera identified during this study, the biggest change was a concomitant increase in the relative abundance of Lactococcus lactis that matched the decrease in Acinetobacter sp. by the end of the depilation treatment, controlling the proliferation of other putrefying organisms. In conclusion, this preliminary study has paved the way for the development of a depilation process that is kind to the environment, but more research is needed to investigate its potential use with other animal skins.
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    Unravelling the molecular contributions to collagen higher order structure : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2019) Visser, Danielle Renee
    Abnormal levels of cross-linking in fibrillar collagen strands have been shown to cause a number of human and animal diseases. Cross-linking is a vital step in fibrillogenesis and contributes greatly to the structural integrity of collagenous tissues. Conversely, defects in cross-link formation can significantly alter fibrillar organisation and lead to pathogenesis. Because collagen cross-links form on collagen-specific hydroxylated lysine residues, an understanding of the link between hydroxylysine and cross-link concentrations is needed to determine whether the level of hydroxylysine, the stereochemistry of these hydroxylysine residues, or other post-translational modifications such as glycosylation affect the level of cross-linking in tissue. While some research has been done to elucidate the connection between the two in different tissue types from the same animal, little has been undertaken to relate hydroxylation and glycosylation of lysine and hydroxylysine to the concentration and types of cross-links in different species. Furthermore, no research has been done to compare the relative distribution of diastereomers of hydroxylysine even within the same species. In order to make a valid comparison, collagen needs to be purified from skin to a high degree and separated into different collagen types and sub- structures as much as possible. To achieve this, the extraction and purification of collagen from the skins of four different mammalian species displaying different skin tensile strengths has been optimised. Different extraction methods were used to prevent the loss of specific features of the collagens that were characterised that may otherwise be lost. Amino acid analysis revealed that while the ratios of the two hydroxylysine diastereomers differed between different animals and extraction methods, the differences were not significant. Mass spectral analysis of cross-links showed that goat skin differed from the other three animals in its cross-link profile. Amino acid analysis combined with mass spectral analysis revealed that on average 70% of proline residues were hydroxylated, a figure much higher than previously thought. Mass spectral analysis also revealed that there are some differences between the glycosylation pattern of different animals, and the ratios of the different types of collagen which are extracted from each animal. While these findings need to be confirmed, they challenge some long held beliefs about the collagen molecule and provide a firm foundation for future work.