published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

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Transplantation is an acceptable treatment for end-stage lung and liver disease patients. In lung transplantation, long-term survival is limited due to Bronchiolitis Obliterans Syndrome (BOS) and in liver transplantation, Hepatitis C Virus (HCV) disease recurrence significantly impacts long-term survival. Treatment options are limited and often not successful. It is therefore important to conduct research on the factors contributing to the pathogenesis and disease severity of BOS and HCV to improve our understanding of the mechanisms and potentially reduce morbidity and mortality. Several factors may play a role. The focus of this thesis is to assess the role of Oxidative Stress (OxS) and nutrition on these patient populations. BOS is a frequent complication of lung transplantation. OxS may contribute to its pathogenesis and induce further tissue injury and inflammation. OxS can be influenced by several factors including nutrition. The cross-sectional study showed that BOS lung recipients have elevated markers of OxS in their Bronchoalveolar Lavage Fluid (BALF) compared to those without BOS. However, there was no difference in nutritional factors potentially affecting OxS. HCV reinfection post transplant is universal, significantly increasing morbidity and mortality. OxS is involved in the pathogenesis of chronic HCV but its role in HCV disease recurrence is unknown. A first study determined whether HCV liver recipients (HCV-LT) were more oxidatively stressed when compared to controls or HCV non-transplant patients. A second study assessed OxS at six-and 12 months post transplant and compared results between those with and without recurrence. The results showed that HCV-LT were more oxidatively stressed, vitamin A intakes were significantly lower and plasma gamma- tocopherol was significantly higher in HCV-LT. Additionally, those with recurrence were more oxidatively stressed at six-months (before recurrence) and 12 months compared to those without recurrence. No differences were seen regarding nutrition parameters. These results suggest that OxS is present in transplant recipients but that nutritional factors do not play a significant role. Other causes of OxS likely play a more significant role such as the presence of inflammation due to immunological reactions associated with BOS and the generation of reactive oxygen species (ROS/RNS) seen in patients with HCV disease.

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It is known that the birth process and initial life exposures, such as feeding, may have an important impact on the acquisition of bacterial communities throughout the human body, including the gut. Preterm infants usually have special dietary needs and undergo increased oxidative stress related to intensive care, which can ultimately impair their gastrointestinal microbial colonization and microbial diversity in the bowel. Using molecular techniques, we analyzed the fecal microbiome of 20 preterm infants and tested the association between bacterial communities and feeding type, as well as levels of F2-isoprostanes. We found that feeding influences the fecal microbiome of preterm infants, however more research is needed to clarify the role of human milk fortifiers in this process. We also observed preliminary evidence of an association between microbial composition and oxidative stress, indicating that future studies in this area should be conducted. / February 2017

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy stress that, once activated, promotes ATP-producing process while it switches off ATP-consuming pathways, in order to restore the cellular energetic balance under conditions of stress. Activation of AMPK is dependent on the phosphorylation of the residue Thr172 in its α subunit. This phosphorylation is generally mediated by the known tumour suppressor LKB1, but also CaMKKβ has been shown to phosphorylate AMPK. As its name suggests, AMPK is also activated by the binding of AMP to its γ subunit. This binding causes a >10 fold allosteric stimulation, promotes phosphorylation of Thr172 by upstream kinases and protects AMPK from dephosphorylation of Thr172 by protein phosphatase(s). In 2010 it was reported that oxidative stress mediated by H₂O₂ activated AMPK by increasing the cellular AMP:ATP and ADP:ATP ratios (Hawley et al, 2010). However, the same year another work suggested that the mechanism of activation of AMPK by H₂O₂was direct, independent of AMP and involved the oxidation of two cysteine residues in the α subunit of AMPK (Zmijewski et al, 2010). Given this discrepancy, here we provided evidence that H₂O₂, generated by addition of glucose oxidase in the cell medium, activates AMPK mostly through an increase of AMP:ATP and ADP:ATP ratios, as previously suggested in our laboratory. However, it seems that there might be a second, minor mechanism of activation that is independent of the changes in cellular nucleotides. This second mechanism was not identified in our previous work because we were not aware of how rapidly a single bolus of H₂O₂ can be metabolized by the antioxidant defences of the cell. We could not identify the alternative mechanism of activation by H₂O₂but showed that H₂O₂ could protect Thr172 from dephosphorylation, which might suggest a direct effect of H₂O₂ on the phosphatase(s) dephosphorylating AMPK. However, since the identity of this phosphatase(s) remains unclear, we could not rule out the possibility that the protection from dephosphorylation that we observed could still be mediated by the increase in AMP:ATP and ADP:ATP ratios. Moreover, it remains still possible that a direct effect of H₂O₂ on AMPK might be responsible for the small but significant activation we detected in cell expressing a nucleotides-insensitive mutant of AMPK. Recently, a new crystal structure of AMPK obtained by Xiao et al (2013) provided new insights about AMPK structure and regulation. In particular, the authors identified a new binding pocket located at the interface between the N-lobe of the α-kinase domain and the β-CBM of AMPK, which appeared to be the binding site for two direct activators of AMPK: A769662 and 991. Here we confirm that this novel binding pocket is indeed the binding site for both A769662 and 991, and provide evidence that another direct activator of AMPK, MT63-78, also binds at the same site. Mutation of two important residues in this pocket (Lys29 and Lys31 of the α2 subunit) abolished the allosteric stimulation of AMPK by A769662, 991 and MT63-78 while it had no effect on allosteric stimulation by AMP. However, we also showed that the same mutation abolished protection against Thr172 dephosphorylation not only by A769662, 991 and MT63-78, but also by phenformin and H2O2, which are known to activate AMPK by increasing the AMP:ATP and ADP:ATP ratios. These data show that the integrity of this pocket is important for the effect of AMP to protect against Thr172 dephosphorylation, but not for its ability to cause allosteric stimulation. Moreover, in HEK-293 cell stably expressing an α2 subunit carrying the mutation of both Lys29 and Lys31, the basal activity of AMPK due to Thr172 phosphorylation was almost 6-fold less than in cells expressing wild-type α2. This result pointed out for the first time that there might be a natural ligand binding in the newly discovered binding pocket that is not able to bind to the double mutant, explaining the difference in activity observed. However the identity of this possible natural ligand remains unclear and more studies will be necessary to uncover it.

Vitamin E (a-tocopherol) is the major lipid soluble antioxidant in vivo and is important for maintaining the integrity of cell membranes. Oxidative stress, defined as an imbalance between oxidants and antioxidants, has been implicated in the aetiology of numerous diseases. There is, therefore, interest in establishing methods to measure oxidative stress. It has been suggested that metabolites of vitamin E such as atocopheronolactone (a-TL), with an oxidised chroman ring, may be an indicator of in vivo oxidative stress and that the carboxyethyl-hydroxychromans (CEHCs), with a shortened phytyl side chain, may provide a measure of adequate or excess vitamin E status. However, doubts have been raised about the authenticity of a-TL since a-CEHC has been shown to be artefactually oxidised to a-TL in many of the procedures described. In the course of the current study a relatively simple method using gas chromatographymass spectrometry (GC-MS) was developed which allowed the reproducible measurement of a wide range of deconjugated vitamin E metabolites in urine. This method was used to study the urinary metabolites produced by normal subjects before and after supplementation with vitamin E. The CEHCs were confirmed as the major urinary metabolites of vitamin E, a-TL was detected and a novel group of metabolites, the carboxymethylbutyl-hydroxychromans (CMBHCs), was also tentatively identified. A range of conjugated (sulphated and glucuronidated) and free metabolites of vitamin E were synthesised chemically and used to a) confirm the identity of (x-CMBHC, b) provide standards for GC-MS and tandem mass spectrometry, c) elucidate the mechanism of artefactual oxidation and to develop new methods for the precise measurement of endogenously produced a-TL and d) investigate the type of conjugation of the various metabolites of vitamin E in human urine.