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We present compelling evidence for the existence of an evolutionary adaptive response to viral agents such as SARS-CoV-2, that results in the human in vivo biosynthesis of a family of compounds with potential antiviral activity. Using nuclear magnetic resonance (NMR) spectroscopy, we detected a characteristic spin-system motif indicative of the presence of an extended panel of urinary and serum metabolites during the acute viral phase. The structure of eight of nucleoside analogues was elucidated (six of which have not previously been reported in human urine), and subsequently confirmed by total-synthesis and matrix spiking. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ and IL-10, suggest an association with the viperin enzyme contributing to an endogenous innate immune defense mechanism against viral infection., Acknowledgements: Acknowledgements: We thank The Spinnaker Health Research Foundation, WA, The McCusker Foundation, WA, The Western Australian State Government and the Medical Research Future Fund (EPCD000037 and MRF2014349) for financial support. We thank the Department of Jobs, Tourism, Science and Innovation, Government of a Western Australian Premier's Fellowship for RLL and the ARC for Laureate Fellowship funding for EH. JW thanks Ministerio de Ciencia, Tecnología e Innovación (Minciencias), Ministerio de Educación Nacional, Ministerio de Industria, Comercio y Turismo e ICETEX (792–2017) 2a Convocatoria Ecosistema Científico - Colombia Científica para la Financiación de Proyectos de I + D + i), World Bank and Vicerrectoría de Investigaciones, Pontificia Universidad Javeriana, Bogotá, Colombia (contract no. FP44842 - 221-2018). We gratefully thank the Werner Siemens Imaging Center under direction of Prof. Bernd Pichler for supporting this project as well as Aditi Kulkarni and Daniele Bucci for excellent technical assistance. JMW, GBE and LDH thank the New Zealand Ministry of Business Innovation & Employment for support (Endeavour Fund program contract UOOX1904 (NZ)).

Background: Public awareness of pharmacy services designed to support use of medicines is low, yet little is known about how the public view promotion of these services or their preferences for the attributes of pharmacies from which they would like to receive them.\ud Objective: To compare the public’s preferred attributes of pharmacies and methods for promoting medicine-related services with community pharmacists’ perceptions of their customers’ views.\ud Method: Parallel surveys of the general public, using a street survey, and community pharmacists, via a postal survey in South East England. \ud Results: Response rates were: public 47.2% (1000/2012) and pharmacists 40.8% (341/836) respectively. Pharmacists’ perceptions of customer preferences for using the same pharmacy, independent ownership and personal knowledge of the pharmacist were higher than actual public preferences. More pharmacists also thought approachability and previous good service would be important than the public. The public’s desires for long opening hours and for a pharmacy with a good relationship with their doctor’s surgery was higher than pharmacists believed. The majority of the public prefer not to interrupt a pharmacist busy in the dispensary, which was not perceived by pharmacists as a factor. Pharmacists’ perceptions aligned more with the preferences of regular medicines users and frequent pharmacy users. Both groups viewed direct recommendation as the most effective approach for promoting pharmacy services, particularly by doctors and pharmacy staff. Pharmacists’ expectations of the effectiveness of posters and mass media methods were much higher than those of the public. \ud Conclusions: Pharmacists and pharmacy owners must ensure good relationships with local medical practices to enable them to maximise opportunities for using the promotional methods judged most effective in encouraging use of medicine-related services. Staff must be approachable and enable access to pharmacists ensuring perceptions of pharmacist busyness are not a deterrent.

We propose a novel statistical approach to improve the reliability of (1)H NMR spectral analysis in complex metabolic studies. The Statistical HOmogeneous Cluster SpectroscopY (SHOCSY) algorithm aims to reduce the variation within biological classes by selecting subsets of homogeneous (1)H NMR spectra that contain specific spectroscopic metabolic signatures related to each biological class in a study. In SHOCSY, we used a clustering method to categorize the whole data set into a number of clusters of samples with each cluster showing a similar spectral feature and hence biochemical composition, and we then used an enrichment test to identify the associations between the clusters and the biological classes in the data set. We evaluated the performance of the SHOCSY algorithm using a simulated (1)H NMR data set to emulate renal tubule toxicity and further exemplified this method with a (1)H NMR spectroscopic study of hydrazine-induced liver toxicity study in rats. The SHOCSY algorithm improved the predictive ability of the orthogonal partial least-squares discriminatory analysis (OPLS-DA) model through the use of "truly" representative samples in each biological class (i.e., homogeneous subsets). This method ensures that the analyses are no longer confounded by idiosyncratic responders and thus improves the reliability of biomarker extraction. SHOCSY is a useful tool for removing irrelevant variation that interfere with the interpretation and predictive ability of models and has widespread applicability to other spectroscopic data, as well as other "omics" type of data.

Metabolic phenotyping of humans allows information to be captured on the interactions between dietary, xenobiotic, other lifestyle and environmental exposures, and genetic variation, which together influence the balance between health and disease risks at both individual and population levels.We describe here the main procedures in large-scale metabolic phenotyping and their application to metabolome-wide association (MWA) studies.By use of high-throughput technologies and advanced spectroscopic methods, application of metabolic profiling to large-scale epidemiologic sample collections, including metabolome-wide association (MWA) studies for biomarker discovery and identification.Metabolic profiling at epidemiologic scale requires optimization of experimental protocol to maximize reproducibility, sensitivity, and quantitative reliability, and to reduce analytical drift. Customized multivariate statistical modeling approaches are needed for effective data visualization and biomarker discovery with control for false-positive associations since 100s or 1,000s of complex metabolic spectra are being processed.Metabolic profiling is an exciting addition to the armamentarium of the epidemiologist for the discovery of new disease-risk biomarkers and diagnostics, and to provide novel insights into etiology, biological mechanisms, and pathways.

The metabolic effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on human blood plasma were characterized using multi-platform metabolic phenotyping with Nuclear Magnetic Resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). Quantitative measurements of lipoprotein sub-fractions, alpha-1-acid glycoprotein, glucose and biogenic amines were made on samples from symptomatic coronavirus disease 19 (COVID-19) patients who had tested positive for the SARS-CoV-2 virus (n = 17) and from age and gender-matched controls (n = 25). Data were analyzed using an orthogonal-projections to latent structures (O-PLS) method and used to construct an exceptionally strong (AUROC=1) hybrid NMR-MS model that enabled detailed metabolic discrimination between the groups and their biochemical relationships. Key discriminant metabolites included markers of inflammation including elevated alpha-1 acid glycoprotein and an increased kynurenine/tryptophan ratio. There was also an abnormal lipoprotein, glucose and amino acid signature consistent with diabetes and coronary artery disease (low total and HDL Apolipoprotein A1, low HDL triglycerides, high LDL and VLDL triglycerides). Plus, multiple highly significant amino acid markers of liver dysfunction (including the elevated glutamine/glutamate and Fischer’s ratios) that present themselves as part of a distinct SARS-CoV-2 infection pattern. A multivariate training-test set model was validated using independent samples from additional SARS-CoV-2 positive patients and controls. The predictive model showed a sensitivity of 100% for SARS-CoV-2 positivity. The breadth of the disturbed pathways indicates a systemic signature of SARS-CoV-2 positivity that includes elements of liver dysfunction, dyslipidaemia, diabetes, and coronary heart disease risk that are consistent with recent reports that COVID-19 is a systemic disease affecting multiple organs and systems. Metabolights study reference: MTBLS2014.

The aim of this thesis was to investigate the use of effort-based intensity prescriptions as a method to reduce individual variability and the occurrence of training non-response. Specifically, this thesis explores the physiological, psychological, and metabolomic responses to the interaction of training duration and effort-based intensity on a single-bout basis, whole session basis, and during chronic training intervention.\ud \ud The first experimental study (Chapter 4) investigated individual variability during self- paced exercise bouts at a rating of perceived exertion (RPE) of either 9, 13, or 17, conducted for either 1, 4, or 8 min. The study found that effort-based intensity prescriptions at higher RPEs and shorter durations result in lower levels of individual variability. The second study (Chapter 5) investigated individual variability during maximal isoeffort training sessions in either short interval (30 sec), long interval (5 min), or continuous exercise matched for total training duration. Long intervals displayed lowest variability in both how the session was performed as well as physiological response compared to short intervals and continuous sessions. The third study (Chapter 6) comprised a 6-week training intervention using maximal isoeffort intensity prescriptions using short (30 sec) or long interval (5 min) session formats, as well as a control group. Whilst short intervals resulted in higher levels of individual variability, a greater training response was found following this session format compared to both the long interval and control groups. The fourth study (Chapter 7) investigated the metabolomic differences between session formats, and between variable and consistent responding participants from Chapter 5. Distinct metabolomic differences were found between all session formats, and key metabolites were found relating to energy turnover, purine metabolism, and amino acid metabolism based on whether individuals were consistent or variable in session performance. The fifth study (Chapter 8) investigated the chronic changes in the urinary metabolome following the training interventions as described in Chapter 6. Several metabolomic markers differentiated between training responders and non-responders, in addition to metabolites associated with increased MMP or V̇O2max across all training groups.\ud \ud The main finding of this thesis was that the use of higher intensity effort-based targets and shorter interval durations has potential in reducing the occurrence of non-response to training.