NEW METHODOLOGIES FOR THE DETERMINATION OF AMMONIUM AND SYNTHETIC CANNABINOIDS IN FORENSIC SAMPLES

Forensic science is a discipline that combines the knowledge of chemistry, biology, pharmacology, statistics, mathematics, physics, and medicine, among others. The present doctoral thesis was focused in two areas of this wide discipline. In post-mortem chemistry, the estimation of the post-mortem interval (PMI) is done through the determination of potassium with capillary electrophoresis (CE). However, it is limited up to 100 hours, after which potassium levels reach a plateau. In forensic toxicology, the elucidation of metabolic pathways of emerging Novel Psychoactive Substances (NPS) is highly important to include those substances in screening methods that prove their consumption. Also, the consideration of different matrices for rapid NPS screening is another interesting topic for when the most common matrices, such as blood and urine, are unavailable for testing or are not enough. Therefore, in the present PhD thesis, three new methodologies are presented for the analysis of ammonium and synthetic cannabinoids in a variety of forensic samples. \ua8 The objective of the first project was the validation of a capillary electrophoresis (CE) method for the determination of ammonium in vitreous humour and the improvement of the estimation of the post-mortem interval (PMI). Vitreous humour samples were collected from a total of 38 medico-legal autopsies or external examination of corpses of violent or sudden deaths, in which the exact time of death was known. The vitreous humour samples were prepared by diluting 1:20 with the IS solution prior to analysis with capillary electrophoresis instrument. The results were analysed and interpreted on the basis of different statistical analyses: linear and polynomial regression and artificial neural networks (multivariate statistics). The findings show a wider PMI window of up to 168 hours which is useful for a much wider time span than that offered by potassium analysis (limited to about 90\u2013100 hours). Furthermore, the results show the potential usefulness of using ammonium and potassium in combination with ANN for inferring the PMI. \ua8 The aim of the second project was the elucidation of the 5F-APINAC metabolic pathway, which was a recently emerged NPS. Firstly, the possible metabolites were theoretically sketched using ChemDraw and scientific articles about substances with similar chemical structures. Secondly, in vitro experiments with HLMs and in vivo ones with rat urine after intravenous administration of the drug were carried out. Thirdly, samples were prepared for analysis with the Toxtyper instrument (LC-IT-MS) and results were confirmed by LC-QTOF. The theoretical metabolites were confronted with the experimental results and the metabolic pathway was elucidated. The metabolic pathway of 5F-APINAC was elucidated finding 15 different metabolites. It was found out that ester hydrolysis was the predominant metabolic reaction found in both in vitro and in vivo experiments. The discovered metabolites may serve for future studies and are likely to be incorporated into routine analytical screening methods as urine markers of 5F-APINAC consumption. \ua8 The objective of the last project was the development and validation of a fast and sensitive method for the screening of 13 new synthetic cannabinoids in human hair. The method was developed using the Toxtyper\uae LC/IT\u2013MS system with different sources (ESI, APCI, and IonBooster) and validated in terms of precision, bias, sensitivity, and matrix effects. The blank hair samples were spiked with 13 synthetic cannabinoids and underwent ultrasonic extraction. In addition, real hair samples from patients that have tested positive for other illegal drugs were tested. The results show the suitability of the developed methodology for the screening of 13 synthetic cannabinoids in human hair. In addition, it is high throughput and can be easily updated with the appearance of new synthetic cannabinoids in the market

Nature-based Interventions and the Environment-Microbiome-Health Axis

This interdisciplinary PhD is an innovative socioecological investigation into two key phenomena: 1. nature-based interventions, which are structured nature-based activities that aim to facilitate behavioural changes for the benefit of health and wellbeing; and 2. the relationship between the environment, the microbiome and human health. The aims of the PhD study include: o Determining the distribution of, and socioecological constraints and opportunities associated with nature-based interventions. o Ascertaining how engagement with nature may have supported population health during the COVID-19 pandemic, and whether the pandemic affected nature engagement. o Investigating the environment-microbiome-health axis, including a review of potential anthropogenic disruptions to this relationship, and determining the spatio-compositional and ecological factors that affect exposure to the aerobiome (microbiome of the air) in urban green spaces. o Determining whether relationships with and knowledge of biodiversity affect attitudes towards microbes. o The development of novel conceptualisations that transcend the boundaries of current knowledge, including Microbiome-Inspired Green Infrastructure (MIG) – multifunctional green infrastructure that aims to enhance ecosystem functionality and human health through considerations for the microbiome; the Lovebug Effect – microbially-mediated nature affinity; and, the Holobiont Blindspot – a newly proposed cognitive bias that may result from the failure to recognise the microbiome’s role in behaviour and health. This is a PhD by publication comprising 9 chapters and 12 publications. Chapter 2 presents publications on nature-based interventions (Robinson and Breed, 2019; Robinson et al. 2020a), and the potential health benefits of engaging with nature are presented in Chapter 3 (Robinson et al. 2020b). This is followed by an overview of the environment-microbiome-health axis (Robinson and Jorgensen, 2020) and how nature engagement may affect our attitudes towards microbes in Chapter 4 (Robinson et al. 2021e). This is followed by the aerobiome studies in Chapter 5 (Robinson et al. 2020c; Robinson et al. 2021b) and potential disruptions to the environmental microbiome and human health in Chapter 6 (Robinson et al. 2021c). Chapter 7 presents Microbiome-Inspired Green Infrastructure (Robinson et al. 2018; Watkins and Robinson et al. 2020), and finally in Chapter 8, novel conceptualisations are presented, including the Lovebug Effect (Robinson and Breed, 2020) and the Holobiont Blindspot (Robinson and Cameron, 2020). The methods used in the nature-based intervention studies included geospatial analysis using geographic information systems (GIS) and online questionnaires. The methods used in the microbiome studies included the creation of novel sampling methods to collect bacterial DNA in urban green spaces, DNA sequencing, and the application of bioinformatic tools. Although systematic reviews were not included, the reviews in this thesis did follow the preferred reporting system for systematic reviews (PRISMA) method to ensure robust data collection. This PhD makes several important contributions to knowledge. For example, constraints to nature-based interventions were identified, and these show that enhanced transdisciplinary collaborative pathways and efficient infrastructure are needed. The research also identified that people significantly changed their patterns of nature engagement during the COVID-19 pandemic and many visited nature for important wellbeing and coping benefits. This highlights the immense value of nature to health and wellbeing during unprecedented times. The research also revealed for the first time that attitudes towards microbes might be influenced by nature engagement and biodiversity literacy, and that anthropogenic pollution (light and sound) could affect the microbiome with important ecological and health implications. In the aerobiome studies, the cutting-edge discovery of ecological factors that drive aerobiome assembly and potentially affect exposure types and routes could have important implications for public health and landscape management. Nature-based interventions have the potential to enhance human and environmental health, and the environmental microbiome will likely play an important role. The novel conceptualisations developed during this PhD add new knowledge to the fields of landscape design, microbiome science, and environmental psychology and have stimulated new research agendas across the world. The work in this thesis is influenced by the emerging planetary health paradigm, the ‘interconnectedness’ and intrinsic value narrative associated with Indigenous cultures, and ‘systems thinking’ (a holistic analytical approach that focuses on the way a system’s constituent parts – – and how systems themselves –– interrelate). Fostering deep reciprocity with the natural world to enhance personal and planetary health has never been more important. We have a key opportunity to redefine our relationship with the wider biotic community by establishing a new appreciation of our ‘microbial friends’ and the profound interconnectedness between the environment, the microbiome and human health. List of publications in this PhD thesis: 1. Robinson, J.M., and Breed, M.F. (2019). Green Prescriptions and Their Co- Benefits: Integrative Strategies for Public and Planetary Health. Challenges. 10, pp. 1-14. Part of the Special Issue - The Emerging Concept of Planetary Health: Connecting People, Place, Purpose, and Planet. 2. Robinson, J.M., Jorgensen, A., Cameron, R., and Brindley, P. (2020). Let Nature Be Thy Medicine: A Socioecological Exploration of Green Prescribing in the UK. Int J Environ Public Health. 17, pp. 1-24. Part of the Special Issue "Planetary Health: From Challenges to Opportunities for People, Place, Purpose and Planet” 3. Robinson, J.M., Brindley, P, Cameron, R., MacCarthy, D., and, Jorgensen, A. (2021). Nature’s Role in Supporting Health During the COVID-19 Pandemic: A Geospatial and Socioecological Study. Int J Environ Res Public Health. 18, pp. 1-21. 4. Robinson, J.M., and Jorgensen, A. (2019). Rekindling Old Friendships in New Landscapes: The Environment-Microbiome-Health Axis in the Realms of Landscape Research. People Nat. 2, pp.339-349. 5. Robinson, J.M., Cameron, R., and Jorgensen, A. (2021). Germaphobia! Does our Relationship with, and Knowledge of Biodiversity, Affect our Attitudes Towards Microbes? Front Psychol, 12, pp. 1-10. 6. Robinson, J.M., Cando-Dumancela, C., Liddicoat, C., Weinstein, P., Cameron, R. and Breed, M.F. (2020). Vertical Stratification in Urban Green Space Aerobiomes. Environ Health Persp, 128, p.117008. 7. Robinson, J.M., Cando-Dumancela, C., Antwis, R.E., Cameron, R., Liddicoat, C., Poudel, R., Weinstein, P., and Breed, M.F. (2021). Urban Green Space Aerobiomes: Exposure to Airborne Bacteria Depends Upon Vertical Stratification and Vegetation Complexity. Sci Rep, 11, pp. 1-17. 8. Robinson, J.M., and Parker, B. (2021). The Effects of Anthropogenic Sound and Artificial Light Exposure on Microbiomes: Ecological and Public Health Implications. Front Ecol Evol. 9, pp. 1-7. 9. Robinson, J.M., Mills, J.G., and Breed, M.F. (2018). Walking Ecosystems in Microbiome-Inspired Green Infrastructure: An Ecological Perspective on Enhancing Personal and Planetary Health. Challenges. 9, pp.1-15. 10. Watkins, H., Robinson, J.M., Breed, M.F., Parker, B. and Weinstein, P. (2020). Microbiome-Inspired Green Infrastructure: A Toolkit for Multidisciplinary Landscape Design. Trends in Biotech. 38, pp.1305-1308. 11. Robinson, J.M., and Breed, M.F. (2020). The Lovebug Effect: Is the Human Biophilic Drive Influenced by Interactions Between the Host, The Environment, and the Microbiome? Sci Tot Environ. 720, p.137626. 12. Robinson, J.M., and Cameron, R. (2020). The Holobiont Blindspot: Relating Host-Microbiome Interactions to Cognitive Biases and the Concept of the “Umwelt”. Front Psychol. 11, p.591071