Over the past several years, fungal species’ (spp.) with the capability to infect humans have caused an estimated 1.6 million deaths annually, and are threatening bats, amphibians, and reptiles with extinction (Frick et al., 2010; Brown et al., 2012; Casadevall, 2017). Additionally, contamination of crops by fungal pathogens causes a loss of a third of all of the crops grown annually, thereby causing billions of dollars in economic losses on an annual basis (Fisher et al., 2012; Almeida et al., 2019). A common crop contaminant is the fungal genera comprised of many fungal pathogens, Alternaria. The overall objective of the present thesis was to use untargeted LC-MS metabolomics to further our understanding of their pathogenicity. Specifically, a robust profile for 60 strains of uncharacterized Alternaria spp. was generated which, when mined, revealed the presence of several unique secondary metabolites (SMs). Subsequent isolation, purification, and structural elucidation of those SMs revealed them to be the family of curvularins. Mining the genome of the isolates producing those curvularins revealed the biosynthetic gene cluster (BGC) necessary for the production of the curvularins. Generating sequence similarity networks (SSNs) allowed for the investigation of the similarity of the BGC necessary for the synthesis of the curvularins, across several pathogenic fungal spp.

No description available.

Metabolites are a potentially useful source of detecting and identifying disease specific biomarkers. This thesis investigates the possibility of using metabolomics applications to detect Alzheimer’s disease associated metabolite peaks in patients and to detect longitudinal changes of the disease. Serum samples and clinical data were collected from 60 healthy controls and 60 Alzheimer’s disease patients (60 at baseline and 60 at 12 month follow-up). The metabolic fingerprinting of serum samples using the FT-IR lacked discriminatory power to discriminate Alzheimer’s disease and non-disease samples due to the similar magnitude of biological and analytical variation. The metabolic profiling of serum samples using the GC-ToF-MS did not reveal any significantly altered metabolite peaks between the Alzheimer’s disease and non-disease groups. Metabolic profiling of serum samples using the UPLC-LTQ/Orbitrap-MS operated in the positive ionisation mode did not reveal any significantly altered metabolite peaks between the disease and non-disease groups. Up to twelve metabolite peaks were significantly altered in the Alzheimer’s disease baseline and follow-up samples, indicating a potential association with disease progression. Metabolic profiling of serum samples using the UPLC-LTQ/Orbitrap-MS operated in the negative ionisation mode did not reveal any significantly altered metabolite peaks between Alzheimer’s disease and non-disease groups. Three metabolite peaks were significantly altered in the Alzheimer’s disease baseline and follow-up samples, indicating a potential association with disease progression. Metabolic profiling of serum samples with the UPLC-LTQ/Orbitrap-MS may potentially be used to detect disease and disease progression associated metabolite peaks. The metabolite peaks require identification followed by a validation experiment.

Oral cancer in early stages (I and II) may be curable by surgery or radiation therapy alone but advanced stage disease (III and IV) has a relatively low survival rate. The pathogenic pathways that contribute to Oral Squamous Cell Carcinoma (OSCC) remain poorly characterised and the critical factor in the lack of prognostic improvement is that a significant proportion of cancers initially are asymptomatic lesions and are not diagnosed or treated until they reach an advanced stage. Hence, a clinically applicable gene expression signature is in high demand and improved characterization of the OSCC gene expression profile would constitute substantial progress. For OSCC, possible themes that might be addressed using microarray data include distinguishing the disease from normal at the molecular level; determining whether specific biomarkers or profiles are predictive for tumour behaviour; and identifying biologic pathways necessarily altered in tumourigenesis, potentially illuminating novel therapeutic targets. However, OSCC is a heterogeneous disease, making diagnostic biomarker development difficult. Although this phenotypic variation is striking when one compares OSCC from different geographic locales, little is known about the underpinning biological mechanisms. Cancer may be accompanied by the production and release of a substantial number of proteins, metabolites and/or hormones into the blood, saliva, and other body fluids that could also serve as useful markers for assessing prognosis, metastasis, monitoring treatment, and detecting malignant disease at an early stage. The primary aim of this thesis is to investigate metabolomic and transcriptomic profiles using multiple bioinformatics approaches and biological annotation tools in an attempt to identify specific biomarkers and prediction models for OSCC from each profile as well as from the interface outcomes of integrating the two platforms. Additional aims of the thesis go further to identify the mechanisms underlying the biological changes during tumorigenic transformation of OSCC, as well as to determine biological processes underpinning the heterogeneity of the disease among populations. Two review studies were carried out in this thesis. The review study of published transcriptomic profiles of OSCC specified several genes and pathways exhibiting substantially altered expression in cancerous versus noncancerous states across studies. However, the result of the review suggests not relying on the final set of genes published by the individual studies, but to access the raw data of each study and start subsequent analysis from that stage using unified bioinformatics approaches to acquire useful and complete understanding of the systems biology. The other review study focused on the metabolic profiles of OSCC and revealed a systemic metabolic response to cancer, which bears great potential for biomarker development and diagnosis of oral cancer. However, the metabolic signature still needs to improve specificity for OSCC from other types of cancer. In an attempt to detect a robust gene signature of OSCC overcoming the limitation of the transcriptomic review in accessing the raw data from the previous works, four public microarray raw datasets (comprising 365 tumour and normal samples) of OSCC were successfully integrated using ComBat data integration method in R software, determining the common set of genes, biomarkers, and the relative regulatory pathways possibly accountable for tumour transformation and growth in OSCC. Examination of the meta-analysis datasets showed several discriminating gene expression signatures for OSCC relative to normal oral mucosa; with a signature of 8 genes (MMP1, LAMC2, PTHLH, TPBG, GPD1L, MAL, TMPRSS11B, and SLC27A6) exhibiting the best discriminating performance and show potential as a diagnostic biomarker set. In addition, 32 biomarkers specific to OSCC and HNSCC were identified with the majority involved in extracellular matrix (ECM), interleukins, and peptidase activity where around 2/3 of them are located in the extracellular space and plasma membrane. Additionally, investigation of the interactive network created by merging metabolic and transcriptomic profiles highlighted the significant molecular and cellular biofunctions, pathways, and biomarkers distinguishing OSCC from normal oral mucosa. The results highlighted interactions of significantly altered expression of Dglucose, ethanol, glutathione, GABA, taurine, choline, creatinine, and pyruvate metabolites with the expressed PTGS2, IL1B, IL8, IL6, MMP1, MMP3, MMP9, SERPINE1, COL1A1, COL4A1, LAMC2, POSTN, ADAM12, CDKN2A, PDPN, TGM3, SPINK5, TIMP4, KRT19, and CRYAB biomarkers of OSCC. Such a pattern may represent a clinically useful surrogate for the presence of OSCC which might help in deciphering some of the obscure multifaceted mechanisms underlying carcinogenesis of OSCC which emerged from dysregulated genetic and metabolic system of the body. In an attempt to define pathways of importance in two phenotypically different forms of OSCC, transcriptomic analysis of OSCC from UK and Sri Lankan patients was undertaken. The development of OSCCs in UK and Sri Lankan populations appears largely mediated by similar biological pathways despite the differences related to race, ethnicity, lifestyle, and/or exposure to environmental carcinogens. However, results revealed a highly activated “Cell-mediated Immune Response” in Sri Lankan tumour and normal samples relative to UK cohorts which may reflects a role in resistance of patients to invasiveness, metastasis, and mortality observed in Sri Lankan relative to UK patients. In conclusion, multiple molecular profiles were able to identify a unique transcriptomic profile for OSCC and could further discriminate the tumour from normal oral mucosa on the basis of 8 genes. Altered expression of several metabolic and transcriptomic biomarkers specific for OSCC were identified, along with several dysregulated pathways and molecular processes found common in patient with oral cancer. Integrating both metabolomic and transcriptomic signatures revealed a promising strategy in analysing the concurrent perturbation in both genetic and metabolic systems of the body. Additional results revealed possible impact of specific supplementary dietary components in boosting the immune system of the body against invasion, progression, and metastasis of the disease. Further clinical studies are required to confirm and validate the current results.

Cannabis is widely consumed medically and recreationally due to the presence of cannabinoids, but the phytochemical complexity of different varieties and preparations is a major knowledge gap. This thesis investigated the phytochemicals present in thirteen different cannabis strains using untargeted and targeted phytochemical analysis to determine “strain” differences in cannabis tinctures and oils. In addition, the phytochemical differences between different oil products, namely oils extracted by ethanol and CO2 supercritical fluid, were also determined to evaluate different processing methods. It was found that inter-strain variability was more significant than the preparation methods due to the strain-specific presence of major cannabinoids, specifically THCA and CBDA. Furthermore, a processing step like drying removed phytochemicals contributing to strain differences, most notably terpenes. The results suggested that consumers can expect different strains and products to have different chemical profiles, as CO2 oils were found to be more chemically consistent across products than tinctures. Cannabis can be consumed in many different ways, and one popular mode of delivery is vaporization. Vaporization extracts active principles of cannabis with heated gas and could lead to a different phytochemical profile compared to the original flower counterpart. Consequently, the product label based on the raw material may not be representative of what is phytochemically available during consumption. The results of this study showed a reduction in available chemicals after vaporizing flower and oils, and little new chemical formation through this process. Decarboxylated cannabinoids were the most significant contributors to differences between pre and post-vaporized samples, and different phytochemistry composition was observed after vaporization. The results also demonstrated that vaporization reduces inter-strain and inter-product chemical diversity, but the content of the vapor can still be affected by the strain used. Furthermore, it showed that vaporization could extract phytochemicals differently from oils than flower material. This thesis provides a new understanding of phytochemical differences, extraction and vaporization processes of cannabis, and provides novel insights into cannabis for producers and consumers. Understanding the differences in chemical content of different types of concentrates can better inform producers and consumers about the products they make, sell and use. In addition, this thesis supports the use of vaporization as a harm reduction method for the consumption of cannabis, and increases understanding of cannabis vaporization. The information from this thesis contributes novel insights into cannabis research and provides a foundation for further studies.

Hagfish are the plesiomorphic sister group of all vertebrates. They are scavengers and many live at depths reaching thousands of meters. In addition, hagfish show the lowest metabolic rate as well as cardiac performance in vertebrates. This dissertation evaluated the biochemical characterizations of hagfish skeletal muscles related to the feeding apparatus and hagfish cardiac muscle associated with cardiac performance and deep-sea effects at the proteomic and metabolomic levels. In Chapter one and two, I found creatine kinase over-expressed in hagfish somatic muscle and deep-sea hagfish cardiac muscle, I suppose that this enzyme was important for utilization of stored phosphocreatine in deep-sea animals¡¦ somatic muscle and cardiac muscle. Over-expressed glycogen phosphorylase in hagfish dental and deep-sea hagfish cardiac muscle supposes these two types of muscles undergoing the anaerobic glycolysis. Compared to teleosts (cobia and tuna), TMAO and urea were higher in hagfish suggest their functions in hagfish cardiac muscle as osmolytes, however, higher TMAO but not urea in deep-sea hagfish, I suggest TMAO functions not only as an osmolyte but also physiological impacts in hagfish cardiac muscle for depth-related adaptations. I also found higher nebulin express in hagfish cardiac muscle and higher tropomyosin express in cobia and tuna cardiac muscles, thus their contractile differentiations were resulted from the protein-protein mechanism. This dissertation provides candidate proteins and metabolits involved in ecophysiological adaptation of hagfish skeletal and cardiac muscles.

1H nuclear magnetic resonance (NMR) metabolomics was used to determine the response of earthworm exposure to polycyclic aromatic hydrocarbons (PAHs) in contact and soil tests. Eisenia fetida is recommended for toxicology testing, but to date this species has not frequently been used in environmental metabolomic studies. The metabolic profile of E. fetida was characterized with the goal of using this species in metabolomic studies. Testing several individual solvents for earthworm tissue extraction indicated that D2O buffer extracted the highest concentration of the widest variety of earthworm metabolites. Sample preparation methods were evaluated to reduce variability and achieve reproducible control groups for use in metabolomic studies. 96h depuration and intact lyophilization of earthworms before homogenization resulted in the least variation between sample extracts. This sample preparation method was used to compare E. fetida and two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) and E. fetida had the most reproducible 1H NMR spectra. E. fetida was then used to identify the metabolic response after exposure to several concentrations of the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene and pyrene, individually and in mixtures. With exposure to individual PAHs in contact tests, earthworm responses were both PAH- and concentration- dependent. In earthworms exposed to PAH mixtures in contact tests, an increase in amino acids was measured. Furthermore, an increase in specific amino acids and a decrease in maltose were identified as potential indicators of sub-lethal phenanthrene exposure in soil. Lastly, the relationship between earthworm response and contaminant bioavailability in soil was tested. Contaminant bioavailability is typically assessed using indirect methods [e.g., ‘soft’ extraction techniques like hydroxypropyl cyclodextrin (HPCD) extraction]. However, it was found that the directly measured response of earthworm exposure to sub-lethal concentrations of phenanthrene in soil is related to both the total and bioavailable phenanthrene. This suggests there is potential for the use of 1H NMR metabolomics for the assessment of contaminant bioavailability. This thesis has demonstrated that E. fetida are suitable for metabolomic studies and has indicated that 1H NMR metabolomics may have potential for measuring and monitoring earthworm exposure to sub-lethal concentrations of PAHs.

1H nuclear magnetic resonance (NMR) metabolomics was used to determine the response of earthworm exposure to polycyclic aromatic hydrocarbons (PAHs) in contact and soil tests. Eisenia fetida is recommended for toxicology testing, but to date this species has not frequently been used in environmental metabolomic studies. The metabolic profile of E. fetida was characterized with the goal of using this species in metabolomic studies. Testing several individual solvents for earthworm tissue extraction indicated that D2O buffer extracted the highest concentration of the widest variety of earthworm metabolites. Sample preparation methods were evaluated to reduce variability and achieve reproducible control groups for use in metabolomic studies. 96h depuration and intact lyophilization of earthworms before homogenization resulted in the least variation between sample extracts. This sample preparation method was used to compare E. fetida and two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) and E. fetida had the most reproducible 1H NMR spectra. E. fetida was then used to identify the metabolic response after exposure to several concentrations of the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene and pyrene, individually and in mixtures. With exposure to individual PAHs in contact tests, earthworm responses were both PAH- and concentration- dependent. In earthworms exposed to PAH mixtures in contact tests, an increase in amino acids was measured. Furthermore, an increase in specific amino acids and a decrease in maltose were identified as potential indicators of sub-lethal phenanthrene exposure in soil. Lastly, the relationship between earthworm response and contaminant bioavailability in soil was tested. Contaminant bioavailability is typically assessed using indirect methods [e.g., ‘soft’ extraction techniques like hydroxypropyl cyclodextrin (HPCD) extraction]. However, it was found that the directly measured response of earthworm exposure to sub-lethal concentrations of phenanthrene in soil is related to both the total and bioavailable phenanthrene. This suggests there is potential for the use of 1H NMR metabolomics for the assessment of contaminant bioavailability. This thesis has demonstrated that E. fetida are suitable for metabolomic studies and has indicated that 1H NMR metabolomics may have potential for measuring and monitoring earthworm exposure to sub-lethal concentrations of PAHs.

No description available.

No description available.