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The purpose of the research outlined here is to study human exposure to cadmium from rice products. The origin of rice products is an important factor in determining the content of cadmium in rice, and cadmium in rice can be greatly reduced by combining procurement and post-harvest processing. The difference in the cadmium content of different types of rice products is also obvious. Choosing rice products with lower cadmium content in food purchase is useful for reducing the body's cadmium intake amount. Groups which are vulnerable to exposure risk by cadmium also deserve attention, such as infants and children. It should be noted that the interest of food safety agencies and experts all over the world in the study of cadmium exposure risk has been increasing. Compared with arsenic, at the same time, the scope of the investigation on the content of cadmium in rice is narrow, and the amount of data is small, and the samples are not diverse enough. We will investigate the content of cadmium in rice worldwide at first, and consider solutions to reduce human cadmium intake amount and rate after obtaining sufficient data. Besides, there are effective methods to reduce the content of cadmium in rice products during not only the rice growth stage but also product processing stage because of the high accumulation efficiency of cadmium in rice plants.

Rice is the staple food of many Asian countries including Sri Lanka. Arsenic and other micropollutants accumulation in rice grain is becoming a serious issue due to several reasons. Some of these micropollutants are highly carcinogenic even in trace amounts. Moreover, People who use rice as their staple food has a high per capita use and therefore there is a tendency to get accumulated those micropollutants in considerable amounts in consumer's body and cause various health problems. On the Other hand, rice is the major source of essential micro elemental nutrients for those people. Mobility and the availability of those trace elements, uptake of trace elements by rice plants and transferring them into rice grain is not fully understood yet. This study was mainly focused on the variability of metal(loid)s in rice, based on the growing regions linking with edaphic and microbial factors that would regulates those variabilities. Paddy soils and associated paddy samples were collected from various geographical locations ana analyzed for trace elements and As species in those samples. Soil and porewater chemical parameters such as pH, Eh, SOC were measured and kinked with element concentrations in soil and porewater. Moreover, bacterial DNA was extracted from paddy soils and log2 copy number of 16S rRNA and arsM genes were detected using qPCR. Those results were linked with the soil chemical parameters. The results of this study suggested that edaphic factors such as pH and Eh control the availability and mobility of trace elements in paddy soil. Furthermore, this study revealed that 16S rRNA and arsM copy number depend on the edaphic factors as well as paddy management practices.

Paddy management practices greatly influence arsenic biogeochemistry. For sustainable rice production, rice needs to be free from arsenic. Different soil amendments such as animal manure in flooded paddy soil could enhance arsenic reductive mobilization, biomethylation, and volatilization which could be a useful option for the arsenic detoxification process. Also, arsM genes in composting manure could enhance arsenic methylation. Furthermore, cattle manure also enhances arsenic mobilization and increased arsenic assimilation in rice grain in some regions. However, the application of biomass ash, a source of silicon (Si) could be beneficial for rice growth, as silicate competes with arsenite for root uptake and hence has the potential to decrease translocation of arsenic into the rice grain. In contrast, the application of chemical fertilizer is not sustainable and could lead to long-run problems. As such, it is hypothesized "do different fertilizers affect the rice paddy microbiome and influence arsenic biogeochemistry, arsenic speciation, methylation and ultimately assimilation of arsenic into rice grain?" Thus, the objectives of this research were to ascertain the effect of different soil amendments on arsenic methylation and mobility in paddy soil and rice arsenic accumulation over time. In addition, how soil amendments affect the abundance and diversity of the arsM, 16S rRNA bacterial and ITS1 fungal genes in the rice microbiome was also investigated. Therefore, the research approach was to set up a rice microcosm experiment in a growth cabinet with a rice cultivar, 'Yongyou12' (a Japonica indica hybrid) with application of five different treatments- mineral fertilizers, full-slurry, half-slurry, ash and no application (four replicates per treatment), were applied to study the arsenic biogeochemistry and microbial population in rice paddy microbiome. Rice soil porewater chemical characterization at 7, 14, 28, 56, 84 d, during heading (99-126 d), grain fill (107-134 d) and dry stage (145-170 d) showed a highly significant effect of time on overall soil and arsenic biogeochemistry, while different soil treatments resulted in a significant response only for pH, Eh, Asi, B, Zn, Rb, Pb. Rice porewater arsenic species (Asi and MMA, DMA and TMAO) showed a strong correlation with different porewater properties, B, P, Mn, Fe, Co, Cu, Zn, Se, Rb, Sr, Mo, Cd and Pb. In rice shoot, overall treatments had a significant response for B, P, Cr, Mn, Fe, Co, Ni, Zn, Rb, Cd, S, K, Na and Sn; while in rice grain, the effects were not pronounced. The 16SrRNA and arsM bacterial abundance in rice rhizosphere soil at 14 d, 56 d, grain fill (107-134 d) and dry stage (145-170 d) revealed a significant increase in both arsM and 16S rRNA gene copy number over time. The highest bacterial abundance was observed during the grain fill (107-134 d) compared to other time points. For treatments, a significant increase in 16S rRNA gene copy number was identified in response to the full slurry treatment. Amplicon sequencing at the grain fill stage of both the bacterial 16S rRNA and fungal ITS1 genes showed that diversity significantly varied among the three compartments, rice rhizosphere soil, root and shoot. Soil showed the highest level of richness and diversity, followed by the rice root, while a low level of richness and diversity was seen in the shoot. The most pronounced treatment effects on microbial operational taxonomic units (OTU's) were observed for soil bacterial OTU's. In most instances, copy number was increased in response to full-slurry and/or half-slurry treatment or decreased in response to ash, full-slurry and/or half-slurry, while mineral fertiliser had no or little effect. For bacterial OTU's, Clostridium was the most relatively abundant genus in rhizosphere soil and shoot and Geobacter was the most relatively abundant in rice root. Geobacter, Clostridium, Anaeromyxobacter, Opitutus, Desulfocapsa, Methylocystis and Gemmatimonas were relatively abundant in all three compartments. Some of these have arsenic detoxification and methylation and could control the N, C and Fe cycling. The statistical analysis identified a significant increase in response to the manure treatment for several genus-level bacterial OTU's in soil (Sarcina, unclassified genera within the phylum Cyanobacteria, Bacteroidetes, the order Clostridiales and the family Rhodothermaceae). These could be involved in arsenic methylation and volatilization as found in the literature. For fungal ITS1, Ascomycota annotated OTU's was identified as the most abundant phylum in all three compartments. Pseudeurotium, Talaromyces, Cladosporium and Mortierella were relatively abundant genus in rhizosphere soil, Cladosporium, Fusarium, Pseudeurotium, Sarocladium, Talaromyces and Zopfiella in root and Cladosporium, Penicillium, Pseudeurotium in the shoot. Statistical analysis identified only a small number of fungal OTU's with significant treatment effect. Application of full-slurry, half-slurry, and ash showed much stronger effect compared to the chemical fertilizer in controlling rice arsenic chemistry and could be applied after filed trial. It is clear that the manure, ash, and half-slurry had some significant effect and could be beneficial to increase arsenic methylating bacteria and fungi in rice microbiome.

Presence of arsenic in paddy soils in Bangladesh has become a major concern, with Holocene soils reported to have higher levels of arsenic compared to Pleistocene. However, to date there is limited data on how geomorphology, land use and soil microbes drive bioavailability of arsenic in the paddy soil in Bangladesh. In the present study, 16S rRNA amplicon sequencing and metagenomics (Illumina), alongside chemical analysis in paddy and nonpaddy soils, from Holocene and Pleistocene regions, of Bangladesh to investigate microbial composition and functional diversity of arsenic metabolism genes and how this may be related to differences in arsenic dynamics within these soils. Compared to Pleistocene soils, the Holocene soils had higher concentrations of arsenic, other elements and pore water arsenic species, the latter dominated by iAs. Further to that, paddy soils were shown to be elevated in arsenic and soil solution arsenic species compared to nonpaddy soils. Based on 16S rRNA amplicon sequencing and metagenomics the dominant bacterial phyla common to all soils were Proteobacteria, Acidobacteria, Actinobacteria, Chloroflexi and Firmicutes but the overall diversity of bacteria was shown to be significantly higher in Holocene compared to Pleistocene soil. Metagenomics indicated that Geobacter, an anaerobic arsenic resistant organism, was most abundant in Holocene paddy soil. The functional genes related to arsenic reduction and resistance (arsC, araA/arsB, ACR3) were also shown to be most widely distributed in Holocene paddy soil. The anaerobic condition and higher level arsenic in the Holocene paddy soil may hence explain the increased abundance of Geobacter as well as of genes involved in arsenic reduction and resistance. The physiochemical properties of soil (pH and Eh) were the most dominant factors influencing arsenic speciation and microbial and functional gene abundance and diversity in paddy soil. The present study provides an insight into the microbial composition and functional gene diversity and their role in biogeochemical cycling of arsenic in paddy soil.

Paddy rice is a major source of exposure, globally, to the carcinogen and nephrotoxin cadmium and the carcinogen inorganic arsenic. As rice is a dietary staple, this makes rice production highly problematic. Thus, if cadmium and inorganic arsenic can be reduced in rice, even in rice based processed products such as flour, as opposed to rice grain, per se, then large production regions rice output can be made safer. The study presented developed robust, low-technology approaches to rice post-harvest processing, through to food-preparation, to lower rice cadmium and arsenic species levels. Here it was identified that the food safe and common cadmium chelator, citric acid, efficiently removed cadmium via pre-soaking rice, while also reducing arsenic species. A twostep pre-soaking stage was developed, such that when 10 different individual types of rice were processed in such a way, this resulted in removal rates of 79% for cadmium and 81% for inorganic arsenic. Also, the use of food waste materials, orange and lemon peels, were evaluated for their adsorption capacities by pre-soaking specified amount of adsorbent and water in the rice grain. Both materials resulted to be efficient for the adsorption of both cadmium and arsenic, although to varying degrees, with lemon peel being responsible for an average of 60% cadmium removal, with up to 88% cadmium removal in one rice type, and 38% average arsenic removal. It was also ensured that, because rice could be laden with the pathogenic bacteria, Bacillus cereus, any interventions being considered did not aid the proliferation of the bacteria in rice. These technologies are particularly suitable for bulk food processing, while putting food waste materials (lemon and orange peels) to good use and could be deployed in the most cadmium and arsenic impacted regions where rice is a staple.

Precision medicine is improving health outcomes by tailoring patient care to specific individuals. The improved patient stratification needed to support further implementation of precision medicine will require developments in molecular diagnostics and identification of novel biomarkers. Small non-coding RNAs, specifically miRNAs, have been shown to exist extracellularly and are stable in biofluids, including blood and urine. The expression profile of extracellular miRNAs is also altered in a number of diseases making them attractive candidates for novel, non-invasive biomarkers. Although RT-qPCR is considered the gold standard for detection of individual miRNAs, RNA-Seq is a next generation sequencing technology that provides greater potential to identify novel extracellular miRNA biomarkers because it provides a global profile of all small RNAs present. However, this approach can be challenging when working with biofluids, such as human plasma, due to low RNA content. Small RNA-Seq is also hampered by sequencing bias introduced during library preparation that can result in inaccurate quantification of miRNAs. The first aim of this thesis is to demonstrate the feasibility of routine miRNA profiling in blood plasma by identifying an optimal small RNA-Seq protocol. The second aim is to then evaluate the ability of global miRNA profiling to differentiate between disease states by implementing this workflow in several patient cohorts, including cardiovascular diseases and age-related macular degeneration (AMD).Methods - RNA was extracted from plasma using MagnaZol cfRNA isolation (Bioo Scientific) and miRNeasy serum/plasma (Qiagen) kits. RT-qPCR was used to quantify and validate expression of individual miRNAs. Small RNA libraries were prepared with CleanTag (TriLink), NEXTflex (Bioo Scientific) and QIAseq (Qiagen) library kits. Sequencing was performed on MiSeq or NextSeq Illumina systems. Data analysis was performed using both commercial (e.g. CLC Genomics Workbench, Qiagen) and publicly (e.g. STAR aligner and HTSeq) available tools for fastq processing, alignment to reference genome and quantification of miRNAs. A number of packages were used within R including EdgeR for differential expression analysis and heatmap3 for hierarchical clustering. Novel algorithms for the quantification of isomiRs were also implemented in R and machine learning models were applied to the outputs of these algorithms to identify key discriminators between disease groups. Results - The small RNA-Seq workflow was optimised by comparing a number of commercially available RNA extraction and library preparation methods. Small RNAs were consistently detected using all approaches, however differences in miRNA diversity and differing detection efficiency were apparent between alternative methods. MagnaZol RNA extraction was shown to increase the proportion of reads mapping to miRNAs and QIAseq library prep was shown to introduce the least sequencing bias during processing and correlate most closely to independent quantification using RT-qPCR. miRNAs were shown to be significantly differentially expressed between case and control groups within disease cohorts. miR-185-3p was significantly downregulated in individuals with atrial fibrillation (AF) compared to healthy controls and improved on the discriminatory power of current AF markers B-type natriuretic peptide (BNP) and collagen 3 synthesis marker PIIINP. miRs 144-3p, 142-5p and 660-5p were shown to be significantly downregulated in individuals with AMD compared to healthy controls. Hierarchical clustering analysis of isomiR data did not improve separation of samples into defined groups however did identify potential batch effects introduced during library preparation, suggesting that isomiR analysis is more sensitive to technical variation and could be applied as a quality control measure during analysis. From the machine learning model, isomiRs were shown to make up >60% of the key features distinguishing between disease groups. Conclusion - Small RNA-Seq has been shown to be a reliable method for quantifying circulating miRNAs and identifying differential expression between disease and control groups. However, global miRNA profiling is unlikely to become a routine diagnostic procedure at present due to variable reliability and consistency stemming from the lack of a clear set of recommended experimental guidelines. A comprehensive, multicentre centre comparison of all available methods is required to facilitate adoption of a consistent and universally accepted set of guidelines regarding RNA extraction and library preparation workflow for miRNA detection from plasma. If this can be achieved, small RNA-Seq would provide an excellent platform for the discovery of novel biomarkers of diagnostic value that can subsequently be measured clinically using RT-qPCR.

Cyanobacteria form an essential part of many freshwater ecosystems. However, eutrophication contributes to the growing presence of cyanobacteria worldwide. When the conditions are favourable, these photosynthetic prokaryotes proliferate intensively and can form harmful algal blooms. Not only do such blooms block vital sunlight, cyanobacterial proliferation also leads to deprived oxygen levels. Alarmingly, some cyanobacterlal species produce compounds called cyanotoxins, which form a threat to both aquatic and human life. Microcystin-LR is believed to be one of the most harmful cyanotoxins, exerting its toxicity through the inhibition of several protein phosphatases. Its primary target organ is the liver, though microcystin-LR also exerts toxicity in other organs. Multiple toxicology studies performed in rodents discovered that the toxin causes hepatocellular damage. Furthermore, many -omics publications found multiple biomarkers of microcystin-LR exposure after using proteomic, transcriptomic and metabolomic screening tools. What most of these studies have in common, however, is that they all applied the toxin In rodents and used dosing strategies that lead to a high bioavailability of the toxin. While this is useful for obtaining more information about microcystin-LR's mode of action, it is less suitable for human risk assessment, simply because humans are never exposed to such high toxin levels. The overall aim of this thesis is to get a better understanding of the toxic effects of microcystin-LR in the context of human exposure. The work presented here took a novel approach by using a systems biology approach to study the effect of microcystin-LR, where dosing strategies were chosen that best mimicked real-life human exposure conditions. The translational model used was the pig, an animal that is closer to humans than rodents. This makes the pig more suitable for interpreting the results with a human perspective. The thesis begins with a literature review on what is currently known about microcystin- LR toxicity, and subsequently provides an overview of all consistent biomarkers of exposure found on the proteome, transcriptome and metabolome levels in at least two publications (Chapter 1). After describing the method development for the -omics tools used in this thesis (Chapter 2) and the rationale for each animal experiment performed (Chapter 3), the effects of low dose microcystin-LR exposure on liver health in pigs are discussed (Chapter 4). Using biomarker screening tools on blood and liver samples, this thesis provides evidence that microcystin-LR may not be as toxic as widely believed, at least in terms of liver disease. Because microcystin-LR has been implicated with colorectal cancer, faeces samples underwent metabolomic analyses (Chapter 5). Preliminary results indicated a toxin effect in the faecal metabolome, suggesting oral exposure to microcystin-LR might affect gut health. Finally, the results of this thesis are reflected upon and future perspectives are provided, as well as some conclusions (Chapter 6).

Cow milk is a primary source of trace elements iodine and selenium. Deficiency of iodine and selenium in the diet can result in various endemic diseases. Furthermore, literature has stressed that dietary intakes of selenium and iodine are insufficient in Ireland and the UK. In addition, it is established that pathogenic and spoilage bacteria are present in milk. However, milk also contains beneficial bacteria including probiotic bacteria important for the development of dairy products. The aims of this project were to characterise Northern Ireland milk by using combined element and microbial analysis, and to link these to Northern Irish environment and husbandry, to enable the milk industry to proactively use this information to maximize the promotion of Northern Ireland milk and dairy products, to address opportunities arising from Global Food Security Challenges and or Food safety risk management. Three different farm- based sampling campaigns were completed. Samples of milk, soil, silage and grass have been collected from farms across Northern Ireland and analysed. Study one was completed in 2016, samples were collected from 71 farms across Northern Ireland during winter. Study two was completed in 2017, samples were collected from 48 farms across Northern Ireland during summer. Study three investigated farming practice; samples of milk, grass and silage were collected every two months, over one year, from farms across Northern Ireland that contrasted in having year-round housing, or not. For these 3 studies, iodine and selenium levels were quantified using inductively couple plasma-mass spectroscopy (ICP-MS). In addition, microbial analysis was completed using a combination of culture dependant and culture-independent approached to determine and compare the microbial diversity in milk samples. This research found that iodine and selenium display very strong relationships in the environment. However, despite the strong environmental relationships there appears to be disconnect between the environment and milk iodine concentrations, as milk iodine and selenium concentrations did not correlate with any of the environmental parameters (p > 0.05). Season had an important determining effect on the iodine and selenium concentrations in milk, with higher concentrations evident during winter months in comparison to summer months.

Food can be contaminated with disease-causing microorganism. With respect to rice and rice-containing products Bacillus cereus group spp. are of particular concern as are frequently found in rice and can cause diarrhoea and emetic episodes. In this study locally purchased samples of rice grain, rice products and paddy soil samples were tested for presence of B. cereus group spp. using selective media, molecular analysis, phenotyping, whole genome sequencing and RNA-seq. B. cereus was successfully isolated from 24% of the rice and rice-containing products and from 100% of the rice paddy soil samples. Ten out of 155 isolates obtained from rice grain or rice products were identified as B. thuringiensis. All isolates were shown to harbor genes associated with virulence, food poisoning outbreaks and environmental persistence. This included genes encoding diarrhoeal toxins but none of the isolates showed presence of the emetic toxin gene. All isolates also showed resistance to multiple antimicrobial compounds, tolerance to low pH and high osmotic stress. All strains investigated could therefore be classified as pathogens that harbor toxin genes as well as genes that facilitate resilience to a range of environmental stresses and could therefore, once established, potentially be difficult to eliminate from food production and clinical environments. Eight strains were selected based on phenotypic characteristics to be genome sequenced and compared to publicly available genome data of B. cereus for identification of virulence and environmental stress resistance genes. Paired lllumina reads were mapped to publicly available B. cereus reference genomes as well as de novo assembled with velvet followed by genome annotation. All eight draft genomes generated showed presence of genes involved in osmotic stress response, antimicrobial resistance and toxin genes in their draft genome assemblies. Following this, RNA sequencing (RNA-Seq, lllumina Hiseq 4000) of one sensitive (F7B1) and one resistant (X22B) B. cereus strain was employed for comparison of gene expression responses to osmotic stress and/or tetracycline. Overall the sensitive F7BI showed a stronger response when exposed to stress, but there were also many overlapping responses and some genes that were only induced in the resistant X22B. The combined results implicate that genes involved in sigma B stress response regulation, I 111 operon, oppA (ABC transporter oligopeptide), ESAT-6 protein secretion system, sortase and capsular polysaccharides biosynthesis present good targets for the further study of the molecular mechanisms behind increased resistance to osmotic and tetracycline stress in environmental B. cereus strains.