Examining the suitability of molecular and metabolomic-based techniques as tools for assessing the effects of pharmaceuticals in the aquatic environment

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Pharmaceuticals represent important and indispensable elements in modern society and their usage is considerable. Post consumption and body-elimination, pharmaceuticals are not completely removed in sewage treatment works (STWs) and as such, have been detected at low levels in STW effluents, surface waters, seawaters, ground waters and some drinking waters. Accordingly, pharmaceutical toxicity has been detected in several aquatic organisms. To date, environmental risk assessments (ERA) examine for toxicity using a series of chronic toxicity assays that examine for standard physiological responses in algae, Daphnia and fish and do not address pharmaceutical mode of action. Therefore, using the fathead minnow (Pimephales promelas) and the β-blocking pharmaceutical propranolol as the test-species and test-drug, respectively, the aim of this study was to establish an intelligent targeted 4-phased ERA using molecular, in vivo exposure, metabolomic and quantitative expression analytical techniques. The first phase established that the fathead minnow expressed the β3bi-adrenergic receptor (AR), which is a target receptor for propranolol in humans. The in vivo pair-breeding assay suggested that at 1mgL-1 and 10mgL-1, propranolol levels in fish blood plasma exceeded the human therapeutic concentration and caused 80% and 100% mortality, respectively. The most likely causes of mortality were liver failure and central nervous system toxicity. It was not possible to identify a robust biomarker of propranolol exposure using proton nuclear magnetic resonance (1H NMR) as there was considerable metabolic variation between male liver tissues within the same treatment groups. β3bi¬¬-AR expression was significantly lower at 1mgL-1 in the brain and liver, which was most likely the result of desensitisation in response to elevated levels of epinephrine and cortisol. β3bi¬¬-AR expression was significantly increased in the heart at the environmentally relevant concentration of 0.001mgL 1, however it was not possible to link β3bi¬¬-AR expression to a toxic response. Propranolol is unlikely to pose a threat to the aquatic environment as the concentrations measured in the environment are approximately 1000-fold lower than those that induced a toxic response. The proposed ERA represents a marked improvement over the existing ERA as it addresses pharmaceutical mode of action and both subtle and physiological toxicity responses, however it still requires further validation studies to address both metabolomic and gene expression variation

Structural studies of MeCP2 in complex with methylated DNA

DNA methylation is a common epigenetic mark that affects gene regulation, genomic stability and chromatin structure. In mammals, methylation is mainly found in the CpG dinucleotides. The CpG methylation signals can be recognised by the Methyl-CpG-Binding Protein (MBP) family which includes MeCP2, MBD1, MBD2, MBD3, MBD4 and Kiaso. MeCP2 and MBD1-4 (except mammalian MBD3) recognise methyl-CpG via their MBD domain whereas Kaiso interprets methylation through its Zn finger DNA binding domain. The TRD domains of MeCP2, MBD1 and MBD2 have been reported to recruit transcriptional co-repressors to the methylated DNA. A thymine DNA glycosylase domain is located at the C-terminal region of MBD4. This study concerns the molecular details of the methyl-CpG recognition by the MBD domain of MeCP2. To achieve this, the MeCP2 MBD domain has been expressed, purified and co-crystallised with a 20 bp DNA fragment from the BDNF promoter. The DNA-protein cocrystal diffracted X-rays to a maximum resolution of 2.5Å using synchrotron sources. It belongs to space group C2 with unit cell dimensions: a = 79.71Å, b = 53.60Å, c = 65.73Å, and β = 132.1°. The X-ray structure of the MeCP2 MBD-DNA complex was solved using the SAD method. Structural analyses of the refined X-ray structure reveal that the methyl groups of the DNA make contact with a predominantly hydrophilic surface that includes tightly bound water molecules. From a structure of the MBD domain in MBD1, established by NMR, the binding specificity of the MBD domain had been thought to depend on hydrophobic interactions between the cytosine methyl groups and a hydrophobic patch within the MBD domain. The findings of this study suggest that MeCP2 recognises the hydration pattern of the major groove of methylated DNA rather than cytosine methylation per se. The X-ray structure also identifies a unique role of T158 and R106, the sites of the two most frequent Rett missense mutations. Both residues stabilise the tandem Asx-ST motif at the C-terminal region of MBD domain. Disruption of this tandem motif destabilises the DNA-protein interaction. The BDNF sequence in this study contains an AT run which displays unique properties of AT tract DNA. Previously, mutation of the AT run has been reported to decrease MeCP2 binding specificity. This study however demonstrated that a significant reduction can only be observed when both AT runs close to the methyl-CpG have been mutated. The X-ray structure of the MeCP2 MBD-DNA complex in this study rationalises the effects of the most common Rett mutations and provides a general model for methylated DNA binding that is dependent on structured water molecules

Free-living nematodes detection using next-generation sequencing technology

The role of nematodes as biological indicators and as key players in nutrient cycling is well recognized. Others have been shown to cause immense losses in food production. Despite their importance, identification of nematodes has been hindered by difficulties in species identification using classical morphology. Alternative molecular-based methods including AFLP, PCR-RFLP and DNA barcoding used alone or in combination with the traditional methods can be time consuming when analysing multiple specimens in a sample. Metabarcoding provides the possibility to identify an array of individuals from many samples simultaneously. The challenge of this approach has been how to identify the most suitable DNA marker(s) as well as the lack of robust analysis pipeline for the sequence data. An evaluation of the performance of four candidate DNA markers (NF1-18Sr2b, SSUFO4- SSUR22, D3Af-D3Br and JB3-JB5) on a mock community of nematodes showed NF1- 18Sr2b is most suitable in terms of coverage and availability of reference sequences. Assessment of the most common bioinformatic tools (QIIME, MOTHUR and USEARCH) showed USEARCH had the best clustering algorithm, was the fastest, had best operational taxonomic units (otus) to actual diversity ratio and ranked the best in userfriendliness. In another mock community experiment, read numbers of taxa showed no correlation with their actual abundance in the community largely due to bias in amplification and copy numbers of the marker region. Analysis of samples collected from a tillage and traffic experiment using morphological approach showed strong inhibition of herbivores by deep tillage and zero traffic. Bacterivores in general were inhibited by traffic and not affected by tillage. Appraisal of the metabarcoding approach using samples from the same experiment showed at broader classification levels (trophic groups and functional guild), abundance biases associated with the mock community experiment were minimal, the broad implication being metabarcoding data may be useful for assessing quality soil based on the structure of its nematode community

Examining the suitability of molecular and metabolomic-based techniques as tools for assessing the effects of pharmaceuticals in the aquatic environment

Pharmaceuticals represent important and indispensable elements in modern society and their usage is considerable. Post consumption and body-elimination, pharmaceuticals are not completely removed in sewage treatment works (STWs) and as such, have been detected at low levels in STW effluents, surface waters, seawaters, ground waters and some drinking waters. Accordingly, pharmaceutical toxicity has been detected in several aquatic organisms. To date, environmental risk assessments (ERA) examine for toxicity using a series of chronic toxicity assays that examine for standard physiological responses in algae, Daphnia and fish and do not address pharmaceutical mode of action. Therefore, using the fathead minnow (Pimephales promelas) and the β-blocking pharmaceutical propranolol as the test-species and test-drug, respectively, the aim of this study was to establish an intelligent targeted 4-phased ERA using molecular, in vivo exposure, metabolomic and quantitative expression analytical techniques. The first phase established that the fathead minnow expressed the β3bi-adrenergic receptor (AR), which is a target receptor for propranolol in humans. The in vivo pair-breeding assay suggested that at 1mgL-1 and 10mgL-1, propranolol levels in fish blood plasma exceeded the human therapeutic concentration and caused 80% and 100% mortality, respectively. The most likely causes of mortality were liver failure and central nervous system toxicity. It was not possible to identify a robust biomarker of propranolol exposure using proton nuclear magnetic resonance (1H NMR) as there was considerable metabolic variation between male liver tissues within the same treatment groups. β3bi¬¬-AR expression was significantly lower at 1mgL-1 in the brain and liver, which was most likely the result of desensitisation in response to elevated levels of epinephrine and cortisol. β3bi¬¬-AR expression was significantly increased in the heart at the environmentally relevant concentration of 0.001mgL 1, however it was not possible to link β3bi¬¬-AR expression to a toxic response. Propranolol is unlikely to pose a threat to the aquatic environment as the concentrations measured in the environment are approximately 1000-fold lower than those that induced a toxic response. The proposed ERA represents a marked improvement over the existing ERA as it addresses pharmaceutical mode of action and both subtle and physiological toxicity responses, however it still requires further validation studies to address both metabolomic and gene expression variation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Helping with inquiries: theory and practice in forensic science

This thesis investigates the reasoning practices of forensic scientists, with specific focus on the application of the Bayesian form of probabilistic reasoning to forensic science matters. Facilitated in part by the insights of evidence scholarship, Bayes Theorem has been advocated as an essential resource for the interpretation and evaluation of forensic evidence, and has been used to support the production of specific technologies designed to aid forensic scientists in these processes. In the course of this research I have explored the ways in which Bayesian reasoning can be regarded as a socially constructed collection of practices, despite proposals that it is simply a logical way to reason about evidence. My data are drawn from two case studies. In the first, I demonstrate how the Bayesian algorithms used for the interpretation of complex DNA profiles are themselves elaborately constructed devices necessary for the anchoring of scientific practice to forensic contexts. In the second case study, an investigation of a more generalised framework of forensic investigation known as the Case Assessment and Interpretation (CAI) model, I show how the enactment of Bayesian reasoning is dependent on a series of embodied, experiential and intersubjective knowledge-forming activities. Whilst these practices may seem to be largely independent of theoretical representations of Bayesian reasoning, they are nonetheless necessary to bring the latter into being. This is at least partially due to the ambiguities and liminalities encountered in the process of applying Bayesianism to forensic investigation, and also may result from the heavy informational demands placed on the reasoner. I argue that these practices, or 'forms of Bayes', are necessary in order to negotiate areas of ontological uncertainty. The results of this thesis therefore challenge prevailing conceptions of Bayes Theorem as a universal, immutable signifier, able to be put to work unproblematically in any substantive domain, Instead, I have been able to highlight the diverse range of practices required for 'Bayesian' reasoners to negotiate the sociomaterial contingencies exposed in the process of its application

Quantum Dot Bioconjugate Platforms for Analysis of Enzyme Activity

Quantum dots are semiconductor nanocrystals with size-dependent optical properties that result from their nanoscale dimensions. These materials are emerging as simpler and more sensitive alternatives to traditional fluorescent small molecules and radioactive reporters in biomarker assays. Quantum dot emission can be modulated via proximal binding of organic dyes or gold nanoparticles, which can form the basis of a sensor. Their multivalency and ease of functionalisation allow for the attachment of multiple biosensing ligands, boosting the detection sensitivity. Quantum dots with different emission wavelengths can be excited simultaneously and distinguished from one another spectrally, a property which can be used for multiplexing. Enzymes mediate the chemical modification of proteins thereby controlling the signalling cascades that regulate cell behaviour. Hence, aberrant activity of enzymes can be associated with the onset of disease. Clinical tests typically determine the total concentration of enzyme in a sample without regard to quantification of activity. In this thesis, the development of generic activity-dependent tests for acetyltransferases, kinases and proteases is described. Enzyme activity is reported via decoration of quantum dots with enzyme substrate peptides and subsequent binding of FRET acceptor dye-labelled antibodies, which mediate changes in quantum dot emission spectra. Using this platform, p300 histone acetyltransferase was detected with a limit of detection comparable to that of radiolabelling assays. Modifications of the platform to detect serine and tyrosine phosphorylation were investigated. The phosphotyrosine assay was combined with a gold nanoparticle-quantum dot assay for the detection of a kinase/protease biomarker pair relevant for the determination of breast cancer prognosis. The modular nature of this assay design allowed for the detection of different classes of enzymes singly and simultaneously, representing a generic platform for high-throughput enzyme screening in rapid disease diagnosis and drug discovery

Fragment based ligand discovery : library design and screening by thermal shift analysis

The central idea in Fragment Based Ligand Discovery (FBLD) is to identify small, low molecular weight compounds (MW < 250) that bind to a particular protein active site. Hits can be used to efficiently design larger compounds with the desired affinity and selectivity. Three approaches to FBLD are described in this thesis. The first topic is the development and assessment of different chemoinformatics procedures to select those fragments that maximally represent the chemical features of a larger compound library. Such a fragment library could be of great value in the so-called “SAR by Catalogue" approach, where the initial stage of fragment growth is by selecting existing compounds that contain sub-structures of the hit fragments. Five schemes implemented in the Pipeline Pilot software are described. The second project was to develop improved approaches to processing Thermal Shift Analysis (TSA) data. The shift in melting temperature can indicate that a ligand binds and thus stabilises a protein. A program, MTSA, has been written which allows more straightforward processing of the experimental data than existing available software. However, detailed analysis of fragment screening data highlighted difficulties in defining the melting temperature and suggest that TSA is not sufficiently reliable for routine screening use. Finally, a number of proteins were assessed experimentally for suitability for FBLD: N-myristoyl transferase (NMT), the bacterial homologue of a GlcNAcase enzyme (BtGH84) and the model system hen egg white lysozyme (HEWL). It was not possible to produce suitable NMT material due to the inherent instability of the protein produced in York. The screening results of HEWL with a new Surface Plasmon Resonance (SPR) assay, a cell based activity assay and TSA were inconsistent and difficult to interpret. However, BtGH84 was suitable for screening by both TSA and SPR. The resulting fragment hits are suitable starting points for further evolution.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Studying sequence effects of mRNA 5' cap juxtapositions on translation initiation rate using randomization strategy of the extreme 5' end of mRNA

Translation initiation is a complex process. The efficiency of translation initiation is determined not just by activity and availability of the translation initiation apparatus, but also the properties of mRNA 5’transcript leaders (5’TL). In most cases of cap-dependent translation, translation initiation begins with the formation of the preinitiation complex (PIC) loading and accommodation onto the m7G capped 5’end of mRNA, facilitated by m7G cap – eIF4F interactions. The PIC accommodation onto the 5’end of mRNA is a point of control in translation initiation where the role of 5’ cap proximal mRNA sequence determinants are poorly understood. To explore the effect of the nucleotides in the extreme 5’ end of mRNA on translation initiation, a library of mRNA molecules was synthesized containing all possible permutations of the first 10 nucleotides, referred to as E5S (Early 5' Sequence). The library was transfected into HEK293T cells. The lysates obtained from transfected cells were separated on a sucrose density gradient to isolate mRNAs bound to polysomes. Based on the assumption that efficiently translated mRNAs are associated with polysomes, the effect of E5S on translation initiation was measured by comparing frequencies of nucleotides (and their combinations) at specific positions in E5S from mRNAs in polysome fractions to their frequencies in E5S of the original library using massively parallel sequencing. The second position of E5S was found to have a markedly higher influence on translation initiation than positions further downstream (for technical reasons it was not possible to estimate the influence of the first position of E5S). In this position G was the most enriched nucleotide, and U was the most depleted nucleotide. Analysis of available ribosome profiling datasets did not reveal a significant association between E5S and ribosome footprint densities at the coding regions. While this work clearly suggests the influence of nucleotide context on translation initiation, it is possible that such as uORFs and RNA secondary structures, have a higher influence on translation initiation than E5S. The E5S is a previously unappreciated determinant of translation initiation, and this work suggests that differences in mRNA 5' end accessibility defined by the cap proximal sequence may be an important determinant in modulating the rate of translation initiation

The epidemiology of trypanosomiasis, a re-emerging zoonosis in Uganda

Sleeping sickness caused by Trypanosoma brucei rhodesiense and transmitted by tsetse flies (Glossina spp.) is a re-emerging zoonotic disease in eastern Africa. The disease may occur in large scale epidemics or be maintained at endemic levels, but in both cases, it is transmitted by the fly vector from the animal reservoir to humans. In Uganda, the principal animal reservoir is domestic cattle.This thesis explores a number of components of T.b. rhodesiense epidemiology in Uganda. Firstly, unrest and cattle raids in some regions of Uganda has led to the depletion of the cattle population, which has subsequently been restocked, resulting in a mobile national cattle herd. Following an outbreak of sleeping sickness in the previously unaffected district of Soroti, eastern Uganda, retrospective data were collected from cattle markets and used to assess the risk, over the period of restocking, of importing cattle infected with T.b. rhodesiense. Secondly, a case-control study is presented, in which the epicentre of the Soroti outbreak and the temporal and spatial changes in the distribution of the affected villages are quantified. Thirdly, molecular epidemiological tools were used to characterise parasite isolates derived from the Soroti outbreak, and, by comparing their genetic structure to parasites collected in other regions, the parasite populations from which they were derived have been determined, and aspects of the population structure of trypanosomes more generally discussed. Finally, a retrospective analysis of clinical data from the 1900-1910 sleeping sickness epidemic in southern Uganda was carried out. There has been some controversy over the species of parasite involved in this outbreak (T.b. rhodesiense or Trypanosoma brucei gambiense), and the analysis of the historical records definitively addresses this question.The risk analysis using the cattle market data shows that in Soroti, 54% of animals traded in the market in question originated from outside the district, from known sleeping sickness risk areas, and that up to 12.5% of the monthly imports from these areas may have been carriers of T.b. rhodesiense parasites. The theoretical impact that the alternative control options of mass chemotherapy or selective treatment following screening by microscopy would have had on reducing this risk are also considered, highlighting the limitations of field microscopy as a diagnostic tool for trypanosomiasis. Results from the case-control study showed that the epicentre of the outbreak which started in 1998 was Brookes Comer cattle market, the site through which most of the cattle in the restocking programme passed. Although residence in a village in proximity to the market was a highly significant risk factor for becoming a sleeping sickness case at the start of the outbreak, the average distance of cases to the market increased with time, indicating that the outbreak is expanding away from that point source. The results of the molecular analyses confirm that the parasites circulating in Soroti were similar to strains from the established sleeping sickness regions further south in Uganda, and shed further light on the nature of trypanosome population structures. T.b. rhodesiense and T.b. gambiense are distinguished primarily by the differences in clinical presentation; comparing the 1900- 1910 sleeping sickness data to a contemporary dataset of T.b. rhodesiense cases shows clearly that the clinical course of the disease in 1900-1910 was identical to that seen at present in Uganda. These results confirm that T.b. rhodesiense was present in Uganda at the timeThe results are discussed in relation to the management of human trypanosomiasis in the cattle reservoir, with particular attention given to the implications of trypanosomiasis control polices aimed at livestock but which also benefit the health of the human population. Policies aimed at controlling disease in this way need to be made at a national level, and the importance of collaboration between medical and veterinary authorities for zoonotic disease control is stressed