Human transthyretin (TTR) is heavily implicated in a range of fatal amyloid diseases. The propensity to form amyloid varies between wild-type TTR and various mutants. In all cases this occurs by tetramer dissociation to a monomeric form, which in turn polymerises into intermediates that ultimately assemble into amyloid fibrils. Numerous X-ray crystal structures of TTR mutants have been determined over the last 40 years. These structures have all turned out to be very similar, shedding little light on the widely varying amyloid-forming properties. The goal of this PhD project was to investigate the neutron crystal structures of these mutants – on the basis that factors that are difficult to identify using X-ray crystallography (protonation states, hydrogen bonding, hydration) may be implicated in the varying TTR stabilities. High-resolution neutron crystallographic studies of two TTR mutants were carried out. These mutants were chosen for their strongly contrasting behaviour. The S52P mutant is highly amyloidogenic, whereas the T119M mutant is resistant to amyloid formation. As part of these studies, a unique way of measuring and exploiting H2O/D2O back-exchange was uncovered – allowing the stability of specific regions in the S52P structure to be probed. A wide range of mass spectrometry measurements were also carried out. These initially focused on the assessment of the impact of deuteration of the proteins produced for crystallographic work. However, in addition, the impact of different mutations on the subunit exchange kinetics of TTR tetramers was also studied. Finally, the impact of binding of a small-molecule ligand, tafamidis, to the S52P mutant was investigated through a combination of structural and biophysical studies. Collectively, these results have been placed in the context of the overall knowledge of TTR amyloidosis, and an exciting perspective for work is given.

Post Mortem Interval (PMI) estimation is a key concern of forensic entomology research. Numerous factors are recognized to affect these calculations, and have shown to potentially introduce error, leading to an incorrect time of death estimation. One such acknowledged factor is the presence of drugs and toxins. A range of Novel Psychoactive Substances (NPS), common adulterants and one illegal drug were tested on two blowfly species, of forensic importance, Calliphora vicina and Lucilia sericata (Diptera: Calliphoridae). An artificial diet was used to enable effective delivery of the substances; the effect on development was studied by observing larval length, weight, instar and time taken to reach pupariation. As a potential alternative for accurate aging, the cuticular hydrocarbon profile was analysed for changes in response to drug presence. Preliminary investigations were also carried out to detect the presence of NPS within larvae, by extraction and derivatisation. The NPS had a profound effect on the development of immature larval samples; rates were mostly accelerated, shown by larval length, weight and an increase in time taken to reach pupariation. A potential PMI overestimation of 48 hours was presented for a number of substances. Paracetamol was the only drug shown to produce developmental delay, up to 48 hours for a higher dose. The effects of drug dosage and potential to use data from chemically similar drugs for PMI estimations are presented; MDA is compared with NPS, 6APB. Noteworthy, the cuticular hydrocarbon profile showed no major changes in response to the drugs, some minor differences were observed but this was less pronounced than the development data and has lesser effect on PMI estimation. Results encourage the use of cuticular hydrocarbon analysis for accurately aging blowfly, despite showing developmental changes triggered by drug ingestion, which may otherwise cause incorrect PMI estimations.

In the research area of travel time prediction, the existing studies mainly focus on aggregated travel time prediction (without distinguishing vehicle types) or travel time prediction for passenger vehicles. The travel time prediction for freight transportation has not received enough attention from researchers. Only a few relevant studies can be found in the literature, and the proposed methods are usually very simple and lack comparisons with more advanced methods. Although many believed that a better prediction model can be developed using more advanced techniques such as artificial neural networks or autoregressive conditional heteroscedastic models, it is usually difficult and costly to train these models and the model interpretability is poor. There is a demand for `off-the-shelf' methods with good performance, ease of implementation and good model interpretability. Thus, the aims of this thesis are: (1) developing some `off-the-shelf' data-driven methods to predict travel time for freight transportation; (2) creating a comprehensive understanding of how the developed methods can be more effectively applied for general travel time prediction problems. Its two main contributions are: (1) it develops data-driven travel time prediction methods for freight transportation by utilising freight vehicles' trajectory data; (2) it investigates the relation between features and performance and discovers the combinatorial effects of features under the effects of different noise processes and different model fitting strategies. The experimental results show that useful features can be mined from the trajectory data to enhance the travel time prediction for freight transportation. The developed methods outperform some of the state-of-the-art data-driven methods.

Parkinson disease (PD) is the second most common neurodegenerative disease affecting 1.8% of population aged over 65 years. The current medications that control the symptoms of the disease are associated with limited efficacy and induction of side effects (dyskinesia) at later stages of the disease. One promising future therapy in PD is cell replacement therapy, however clinical trials declared inconsistent outcomes and developing dyskinesia related to the graft. Studies later suggested suboptimal conditions contributed on these outcomes. This thesis builds on this knowledge endeavouring to support cell transplantian therapy in Parkinson disease in models that are more closely aligned to the clinic thorough considering anti-parkinsonian medications in the model. It is addressed the low survival and efficacy problem of the transplanted cells examining neuroprotective agents that have previously shown the ability to protect nigrostriatal dopaminergic neurons against toxic challenges. In addition, this thesis characterises stem cell transplantation, the potential cell source for transplantation that can overcome the many practical and ethical issues surrounding foetal tissue. In the first part of the thesis, the investigations on finding neuroprotective agents to support graft survival and efficacy was achieved in the unilateral 6-OHDA lesioned rat model, treated with chronic L-dopa, (the gold standard anti- Parkinson medication), prior to, and following, cell transplantation. The results revealed for the first time that Glucagon Like Peptide-1 (GLP- 1) receptor agonists (exendin-4 and liraglutide) were capable of improving graft size and the motor and behaviour recovery results from peripheral administration. Importantly, this protection was affected by the presence or absence of L-dopa treatment, as exendin-4 supported the graft only in absence of L-dopa while liraglutide supported the graft only in the presence of L-dopa. While other neuroprotective agents (ghrelin and ghrelin receptor agonist) failed to support graft survival or efficacy in the same animal model. In the second part of the thesis, the characterisation of different source of stem cells derived dopaminergic neurons revealed for the first time that these cells can survive and function in the striatum of 6-OHDA rat model primed with chronic L-dopa treatment and exposed to L-dopa treatment for 16 weeks after transplantation. I show, for the first time, that these cells are capable of ameliorating L-dopa induced dyskinesia.

Autism spectrum disorders (ASD) are more prevalent in male than in female individuals and are characterised by clinical core symptoms such as impaired sociability, verbal communication and ritualistic behaviours. The gene encoding the cytoplasmic FMR1 interacting protein 1 (CYFIP1) has been associated with ASD in humans. At the molecular level, CYFIP1 has been demonstrated to negatively regulate protein synthesis and actin remodelling. At the neuronal level, Cyfip1 haploinsufficiency leads to defects in synaptic plasticity associated with alteration of dendritic spine morphology, two pathophysiological features found in numerous mouse models of ASD. However, the consequences of Cyfip1 deletion at the behavioural level remains unclear, limiting our understanding of the relationship between pathophysiology and behavioural phenotypes. With this study, we aimed to characterise the behavioural phenotype of Cyfip1+/- mice and then to identify associated cellular phenotypes. The results we obtained revealed sex-specific defects in social interest and motor learning. In addition, motor learning deficits were observed in adult Cyfip1+/- mice but not earlier in development. Associated with motor learning deficits, we identified a brain region-specific neuronal phenotype with decreased dendritic spine densities and increased dendritic spine turnover in Cyfip1+/- mice. The dendritic spine formation and the in vivo protein synthesis rate were intact in Cyfip1+/- mice. These results identified behavioural deficits in Cyfip1+/- mice, which relate to symptoms and comorbidities of ASD in human. The cellular phenotypes indicated an alteration of dendritic spine density and spine turnover, a phenotype found in several mouse models of ASD and in humans affected by the condition. Altogether, these findings indicate that Cyfip1+/- mice can represent a valuable model for the study of ASD pathophysiology and in particular the relationship between specific neuronal phenotypes and behavioural alterations.

This thesis aimed to examine the impact of environmental tobacco smoke on biological markers of ageing in pet dogs. In order to achieve this, community-based dogs and owners were recruited, approximately half of whom lived in smoking homes and half non-smoking homes. Owners were asked to attend 2 appointments, 12 months apart. Questionnaire assessments of dog environmental tobacco smoke exposure were compared to biomarkers in hair of the dogs (Chapter 3). This gave an objective measure of exposure and hair nicotine concentrations reliably reflected information provided by owners. A qPCR technique was optimised to measure telomere lengths (Chapter 4). This was utilised to measure telomere lengths in leukocytes, buccal cells, cremaster muscle, vas deferens and epididymis samples from the study dogs. Owners were offered free-of-charge neutering for their pet, and the spare tissues used for these analyses. A negative relationship in leukocyte telomere length with hair nicotine was observed, among other changes (Chapter 5). mRNA levels of further biomarkers of ageing were measured in testes, as well as leukocyte global DNA methylation percentage (Chapter 6). Again, several significant relationships were found between tobacco smoke exposure markers and the biomarkers of ageing, including a significant negative relationship with CDKN2A expression with increased tobacco smoke exposure. Plasma testosterone and hair cortisol concentrations were measured. In addition, factors which related to weight gain after neutering were examined (Chapter 7). Increased cotinine concentrations in fur were significantly related to increased percentage weight gain. Several avenues for future research were generated, and many areas warrant further investigation.

The transmission of the malaria parasite (Plasmodium spp) is effected by a small proportion of the population that commit to sexual development forming either male or female gametocytes. Currently front-line anti-malarials are developed to treat the asexual life stage parasitising mature erythrocytes. While circulating mature erythrocytes form a relatively homogenous population, it has been recently established that Plasmodium spp can colonise progenitor cells in the erythroid lineage. I hypothesised that these tissue-resident erythroid precursors may form a niche for preferential development of gametocytes, and therefore developed a quantitative approach to enumerate Plasmodium-infected erythropoietic intermediate cells in haematopoietic tissues. We demonstrate that within erythropoietic tissues, early reticulocytes preferentially drive commitment to gametocytogenesis, with the splenic niche contributing the quantifiable majority of gametocytes. Furthermore, this same erythropoetic niche offers the parasite protection against antimalarial drug treatment and, as such, may serve as origins of recrudescent infection and continuing transmission. My data therefore demonstrate that host cell phenotype and location play an active role in determining the developmental fate of malaria parasites and furthermore suggests a mechanism for the persistence of malaria parasites.

Breast cancer (BCa) is the most prevalent cancer among women in the UK. The majority of BCas are endocrine sensitive and develop through the action of oestrogens, facilitated through the transcription factor Oestrogen Receptor alpha (ERα). Treatment for these patients usually involves endocrine therapies (Aromatase Inhibitors and antioestrogens), which are successful in many patients, but therapy resistance represents a major clinical issue. The Androgen Receptor (AR) is a transcription factor that is more highly expressed than ERα in BCa, and mediates the functions of androgens. In early forms of ERα-positive disease, AR is a positive indicator of prognostic outcome and suppresses ERα signalling. However, in ERα-negative disease AR has been demonstrated to drive cancer progression and recent evidence has suggested that AR can drive endocrine resistance. Reporter assays, gene expression analysis and Chromatin Immunoprecipitation assays demonstrated that AR and ERα inhibit each other’s activity and that antioestrogens can reverse this inhibition, resulting in an active AR. Importantly, long term colony formation assays demonstrated that androgen could induce anti-oestrogen resistant growth, but anti-androgens prevented this from developing. Co-treatment of tumours with anti-oestrogens and anti-androgens could therefore be a viable option to block this mechanism of resistance. Cell line models of endocrine resistant disease were used to investigate AR signalling in therapy resistance. The results demonstrated that AR levels were enhanced in several lines and that all cell lines were sensitive to androgen for growth. Importantly, anti-androgens could inhibit androgen-induced growth in all models. Anti-androgens could therefore also be a viable option for the treatment of tumours that have become resistant to endocrine therapies. This study therefore furthers our understanding of the role of the AR in BCa progression and suggests that it is a valid therapeutic target to prevent and/or treat endocrine resistant disease.

DNA profiling has become a universal technique for identifying individuals for evidential use in courts of law. In more complex cases such as sexual assaults, identifying the origin of a stain or sample offers valuable information as to the events that occurred. Currently, many ‘in service’ body fluid identification (BFID) techniques are presumptive, require significant sample volumes and generate false positives. As such, the development of a highly specific and reliable BFID technique would be highly beneficial to forensic scientists in provide more informative and reliable evidence. MicroRNAs (miRNA) are short, stable, non-coding RNA’s which modulate gene expression. Expression of some of these miRNA are body fluid specific, making them a potentially robust tool for BFID. The possibility for the integration of a robust, miRNA based BFID technology for forensic casework employing stem-loop reverse transcription and qPCR analysis was the theme of the research presented here. To be incorporated into the workflow of current forensic laboratories, the protocol must be able to be carried out alongside current techniques with limited addition of cost, equipment, analysts and time. A range of custom designed miRNA markers were analysed on vaginal material, menstrual blood, saliva, venous blood, semen, seminal fluid and skin. Screening indicated specificity of hsa mir-124 to vaginal material, hsa-mir-10a, 135a and 888 to semen, hsa-mir-412 and 507 to menstrual blood, hsa-mir-144-3, 144-5, 142 and 451 to blood and although highly expressed in saliva, hsa-mir-205 was also observed in vaginal material. Universal expression was observed in hsa-mir-93, 508, 1260b and SNORD 47 providing a means of normalisation through the designation of these markers as endogenous controls. A combined panel of markers are presented which were capable of identifying all body fluids, excluding skin from single stains. The panel was successful at identifying certain mixtures such as semen within vaginal material but was unable to confirm saliva presence within vaginal material. Screening of hsa-mir 205 within vaginal material uncovered the observation that hsa-mir-205 was impacted by the use of female contraception. Once a full BFID panel was generated the robustness of the markers was further analysed across the menstrual cycle. No significant difference (p > 0.001) was seen in markers highly expressed in vaginal material during screening (hsa-mir-124, 203a, 205). Expression of non specific markers highlighted the importance of the optimisation of input miRNA. Differential extraction of genetic material was found to be detrimental to miRNA sample integrity. As such, total DNA extraction was employed for vaginal swabs obtained from volunteers following unprotected sexual intercourse, markers hsa mir-10a, 135a and 888 were able to successfully detect semen presence for up to 96 hours. The data generated to date has highlighted a number of miRNA markers that provide a platform for BFID. The developed protocol is reliable and robust; requiring minimal optimisation and is capable of integration with current laboratory workflow with minimum implications to time and cost. The markers identified for BFID have been implemented within studies that are representative of real case scenarios, and have demonstrated their ability to be stable, specific and successful in the identification of certain body fluids. Overall, this research showcases a reliable and body fluid specific protocol capable of being performed alongside DNA profiling.

The current plan for the permanent disposal of Low Level and Intermediate Level nuclear wastes is the cement-based geological disposal where the wastes will be disposed off in geological disposal facility (GDF). The chemical evolution of the GDF is expected to cause cellulosic materials in the waste to degrade into cellulose degradation products (CDP) of which isosaccharinic acids (ISA) are the major components. ISAs are reported to form complexation reactions with radionuclides, potentially enhancing their migration out of the GDF. Recent studies have shown that microbial consortia indigenous to anthropogenic sites can potentially degrade these ISAs but have focused on the use of different nitrogen sources Ca(ISA)2 as analogue for CDP. This study therefore aimed at characterizing novel ISA degrading bacteria and investigate the metabolic potentials of microorganism within soil sediments from Harpur Hill site to biodegrade ISAs and assess the impact of using Ca(ISA)2 or CDP with NH4+ against NH4+ free systems on the chemical and microbial community evolution under conditions representative of the GDF. In a batch/fed microcosms, microorganisms within the soil sediment were able to biodegrade ISAs in pH ranging from 9-11.5 irrespective of the source. The microbial community evolution in these systems were however different suggesting that the type of carbon source and the presence of a nitrogen source impacted on the selection of these communities. The evolution of the microcosms gave rise to a complex methanogenic, polymicrobial communities where the degradation of ISAs led to the formation of acetic acid and gases including CH4, CO2 and H2. The formation of these gaseous products are likely to contribute to the pressurization of the GDF as a result of which the porosity and permeability factors should be taken into account in the formulation of the cement backfill materials. Molecular characterization of the ISA degrading communities and pure isolates (Exiguobacterium sp. strain Hud and Oceanobacillu sp. strain Hud) will allow for studies into genes associated with ISA degradation which is currently lacking in the literature. The Harpur Hill site presents a diverse pool of microorganisms with the metabolic potentials to degrade ISA hence it could be a good candidate site for the GDF.