Theses - Veterinary Medicine
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Item Open Access Comparison of Cone-Beam Computed Tomography (CBCT), Fan-Beam Computed Tomography (FBCT), and Low-Field Magnetic Resonance Imaging (MRI) for Detection of Equine Fetlock LesionsLin, Szu-TingComputed tomography (CT) and magnetic resonance imaging (MRI) have been increasingly applied to diagnosing lameness localised to the fetlock region in standing horses. A full understanding of the benefits and limitations of CT and MRI for detection of bone and soft tissue lesions is essential for the most efficient application in equine practices. The aim of the work presented in this thesis was to compare cone-beam CT (CBCT), 64-slice fan-beam CT (FBCT), and low-field (0.27 Tesla) MRI for detection of equine fetlock lesions. The first part of the work compared imaging diagnosis and measurement of third metacarpal/metatarsal parasagittal groove and proximal phalanx (P1) sagittal groove fissure, P1 dorsoproximal osteochondral defect, palmar/plantar osteochondral disease (POD), and heterotopic mineralisation in 35 equine cadaver limbs between CBCT, FBCT, and MRI using pathological findings as a gold standard (Chapter 3, 5, 6, and 7). The second part of the work retrospectively reviewed and compared CBCT and low-field (0.27 Tesla) MR imaging findings from 24 fetlock regions of clinical patients. The diagnosis of fetlock lesions including fissures, dorsoproximal defects, POD lesions, and heterotopic mineralisation was comparable between CBCT and FBCT, with MRI having lower sensitivity but similar specificity compared to CBCT and FBCT (Chapter 3, 5, 6, and 7). The comparison of diagnoses indicated that for detection of bone lesions and heterotopic mineralisation, CBCT and FBCT are equivalent as the diagnostic tool of choice, and although the likelihood of failing to detect the presence of lesions is higher with MRI, lesions detected on MRI are likely to be genuine. MRI also detected increased fluid accumulation related to bone lesions and soft tissue injuries associated with heterotopic mineralisation, providing unique diagnostic information not available from CBCT and FBCT. The correlation of imaging measurements was strong between CBCT, FBCT, and MRI. The correlation with macroscopic measurements was strong in CBCT and moderate in FBCT and MRI for dorsoproximal defects, and strong in MRI and CBCT and moderate in FBCT for POD lesions (Chapter 5 and 6). The superior correlation with macroscopic measurements in CBCT compared to FBCT could indicate the benefit of superior spatial resolution of CBCT over FBCT for the particular devices used. Retrospective review of images from clinical patients (Chapter 8) found CBCT detected altered bone attenuation and structural changes in bone lesions including POD lesion, fissure, subchondral and trabecular bone injury, P1 dorsoproximal defect, osteoarthritis, and proximal sesamoid bone injury. In contrast to morphological details provided by CBCT, MRI detected the extent of increased fluid accumulation of bone injuries, providing information relevant to pathological status of bone injuries. Detection of soft tissue injury was superior using MRI compared to CBCT, in which the differentiation of soft tissue injury from normal structure was limited by lower contrast resolution. In conclusion, the work presented in this thesis shows that CBCT and FBCT are comparable for detection of bone lesions and heterotopic mineralisation, and MRI detects associated increased fluid accumulation and soft tissue injuries. For clinical suspicion of bone injury and heterotopic mineralisation in the fetlock region, combining CBCT or FBCT with MRI provides the most thorough evaluation of lesions. For clinical suspicion of soft tissue injury, MRI alone is sufficient for a thorough assessment of soft tissue lesions.Item Open Access Recovery of Ambulation in Medically Managed Thoracolumbar Disc Extrusions in Non-Ambulatory Small DogsKhan, SamAcute thoracolumbar intervertebral disc extrusion is a common reason for dogs to present to referral practices the consequences of which range along a spectrum from spinal hyperaesthesia without concurrent neurological deficits to paraplegia with absent pelvic limb and tail deep nociception, something usually termed “deep pain negative”. Although we have been aware of this type of myelopathy as far back as the 1890s, there remains debate regarding the appropriate treatment for affected dogs. Currently available treatments are categorised as either surgical or conservative but to date, these treatment options have never been directly compared in a randomised controlled trial. Furthermore, most studies reporting outcomes following conservative management are either retrospective or small prospective series, leaving limited available evidence to facilitate the use of conservative management in clinical practice. This has led to a consensus that surgical management is superior to conservative management and that it may be the sole route to recovery for non-ambulatory dogs despite several reports of recovery following conservative management. Whilst the prognosis for recovery of function following surgery is undeniably favourable this is not available for many dogs due to financial or geographic restrictions and so it is possible that the consensus is leading to dogs being euthanised who could have been recovered conservatively. To address the paucity of evidence for conservative management I recruited non-ambulatory dogs with thoracolumbar intervertebral disc extrusions and managed them conservatively over a period of 12 weeks. By doing this I was able to demonstrate that many dogs managed conservatively can be recovered medically including many of those who were deep pain negative on presentation confirming that this is a valid alternative to euthanasia. I also undertook morphometric analysis of MR images obtained at the beginning and end of the study period which documented complete regression of some extruded discs as well as some which remained relatively unchanged. I was also able to show that not only can recovery occur in the face of substantial compression but also that for many dogs, recovery is independent of the removal of compressive material. Lastly, through morphometric MRI analysis I documented the importance of the contusive injury and its potential significance in deep pain negative dogs which may help guide future research.Item Open Access Novel Diagnostic Methods for Rapid Detection and Identification of Bacterial Infections in Bovine MastitisBuhl, DanielBacterial infections affect the lives of millions of individuals worldwide. When a patient is diagnosed with an infection, the exact cause is often unknown. Microbial diagnostics rely on methods requiring the growth of a bacterial colony, including lengthy and time-consuming culturing techniques and error-prone manual handling steps. Consequently, an accurate diagnosis may only be made after many days, by which time the patient could have received potentially unnecessary or non-effective antibiotic treatment. In the dairy industry, speeding up this diagnostic process would facilitate considerable improvements in antibiotic stewardship. This thesis describes the development of three methods developed for the rapid diagnosis of different bacterial species in bovine mastitis directly from samples requiring different levels of laboratory equipment, costs and expertise. Laser-assisted rapid evaporative mass spectrometry was used to identify molecular biomarkers in mastitic milk. Diagnostic performance and individual markers were further investigated using transcriptomic studies. The second method used conserved species-specific primers designed from response regulators which were combined with a portable PCR system and a diagnostic dipstick for the rapid detection of species specific DNA. For the third method a whole-cell bacterial biosensor was constructed for different mastitis pathogens, allowing for the detection of quorum sensing signals released by the bacteria without further equipment. All three diagnostic methods were successfully established and tested for the rapid identification of major bacterial species in bovine milk. By combining metabolomic and transcriptomic data, I was able to show that the detected mass spectrometry signals originated from the bacteria as well as the host. All three methods can detect bacteria in less than 30 to 240 min, with more technologically complex methods and less manual handling, yielding higher information content. These methods allow for a diagnosis within the time needed to inform the treatment of the patient, which could help reduce the prescription of unnecessary antibiotics. The general principles of these techniques can be utilised for other pathogens and diseases, broadening the impact and applicability of rapid bacterial identification for antimicrobial resistance prevention.Item Open Access Preclinical Evaluation of 3D Printed Biomaterials for Repairing Critical-Size Bone DefectsElsayed, SaraProblem and study aim: Millions of patients each year suffer from non-healing bone defects secondary to trauma, cancer, osteomyelitis, or congenital anomalies. The standard of care for managing these cases necessitates bone reconstruction surgery. Although using autologous bone (autograft) or banked cadaveric bone (allograft) has been considered the standard of care for decades, both come with significant attendant risks and limitations. Commercially available bone substitute materials overcome some of these challenges but do not necessarily meet the challenging anatomical and mechanical requirements of patients undergoing complex bone reconstruction surgeries. For this reason, there is a clear, unmet need for biomaterials that can support bone healing and act as bone graft substitutes for patients with challenging and non-healing bone defects. The ultimate goal of my work is to offer a solution to non-healing bone defects with a particular focus on developing and characterising an osteoinductive, biocompatible and mechanically robust biomaterial that can act as a bone graft substitute to support bone healing for critical size (non-healing) bone defects and can be customised to patients’ specific needs. Research Methodology: The work in this thesis extends across five fundamental sections:
1- Isolation and characterisation of adipose-derived mesenchymal stem cells (Ad-MSCs) as a source of osteogenic precursor cells.
2- Investigating the potential use of extracorporeal shockwave therapy (ESWT) to stimulate bone healing.
3- Development of a novel 3D printable biomaterial, based on decellularised bone matrix (DCBM), for potential use as bone graft substitute and *in-vitro* characterisation of the scaffolds to ensure complete decellularisation, evaluate mineral content, cytocompatibility, osteoinductivity, microstructure and biomechanical properties.
4- *In-vivo* testing of the local biological effect of candidate biomaterials following subcutaneous implantation in rats.
5- *In-vivo* evaluation of the biomaterials’ ability to support bone healing in a critical-size bone defect (15 mm rabbit’s radius segmental defect). Results and conclusion: Our results show that Ad-MSCs were capable of differentiating towards the osteogenic lineage, as indicated by the expression of markers of both early matrix maturation markers (such as (alkaline phosphatase, RUNX2, collagen type I, osteopontin osteonectin) and late matrix mineralisation markers (such as osteocalcin, Alizarin red staining), as well as exhibiting osteoblastic morphology upon scanning electron microscopy. While ESWT didn’t promote Ad-MSCs proliferation, the results showed that focused ESWT enhances Ad-MSCs osteogenic differentiation ability *in-vitro*. The 3D-printed DCBM scaffold was shown to be cytocompatible and osteoinductive. And to have microstructure, mechanical and material properties that support clinical use as a bone graft substitute. *In-vivo* testing in the rat subcutaneous model established the biocompatibility of the material, and data from the segmental bone defect study in rabbits confirmed that the scaffolds could integrate with native bone and support osteoinduction and osteoconduction by week 12 post-implantation.Item Open Access Zoonotic disease dynamics in displacement: A multisite case study in Sindh, Pakistan and Mafraq, JordanBraam, Dorien; Braam, Dorien [0000-0002-6011-2392]Complex emergencies are causing unprecedented levels of forced migration, with displacement primarily affecting countries where people are dependent on their animals, with livestock a significant contributor to social and economic output. Animal health is fundamental to human health and wellbeing, including through the provision of food, nutrition and livelihoods. Animal and human health are closely interlinked, as the majority of human infectious pathogens originates in animals. These zoonotic diseases are considered a threat to public health in emergencies and displacement, linked to changes in pathogen and vector environment. Although little is known about the exact disease dynamics due to scarce evidence, the fear of zoonoses has resulted in the exclusion of animals from emergency responses. This doctoral research aims to answer the research question of: how can interdisciplinary theoretical and empirical foundations be enhanced and applied to the problem of the simplified narrative around zoonotic disease dynamics associated with displacement of humans and animals? Adopting a multisite case study methodology, I investigated zoonotic disease dynamics across three displacement contexts: among survivors of the 2010 superfloods in Sindh province in Pakistan; environmentally displaced populations during monsoons and following sea intrusion in Sindh; and Syrian refugees in Mafraq Governorate in Jordan. I adopted qualitative methods, including literature reviews, semi-structured expert key informant and household interviews, supported by observations, to allow for a holistic consideration of the factors and processes impacting zoonotic disease dynamics during displacement. Among the participants were 30 expert key informants and 26 households in Thatta and Sujawal districts in Sindh province in Pakistan, and 14 expert key informants and 14 households across Mafraq Governorate in Jordan. Data was analysed using a thematic analysis, to construct themes describing the complex elements determining zoonotic disease dynamics in displacement. The case studies show that zoonotic disease dynamics in displacement, and forced migration itself, are rooted in complex historical, political and socio-economic processes, leading to social marginalisation and vulnerability to disaster and disease. As a result, conventional applications of epidemiological models may be insufficient. Simultaneously, the anthropocentric view on biosecurity and a failure to understand the importance of animals to human lives and livelihoods, has led to their exclusion in humanitarian responses. This thesis proposes a more holistic analytical framework to investigate and address zoonotic disease dynamics in displacement, enabling the examination of these complex processes. This framework includes the consideration of social and economic capital, environmental inequality and policies and practices of exclusion to zoonotic disease dynamics in displacement. The framework may inform future empirical studies to strengthen evidence-informed policy development and humanitarian responses, enhancing the facilitation and protection of animals in emergencies and displacement, ultimately benefitting displaced communities’ health and welfare. As such, this work contributes to the improvement of strategies for comprehensive emergency and recovery responses, recognising animals as important entry points. Existing local and traditional knowledge, resources, and capacity needs to be strengthened, including disease prevention, veterinary services and institutions during displacement. The framework and its implications need to be further developed and tested for implementation to ensure that displaced populations and their animals are truly integrated in policies and service delivery, for the protection of both human and animal health.Item Open Access Identification and recombinant production of antimicrobial peptides from the Black Soldier Fly, Hermetia illucens (L.)Fahmy, LeilaThe need for new antimicrobial therapies to treat drug-resistant bacterial infections is one of the leading risks to global public health. Previous discovery strategies concentrated on the isolation of secondary metabolite antibiotics that are secreted by soil-dwelling bacteria. However, the exhaustion of such discovery platforms has led to an interest in other avenues of research. In recent years, antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional antibiotics that possess potent antimicrobial abilities and are less prone to resistance. AMPs are important components of innate immune systems found ubiquitously in nature. The Black Soldier Fly, Hermetia illucens (L.), has been recognised as a rich source of AMPs and several AMPs have been characterised for their antibacterial activities. This thesis aims to identify and produce AMPs from H. illucens and build on our collective repertoire of antimicrobial strategies against drug-resistant bacterial infections. To achieve this, an approach was taken that amalgamated experimental and theoretical studies and led to the identification of several novel putative AMPs from H. illucens. AMPs are typically small proteins under 10kDa. Low molecular weight proteins were identified by mass spectrometry from the haemolymph of H. illucens larvae. At the same time, AMP sequences were computationally predicted from the coding sequences of the H. illucens genome. Translated protein sequences from 3 of the coding sequences predicted to be AMPs were contained in the haemolymph mass spectrometry data set. These 3 putative AMPs alongside 6 other H. illucens AMPs from published studies encompassed several families of AMPs including attacins, cecropins, and defensins. The 9 candidate putative AMPs were carried forward to in silico and in vitro phenotypic studies. During this project, there have been major advancements in computational protein structure predictions that allowed us to theoretically characterise the 9 candidate putative AMPs. Such analyses led to several important discoveries. The 3 novel putative AMPs (named LF01, LF07, and LF10) were identified as members of paralogous gene clusters with closely related protein structures which is indicative of an evolutionarily conserved family. Also, a predicted attacin AMP (named LF06) was proposed to adopt a homo-trimeric structure similar to a Gram-negative bacterial porin. Genes encoding the 9 candidate putative AMPs were cloned into a pET expression system that was under the control of the inducible T7 promoter. Recombinant proteins were heterologously expressed and purified from Escherichia coli BL21(DE3) cells. Of the 9 proteins, 5 putative AMPs were screened for antimicrobial activities against a panel of 12 bacterial strains. Two of the candidate putative AMPs were recognised for their antimicrobial activities against the serious Gram-negative pathogen Pseudomonas aeruginosa. The 2 AMPs were the LF06 attacin which had been predicted to function as a homo-trimeric porin and LF10 which was one of the novel putative AMPs that was encoded in a paralogous gene cluster. The 2 AMPs were characterised by their activities against 2 strains of P. aeruginosa including the highly virulent Pa14 strain. Membrane damage induced by the AMPs was visualised using fluorescent and transmission electron microscopy, and electrochemical impedance spectroscopy. In summary, this work has used modern and traditional approaches to identify and engineer 2 new AMPs with antibacterial activities. These important AMPs warrant further development and could be used for the treatment of infections caused by the serious pathogen P. aeruginosa.Item Open Access Health and Welfare Implications of Temperament Differences in Dairy CattleBritten, NicholasDairy cattle exhibit consistent variation in behaviour between individuals, referred to as temperament. In humans consistent variation in behaviour is referred to as personality and has significant associations with health and wellbeing. The studies reported in this thesis investigated whether temperament is also associated with health and welfare outcomes in dairy cattle. No universal framework for expressing temperament currently exists in cattle and temperament has previously been evaluated using both behavioural and physiological measurements. In this thesis a framework for expressing temperament, comprised of the five traits Activity, Aggression, Boldness, Exploration and Sociability, was validated in lactating dairy cattle using factor analysis on the results of behavioural testing. The validated framework was used to quantify the temperaments of cattle at 21, 60 and 100 days lactation. Measurements of physiological parameters, hair cortisol concentration, saliva cortisol concentration, heart rate variability and maximum eye temperature, were taken alongside behavioural testing to determine whether they were reliable indicators of temperament. Temperament testing was consistent between 21 and 100 days lactation but not between 21 and 60 days or 60 and 100 days, possibly due to the effect of milk yield on the autonomic nervous system. Cortisol concentrations and eye temperature had no consistent relationship with single temperament traits but heart rate and sympathetic response, quantified by heart rate variability, were reliable indicators of Boldness during a startle test at both 21 and 100 days lactation. The validated framework was used to test a cohort of dairy calves pre-weaning, post-weaning, pre-puberty and post-puberty to determine whether temperament of cattle is consistent during maturation. This study found that behavioural measures of temperament were not consistent between measurements but parasympathetic activity in a crush test and eye temperature response to a novel arena, startle and novel object test were consistent from pre-weaning to post-puberty. The temperament scores and eye temperature measurements were used to quantify associations between temperament and risk of infectious disease. High Activity calves were significantly more likely to be treated for pneumonia and high Activity cows at 21 days lactation were significantly more likely to be treated for an infection from 21-100 days lactation. This work demonstrated that temperament can be measured consistently and is associated with greater risk of infectious diseases in both lactating cattle and youngstock.Item Open Access Intestinal Handling of Food Grade Titanium DioxideMiniter, MichelleFood grade titanium dioxide (fgTiO2) is a biologically persistent microparticle with an average diameter of 130 nm. It is an additive in the human diet and an excipient in some capsules, tablets, and toothpastes with a median intake of ~2.5 mg per person per day in the UK. Its impact, if any, is not understood. In particular, understanding which gut cells are targeted by fgTiO2 and any potential effects of this requires clarification. This thesis reports a detailed understanding of cellular uptake of fgTiO2 in the mammalian gastrointestinal tract. Having reviewed the relevant literature it was clear that methodology for fgTiO2 detection in tissues merited careful attention. This work identifies reflectance microscopy, with a confocal microscope, as a suitable technique for per particle analysis in intestinal tissue and subsequently demonstrates that this can be used quantitatively, and single particle detection was validated against electron microscopy with microanalysis. Employing the same tools, methodology for quantitative immunofluorescence of the immune cell marker PD-L1, also at the per-cell level, was developed to allow co-detection of this and fgTiO2. With appropriate methodology firmly established, I investigate sites of particle uptake in the murine gut and describe the subepithelial dome of the Peyer’s patch as a newly recognised site of accumulation. I then demonstrate the marked similarities between this and fgTiO2 accumulation in human Peyer’s patches through the study of six separate samples from each species. Following this, I sought to determine how a mildly pathogenic Salmonella inoculation would modulate particle accumulation in the Peyer’s patch cells. A loss of fgTiO2 from the subepithelial dome region was demonstrated when compared to non-inoculated control mice; I propose that pathogen-induced particle-cell migration occurs but found no evidence that this was to elsewhere in the patch. Thus, further work should consider mesenteric lymph nodes as a potential target in such situations. Finally, I show that fgTiO2 does not alter subepithelial dome expression of PD-L1 whether in the presence or absence of Salmonella. Overall, I conclude that ingested fgTiO2 selectively migrates to phagocytic cells of intestinal Peyer’s patches, especially in the subepithelial dome region. However, if the local system is perturbed by this accumulation, PD-L1 is not modulated so inflammation is unlikely to occur. Conversely, a mild Salmonella challenge led to particle migration. Thus, establishing where fgTiO2 goes and how it gets there, should form the basis for future research.Item Open Access Oligodendrocyte precursor cell states and their regulationKamen, Yasmine; Kamen, Yasmine [0000-0003-1867-7397]Oligodendrocyte precursor cells (OPCs) differentiate into myelinating oligodendrocytes throughout life, allowing the formation of myelin during early development, but also following learning or demyelinating injuries. A number of factors are thought to regulate OPC proliferation and differentiation, including neuronal activity, which OPCs can sense and respond to through their voltage-gated ion channels and neurotransmitter receptors. Functional expression of these channels and receptors is often described as a defining feature of OPCs, although several reports suggest that OPCs exhibit diversity in their electrophysiological properties. In this thesis, I investigate the diversity in OPC electrophysiological properties by performing whole-cell patch-clamp recordings of OPCs in different brain regions throughout the lifespan. I find that although OPCs first appear as a homogeneous population in the embryo, lacking voltage-gated ion channels and glutamate receptors, they gradually acquire these channels and receptors at different rates and become heterogeneous between and within brain regions. Correlating these electrophysiological data with RNA sequencing data, I propose that OPC diversity represents functional cellular states rather than different cell types. In addition, I investigate how passive membrane properties change with age, and examine how AMPA but not kainate receptors differ with age. I further investigate whether OPCs can transition between different electrophysiological profiles, as would be the case if OPC diversity represented cell states. I focus on G protein-coupled neurotransmitter receptors as potential regulators of state transitions, as they can regulate OPC proliferation and differentiation, and were shown to modulate glutamate receptors in OPCs. I find that clemastine, a muscarinic receptor antagonist currently being tested to enhance myelin repair in Multiple Sclerosis clinical trials, alters glutamate receptors in OPCs, suggesting that cholinergic signalling can modulate OPC states. Moreover, driving G protein signalling in OPCs using Designer Receptors Exclusively Activated by Designer Drugs modifies their voltage-gated ion channels and glutamate receptors. Collectively, these data suggest that OPCs can transition between electrophysiological profiles in response to environmental cues such as G protein-coupled neurotransmitter signalling, and thus, that their diversity may represent functional cellular states.Item Open Access Modelling the spatial and temporal distribution of Bacillus anthracis suitability across Uganda and Kenya: A Bayesian ApproachNdolo, ValentinaModelling the spatial and temporal distribution of Bacillus anthracis suitability across Uganda and Kenya: A Bayesian Approach Valentina Awino Ndolo Anthrax disease, caused by Bacillus anthracis, poses a significant global disease burden, leading to sporadic outbreaks across a variety of climatic regions. Through the action of two virulence plasmids, pXO1 and pXO2, the bacteria can evade host immunity and produce two toxins that can rapidly kill susceptible hosts, which then shed the bacteria back into the environment, resulting in recurrent outbreaks and catastrophic loss of animal life. The location, timing, size, and duration of anthrax outbreaks usually vary from year to year, making the characterization of the spatial-temporal patterns of disease occurrence challenging. It is necessary to consider environmental and host-specific drivers of the spatial distribution of B. anthracis suitability across different contexts. Ecological datasets have become richer, and there are statistical challenges associated with their analysis. Accurate insight and prediction can only be developed using a novel hierarchical approach that deals with structural dependencies in data, and so produces more robust predictions of B. anthracis suitability to guide efficient prioritization of scarce resources for disease prevention. In this thesis, I examine the environmental and socio-economic drivers of B. anthracis suitability, applying both conventional and novel hierarchical modelling approaches. In Chapter 1, I describe the biology, ecology, clinical signs and diagnosis, treatment, prevention, control, and epidemiological burden of anthrax disease. In Chapter 2, I review published literature to understand the type of input data, suitability modelling algorithms, and evaluation methods previously used. I discuss the application of the Integrated Nested Laplace Approximation (INLA), a spatial method that can be applied to structurally complex data, to model the suitability of B. anthracis. In Chapter 3, I apply a common conventional algorithm to analyse a 15- year dataset comprising confirmed and probable wildlife, livestock, and human anthrax cases collected across Uganda to determine the spatial distribution of B. anthracis suitability across the country. In Chapter 4, I apply INLA to develop a model for B. anthracis suitability in Uganda using the same dataset as in Chapter 3. In Chapter 5, I use a spatiotemporal model to investigate the socio-economic and climatic drivers of anthrax occurrence and incidence in Kenya at the national and sub-county levels. In Chapter 6, I assess the knowledge, attitudes, and practices of livestock farmers in Northern Uganda that might influence anthrax prevention and control. In Chapter 7, I summarise my findings and discuss how the new insights into the drivers and the spatial distribution of B. anthracis suitability can avail new opportunities for more robust anthrax prevention and control and future research.Item Open Access Bats as Bushmeat in Ghana(2012-12-10) Kamins, AlexandraDespite major advances in vaccines, antibiotics and antiviral treatments, infectious diseases still kill 15 million people around the world each year - one quarter of global deaths (Fauci and Morens, 2012). Emerging diseases, caused by new or newly expanding pathogens, form a rapidly changing and intensely challenging front of this international battle. Pathogens that can jump from animals into humans, known as zoonoses, cause over 65% of emerging diseases; three quarters of zoonoses originate in wildlife (Jones et al., 2008). One of the most common sources of human-wildlife contact is through hunting wild animals for food. Researchers have estimated that western and central Africans alone harvest five million tons of bushmeat annually (Fa et al., 2002; Ntiamoa-Baidu, 1998). This massive industry raises concerns across disciplines: not only do epidemiologists fear for the transmission of zoonotic diseases (Daszak, 2000), but conservationists fear for the depletion of threatened and endangered species (Milner-Gulland and Bennett, 2003) and development practitioners worry for the welfare of the communities and people that depend on wild meat for protein and income (Davies, 2002). Of the many animals hunted for bushmeat, bats pose a number of unanswered questions. Hosting almost sixty viruses that can or do infect humans (Wong et al., 2007), bats globally also are suffering severe population declines due to overhunting (Streubig et al., 2007; Harrison et al., 2011). Yet their roles as reservoirs of human disease, as sources of valuable products, and in human culture are all understudied and largely unexplained. The newly identified presence of henipavirus antibodies and $\textit{Bartonella}$ bacteria in $\textit{Eidolon helvum}$ fruit bats in Ghana, as well as the potential for commercial hunting of fruit bats, led me to this project in Ghana, West Africa. I set out to: (1) delineate ways in which bats and their pathogens may come into contact with humans; (2) understand how, where and how many bats are hunted, prepared and sold as bushmeat; (3) identify and characterise potentially at-risk populations for bat-borne zoonoses; and (4) determine whether transmission of selected bat-borne pathogens is already occurring within the identified populations. The urban nature of $\textit{E. helvum}$, the high prevalence of raw palm sap drinking and the large bat bushmeat market all serve as modes of contact between humans and bats. Through 551 interviews with Ghanaians as well as more in depth work with 26 vendors and hunters, I identified demographics as well as perceptions of people involved in bat bushmeat trade. Bat hunting, selling and consumption are widely distributed across region and tribal lines, with hotspots in certain locales; butchering is concentrated in females and active hunters. Interviewees held little belief of disease risk from bats, saw no ecological value of fruit bats outside their economic worth and thought that consumption related to specific tribes. Serological and culture evidence for humans in close contact with bats (by living or working around large $\textit{E. helvum}$ colonies; or through hunting or butchering bats) strongly suggests there is no current spillover of bat-borne $\textit{Bartonella}$ infections. However, a low prevalence of positive human sera samples for henipavirus antibodies using a Luminex testing platform suggests that there may be human exposure to a henipa-like virus.Item Embargo Genomic diversity and antimicrobial resistance in Salmonella isolates recovered from diverse sources in NigeriaAchi, ChiomaAntimicrobial Resistance (AMR) jeopardises the gains of modern medicine and is therefore considered a global public health threat. It has been predicted to cause up to 10 million deaths by 2050 if no serious actions are taken. AMR threatens people's lives worldwide but with a significantly higher burden in low and middle-income countries (LMICs), where it is challenging to determine the exact burden of bacterial AMR due to gaps in surveillance. Salmonella is an important zoonotic gram-negative pathogen that may be categorised into serovars and sequence types (STs) based on seven housekeeping genes. The Salmonella isolates that cause problems vary for geographical regions and have different antimicrobial susceptibility patterns. This is likely to be driven by the antimicrobials that are in use in these regions. Fluoroquinolone and cephalosporins are important antimicrobials for treating salmonellosis, but due to growing resistance, the World Health Organisation now considers fluoroquinolone-resistant (FQR) Salmonella a critically important pathogen. The central hypothesis of this thesis is that a better understanding of the genetic diversity, and sources, of antibiotic resistant Salmonella spp in Nigeria, and of markers of emergent antibiotic resistance will inform and encourage the development of integrated interventions to reduce the impact of Salmonella in this country. Whole-genome sequences of the sampled dataset combined with publicly available Nontyphoidal Salmonella (NTS) genomes (Nigeria NTS collection) recovered from various sources revealed the diversity of NTS in Nigeria. One hundred and twenty-one unique serovars belonging to 167 STs were identified within the dataset, with some serovars having more than one ST. An eBURST diagram confirmed that the majority of the Nigeria NTS collection were not closely related. S. enterica Kentucky (ST198) was the most common strain in poultry, while S. enterica Enteritidis (ST11) was the most common human serovar. Phenotypic and genotypic analyses were performed on the isolates. The highest level of resistance (57%) was observed for nalidixic acid, followed by 36.2% and 35% resistance to ciprofloxacin and gentamicin, respectively, on Disc Diffusion (DD); while VITEK-2 identified 32% of the strains to be resistant to enrofloxacin and marbofloxacin and 34% to gentamicin. Multidrug resistance to other antimicrobials was found in some serovars, and poultry isolates had higher resistance than other species, probably due to greater use of antimicrobials in these species. The tet(A) gene was the most common single resistance gene identified in the collection. In strains that showed resistance to the fluoroquinolones, 15% had the qnr gene. FQR in NTS was largely mediated by amino acid substitutions in the clinically significant positions of Ser83 and Asp87 of gyrA. Col(pHAD28) was the most common plasmid identified in the Nigeria NTS collection. My study showed that FQR could not be simply predicted from the presence of resistance genes and demonstrated that SNPs associated with FQR were the primary indicators of phenotypic resistance. FQR was poorly predicted in S. Kentucky, and more sulphonamide genes were predicted compared to phenotypic methods. Three genes conferring resistance to the β-lactams were detected, with TEM-1 β-lactamase being the most common β-lactamase found, and S. Schwarzengrund isolates with a variant of blaTEM-215 were the only isolates with an amino acid change from Histidine to Arginine at position 151 of TEM-1. Antimicrobial stewardship was worryingly low amongst poultry farmers. In particular, critically important antimicrobial such as colistin was reported to be used on farms. The antimicrobial for which resistance was found matched those for which high usage was reported. Findings from this research indicate that the burden of AMR is high in Nigeria as up to 44% of isolates in the collection carried at least one AMR gene. Although the dataset used in this study is only a fraction of the larger Salmonella population and does not consider other bacterial pathogens, the findings here might suggest a broader AMR problem in the larger population. The ease of access and misuse of important antimicrobials might contribute to the high AMR observed. This study also suggests a high-level diversity of NTS in Nigeria identified from multiple hosts, with isolates from Nigeria sharing some similarities with strains from neighbouring African countries, suggesting that trade and travel might play an important role in the transmission of pathogens and possibly resistance. A One-Health approach and regional effort are needed to tackle NTS and AMR's spread within Nigeria and the African region, considering the overlap of strains from various sources.Item Open Access Fatty Acid Regulation of NLRP3 Inflammasome ActivityLiang, JonathanDiseases associated with unhealthy diets and lifestyles, such as type 2 diabetes, atherosclerosis, and fatty liver disease, are driven by sterile tissue inflammation that is driven in part by the NLRP3 inflammasome. This macromolecular complex responds to diverse danger signals by inducing secretion of inflammatory IL-1 family cytokines and promoting a type of cell death called pyroptosis. Saturated fatty acids (SFAs) are known to activate the NLRP3 inflammasome but the intracellular pathways by which this activation occurs are incompletely described. I hypothesized that SFAs activate the NLRP3 inflammasome through different intracellular pathways than “classical” NLRP3 activators. Motivated by preliminary data on the kinetics of NLRP3 activation by the SFA palmitic acid (PA) and the weak effect of the NLRP3 inhibitor MCC950, I found that this process requires not only the canonical inflammasome pathway but also the non-canonical inflammasome components caspase-4/5/11. Disruption of either pathway leads to partial impairments in the inflammasome response to PA, though the canonical pathway generally accounts for more IL-1 secretion while the non-canonical pathway is the primary driver of cell death. PA also has a weaker dependence on reactive oxygen species (ROS) compared to other NLRP3 activators. While classical activators engage multiple ROS sources including both mitochondrial and cytosolic enzymes, PA activation of NLRP3 specifically requires ROS from NADPH oxidases. PA exposure leads to activation of the MAP kinase JNK in the cytosol of macrophages and interference with JNK signaling blocks PA-induced NLRP3 activation. While SFAs activate the NLRP3 inflammasome, unsaturated fatty acids such as arachidonic acid (AA) inhibit NLRP3 activation. AA counteracts the effect of PA on multiple intracellular signaling pathways, especially the JNK pathway. My findings further characterize the mechanisms by which SFAs activate the NLRP3 inflammasome and describe a novel role of JNK as a key mediator for opposing effects of saturated and unsaturated fatty acids on NLRP3. These mechanistic insights highlight pathways that may be targeted to delay or reverse the progression of sterile inflammation in many “lifestyle diseases”.Item Open Access Pain alleviating effects of mesenchymal stem/stromal cells and derived extracellular vesicles in osteoarthritisAi, Minji; Ai, Minji [0000-0002-0614-5265]Musculoskeletal disorders pose significant burdens on both individuals and society. Osteoarthritis (OA) is a common form of musculoskeletal disease affecting around a third of the population aged over 60 years old. It is characterised by debilitating joint pain and stiffness that only partially correlates with joint structural damage. Clinically used OA treatments fail to halt disease progression and yield limited pain relief. During the search for new therapies for OA, mesenchymal stem cells (MSCs) and derived extracellular vesicles (MSC-EVs) have led to promising therapeutic outcomes such as joint function improvement and pain relief. Whether the observed pain relief effects of MSCs and MSC-EVs originate from the direct effect on pain-sensing neurones in OA joints remains unknown. This Thesis aims to uncover the mechanisms of the pain-alleviating effects of MSCs and MSC-EVs in OA joints. To study whether MSCs and MSC-EVs reduce pain through acting on pain-sensing neurones in OA joints, it is essential to examine whether the presence of MSCs and MSC-EVs influence naïve sensory neurone activity. To do this, an in vitro co-culture was established to study the excitability and ion channel activities of dorsal root ganglion (DRG) neurones, the cell bodies of knee-innervating sensory neurones located in the DRG, both in direct and indirect co-culture with MSCs. Results showed that neither the excitability of nor the tested ion channel function of naïve DRG neurone altered when co-cultured with MSCs and the MSCs conditioned medium in vitro. These results provided a proof of concept of using an in vitro co-culture model to study the impact of MSCs on DRG neurone activity. Additionally, understanding the molecular mechanisms of OA pain at different stages of the disease is also critical for unveiling the analgesic mechanisms of MSCs and MSC-EVs. Thus, a surgically induced mouse OA model, destabilisation of the medial meniscus (DMM), was used to identify molecular targets mediating OA pain. DMM operated mice were kept for 8 weeks to develop OA, during which pain-related behaviour changes were monitored by the digital ventilated cage (DVC) system and digging behaviour testing. After 8 weeks, DMM mice presented moderate OA in operated joints but not pain-related behavioural changes. Further characterisation of knee-innervating neurones showed that such neurones from DMM mice were significantly more depolarised than those from Sham mice. Additionally, DRG neurones isolated from 8-weeks DMM mice showed increased Ca2+ influx in response to P2X3R agonist α,β me-ATP, and increased P2X3R immunoreactivity was also seen in the DRG of 8-week DMM, which suggest the potential role of P2X3R in OA pain at this stage of the disease. With the establishment of in vitro and in vivo models for OA pain study, the pain alleviation effects of MSCs and MSC-EV in advanced OA were next investigated. Using the DMM induced OA model, it was found that MSCs and MSC-EVs improve pain-related behaviour changes (i.e. improved motor coordination, digging and sleep) in DMM mice, and such behavioural improvement was not the result of reduced joint damage, but rather normalised excitability of knee-innervating sensory neurones from MSCs and MSC-EVs treated DMM mice. Furthermore, in vitro study showed that MSC-EVs can normalize sensory neurone hyperexcitability induced by nerve growth factor (NGF). Together, these results suggested that MSCs and MSC-EVs improve pain in OA which may be through a direct action on peripheral sensory neurones. Mice have been the predominant species used for OA research in this Thesis. However, the genetic, biomechanical, and anatomical differences between mice and humans can hinder the clinical translation of observed outcomes. Thus, this Thesis further explored the possibility of using sheep as an animal model for OA pain research. An osteochondral defect (OD), a common clinical observation in joints leading to OA, was created in sheep, and the sensory neurone properties of these operated sheep were investigated. Increased sensory neurone excitability and increased transient receptor potential vanilloid 1 (TRPV1) ion channel function were observed in DRG neurones innervating the OD operated joint, which demonstrated the utility of sheep for the study of joint pain mechanisms. Overall, this Thesis discovered that MSCs provide pain relief in OA joints through normalising sensory neurone activity, possibly via the release of MSC-EVs. Additionally, this Thesis also established an in vitro model that can be used for further study of MSCs and sensory neurone interactions and explored the possibility of using sheep as an animal model for orthopaedic pain research.Item Open Access Potential of faeces for non-invasive assessment of Juan Fernandez fur seals and their environmentToro Valdivieso, Constanza ValentinaThe Juan Fernandez fur seal (JFFS) is a marine mammal endemic to the Juan Fernandez Archipelago in the Pacific Ocean. The archipelago is a UNESCO Biosphere Reserve and has been identified as one of the eleven irreplaceable priority sites for marine conservation worldwide. As a result of overhunting between the 17, 18 and 19th centuries, the JFFS was severely reduced. Furthermore, it was presumed extinct by the end of the 19th century. Since its "rediscovery" in the early 60s up to current times, the JFFS population have evidenced a steady recovery. Today, it is an icon for local tourism and a great example of population recovery. Since 1995 the Chilean government has decreed a 30-year hunting ban, enabling an impressive recovery. However, the ban on hunting will only last until 2025, and it is unclear what conservation measures will be put in place after this date. Besides intermittent basic censuses, no further monitoring has been done on this species in the last two decades, to the best of my knowledge. Complicated logistics, inaccessibility, lack of funding, and human resources may partially explain the lack of research. However, in the context of the hunting ban coming to an end in less than five years, there is an urgent need to build as much knowledge as possible in a relatively short period to inform policymakers when deciding on the future protection measures for this species. Studying marine mammals such as the JFFS can bring further benefits. For instance, as high trophic organisms, marine mammals play an essential role in nutrient and energy transfer and regulating other species' abundance. Additionally, their long lifespan and extensive fat storage enable the bioaccumulation and biomagnification of liposoluble toxins. These and other characteristics have made marine mammals an increasing target for understanding and monitoring various changes in oceanic ecosystems, including the behaviour and effects of pollutants. However, adequate knowledge about the target species' behaviour and ecology is required to use these animals as marine bioindicators. To establish a baseline for the study and monitoring of the JFFS, I explored the potential of faecal samples as a non-invasive method to obtain diverse information while lowering sampling cost and logistic complexities. Here, I focused on only three of the multiple topics that can be studied from faecal samples. First, I used 16S rRNA amplicon sequencing to characterise the faecal microbiome (Chapter 3). This first faecal microbiome characterisation evidenced a clear separation of the samples into two clusters. Due to the little information available on the species, it was not possible to provide a clear explanation of the pattern observed here. However, diet and sex (associated with prey selection and, therefore, diet) could be possible explanations. On the other hand, the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) inferred pathways associated with pathogenesis were enriched in cluster 2, which contained only 22 % of the samples. This first insight contributes to understanding the natural microbial diversity in free-free ranging pinnipeds. Next, I used inductively coupled plasma mass spectrometry to evaluate heavy metal exposure (Chapter 4). The results evidenced high levels of Cd and Hg in the JFFS compared to the Antarctic fur seal (AFS), suggesting high exposure. Diet is the most likely source of contamination. Motivated by these results, I analysed Cd in bone samples, evidencing Cd absorption. These samples, however, did not evidence any of the changes usually associated with Cd intoxication in bone, suggesting some degree of adaptation to high levels of this toxic heavy metal. Furthermore, Si levels, an ultra-trace element related to bone health, could be an interesting target for future studies on Cd tolerance in JFFS. Human studies on this topic may also benefit. Finally, in Chapter 5, I focused on optimising a method for collection, storage, and host DNA extraction and amplification of faecal samples. For this optimisation, I targeted the mtDNA control region, five different microsatellite loci and two loci commonly targeted for molecular sex identification. Swabbing the faecal surface was usually associated with less specific PCR products. However, by performing nested PCRs, the specificity of the amplification dramatically improved in samples with poor DNA. On the other hand, when using more sensitive assays such as real-time PCR, which was used for molecular sex identification, a nested PCR approach should only be considered when direct amplification fails to avoid sample contamination. This study showed that working with faecal samples for investigating population genetics requires significant optimisation. However, once methods become optimised, the difficulty of processing these samples reduce while the probability of success increases. Even though not yet complete, this study is a significant contribution that will enable more rigorous monitoring of the JFFS. The information generated from my research is an essential contribution to the knowledge about this species which is urgently needed to inform policymakers for future conservation policies. Here, I have shown that working with faecal samples can be an accessible alternative to studying various aspects of a species.Item Open Access Absorption of Dietary Titanium DioxideBarreto Da Silva, AlessandraFood grade Titanium dioxide (fgTiO2) is used as a colouring additive in the food industry, and it is present in food, capsules, and toothpaste. We are exposed to a median of 2.5 mg fgTiO2 per day in the UK through such sources. These are persistent micro and nanoparticles that have been reported in human tissues, yet how much is absorbed and any health impacts from this are not understood. Published studies suggest that humans absorb fgTiO2 particles when ingested as a powder. This thesis investigated the fgTiO2 bioavailability from ‘real foods’. Having reviewed the literature in Chapter 1, I then, in Chapter 2, developed a highly sensitive and robust method to quantify Ti (TiO2 proxy) in whole blood using Inductively Coupled Plasma Mass Spectrometry (ICP MS/MS). I proved that this would detect 0.012 parts per billion of Ti. I then used this method in Chapters 3 and 4, showing, in the former, that oral exposure to 215.5 mg of fgTiO2 in a hard shell candy (M&M®) did not lead to detectable Ti in the blood of 17 healthy human volunteers. This study had a large control group (n = 26), allowing me to report on the baseline blood levels of Ti in humans. In Chapter 4, I investigated whether fluid food matrix may lead to detectable fgTiO2 absorption. This was a cross over study with 12 healthy volunteers ingesting 253.8 mg of fgTiO2 in a coffee creamer (Walden Farm®). However, again, no increase in blood Ti levels was observed. In chapter 5, I took a fresh approach, using mice fed for 112 days with human-relevant levels of fgTiO2, hypothesising that minimal but continuous absorption would allow me to detect elevated Ti levels in target tissues (i.e., evidence of bioaccumulation). Again, analytical propriety was observed, but elevated Ti was not detected in tissues. Interestingly, for intestinal Peyer’s patches, where spots of TiO2 could be observed by microscopy, there was no detectable signal by ICP MS/MS. I conclude that (a) background Ti levels in blood and organs are very low, (b) any absorption of fgTiO2 from foods ingested by humans must be extremely low and, (c) total analysis techniques, even as sensitive as ICP MS/MS, will fail to detect tiny amounts of absorbed fgTiO2, presumably because the rare particle events are too diluted by their surrounding.Item Open Access Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuniCoates, Ruby; Coates, Ruby [0000-0002-2087-654X]Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. This thesis aims to address this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression and thereby investigate gene function. To achieve this, a “dead” cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176. The tool was validated through successful repression of the arylsulphatase gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was futher validated against the hippicurase gene hipO, and trialled with an Anhydrotetracycline (ATc) inducible promoter, which demonstrated leaky expression in the absence of ATc. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM). Finally, CRISPRi-based repression of the flagella genes fliE and fliQ was attempted to trial targeting of essential genes in C. jejuni. To date, this is the first report of a CRISPR-based interference system demonstrated in C. jejuni.Item Open Access Genomic insights into the host range and interspecies transmission dynamics of Staphylococcus aureus(2022-01-18) Matuszewska, Marta; Matuszewska, Marta [0000-0002-2653-7725]Staphylococcus aureus is an important human bacterial pathogen with a wide host range, including livestock, companion, and wild animal species. Genomic and epidemiological studies show that S. aureus can freely transmit between different species, with some of these transmissions resulting in successful adaptation and ongoing transmission in a new species. Given an ever-increasing risk of zoonotic disease emergence due to changes in the humananimal interface, it is increasingly important that we understand the capacity for human pathogens such as S. aureus to move between and adapt to different host species. In this thesis, I apply comparative genomic analyses to investigate the host range, transmission dynamics and host adaptation of S. aureus in livestock, companion animal, and wild animal populations. Chapters 2 and 3 consider the role of horizontal gene transfer in the adaptation of S. aureus clonal-complex (CC) 398 to livestock. CC398 is the dominant S. aureus lineage in European livestock and is implicated in increasing numbers of human infections. I found that the emergence of livestock-associated CC398 coincided with acquiring a Tn916 transposon carrying a tetracycline resistance gene, which has now been stably maintained for 57 years. This was followed by the acquisition of a SCCmec type V that carries methicillin, tetracycline, and heavy-metal resistance genes, which some lineages have maintained for 35 years. In contrast, a phage containing human immune evasion genes is dynamically gained and lost. These contrasting dynamics result in no loss of antimicrobial resistance but the rapid acquisition of the human adaptive element when methicillin-resistant S. aureus (MRSA) jumps from livestock into humans. In later chapters, I address a gap in our understanding of host specificity of S. aureus through using unbiased sampling of both methicillin-sensitive S. aureus (MSSA), and MRSA isolates from both an unsampled wild animal population and an undersampled companion animal species. Chapter 4 investigates the prevalence and risk factors associated with S. aureus carriage in cats in south-eastern Poland. Results suggest that older cats, sick cats, and cats with an S. aureus colonised owner increase the risk of carriage. I then used whole-genome sequences to investigate population structure, showing that S. aureus in cats is diverse and has no dominating lineage. Chapter 5 demonstrates that transmission between cats and their owners can occur within households, and I characterise potential catassociated lineages. In chapter 6, I found no S. aureus isolates in wild populations of brown trout in the Scottish Highlands. This population is phylogenetically divergent from the host species S. aureus is most associated with and is geographically and ecologically isolated. Together these results highlight the considerable transmissibility of S. aureus between host species. Most transmission events that I identify reflect spillover events that are self-limiting in their new populations. Nevertheless, I demonstrate that some level of persistence is common, enough that intermediary hosts can transmit to new species. This highlights the connectedness of S. aureus among different host species and that antimicrobial use in other hosts may impact human health.Item Open Access Genetics of transmissible cancers in the Tasmanian devil (Sarcophilus harrisii)Stammnitz, Maximilian Ruprecht; Stammnitz, Maximillian [0000-0002-1704-9199]Transmissible cancers are clonal lineages that spread through populations via contagious cancer cells. Such epidemics have only ever been observed to affect two mammals in nature, yet two distinct clones known as devil facial tumour 1 (DFT1) and devil facial tumour 2 (DFT2) have arisen within populations of the same marsupial carnivores: Tasmanian devils. As a consequence of these aggressive infectious cancers and their rapid spread, the species has been undergoing a substantial decline since the mid-1990s. In my here presented PhD dissertation research, conducted between 2015 and 2020, I provide the first genetic and functional comparisons of these two transmissible Tasmanian devil cancer lineages. My results suggest that both DFT1 and DFT2 emerged recently and independently in different devil subpopulations, yet from similar Schwann cell-related progenitor cells with comparable molecular properties. Through deep sequencing of whole genomes from tumour cohorts of 65 DFT1 and 41 DFT2 cases as well as a normal panel of 80 animals, I reconstruct the mutational trajectories of both clones in space and time. These somatic phylogenies allow for a detailed longitudinal analysis of lineage-specific cell heterogeneity, mutation rates, Darwinian selection and immunological adaptation events. A parallel line of my research was dedicated to the production of data resources for the wider genetics and animal conservation community. Primarily, this comprises ultra-long nanopore DNA sequencing for the assembly of a high-quality Tasmanian devil reference genome, together with a devil tissue-specific gene expression atlas. In this thesis, I show how these can be integrated to deliver valuable new insights into the genetics of the Tasmanian devil population and their transmissible cancers.Item Open Access Understanding the mechanisms of phosphatidylserine exposure in sickle cellsWadud, RasiqhSickle cell disease (SCD) is the most common severe inherited disorder affecting millions of people worldwide. HbS polymerisation leads to a change in red blood cells’ (RBCs) membrane permeability, high phosphatidylserine (PS) exposure, altered RBC rheology and fragility. The high PS exposure is considered to cause some of the hallmark complications of the disease such as vascular occlusion, anaemia and inflammation. This study investigates the possible physiological and cellular signalling pathways involved in PS exposure in RBCs from SCD patients. In the first part, conditions specific to the renal medulla were examined as the majority of SCD patients suffer from nephropathy early in life. It is hypothesised that the ambient conditions of the renal medulla promote polymerisation of RBCs and PS exposure, and so contribute to the detrimental effects of SCD. Thus, the impact of the medullary environment, which is hypoxic, acidotic, hyperosmotic and hypertonic, on RBCs of SCD patients was investigated. In the second part of the thesis, intracellular signalling pathways which could cause high PS exposure were investigated. The study aimed to establish the molecular identity of Psickle and strengthen the link between Psickle and PS exposure. It was hypothesized that PIEZO1, a mechanosensitive ion channel, is a potential candidate for Psickle and a major channel for Ca2+ entry in RBCs. Thus, drugs acting on PIEZO1 such as Yoda1, Dooku1 and GsMTx4 were used to examine their effects on Ca2+ entry and PS exposure. The results from the first part of the thesis suggest that while the hypoxic, hyperosmotic and hypertonic environment have a substantial effect on sickling and PS exposure, the effect of pH is minimal. Furthermore, the experiments have also shown that urea inhibits both sickling and PS exposure (highly significantly in all the above conditions). Results from the second part of the thesis strongly suggest that PIEZO1 can be a major channel for Ca2+ entry leading to PS exposure, together with a Ca2+-independent pathway leading to PS exposure, which is reliant on protein kinase C (PKC). Furthermore, the sphingomyelinase (SMase) signalling pathway was also explored to identify its role in PS exposure. Results with a SMase inhibitor and ceramide on RBCs of SCD patients showed a strong correlation between SMase activity and PS exposure. Moreover, experiments revealed that urea inhibited SMase strongly and might be potentially working through this pathway to reduce PS exposure. These findings further increase our understanding of the conditions and mechanisms, which promote PS exposure and suggest potential future therapeutic targets.