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Drifting pollen geographies: an amateur’s engagement with palynological practice and a drifting self
Get PDFThis thesis aims to explore how pollen science may be used as a tool to understand myself in the world through its history and practices of said history. The thesis uses palynology to explore the relationships between the material and temporal and seeks to unpack the role ‘science’ and ‘art’ play in constructing these relations. I situate the thesis within a growing literature of plant geography with an underlying foundation in the idea of a shimmering relationality being key to unveiling a myriad of assemblages which carry with them temporal and material relationships. This is done through my own personal experiences with researching palynology and participating in palynological field and lab work, as well as my own encounters with more creative expressions and investigations including poetry, photography and a foray into more ‘experimental’ ways of engaging with plants. Moreover, the thesis takes seriously the implications of how a colonial past has shaped my understanding of self and science, and how pollen, much like my own body, becomes a story because of its temporal and material assemblages
Real-time recursive risk assessment for autonomous vehicles
Get PDFThe existing risk assessment coverage for Autonomous Vehicle (AV) deployment is insufficient for AV operations. The existing risk assessment is based on static processes, such as Hazard Analysis and Risk Assessment (HARA), which are performed during AV development. The hazard identification is based on prior lessons learned and know-how. The current risk assessment primarily focuses on vehicular malfunctions and does not assess AV's safety actions when hazards are detected during real-time operations. The static risk assessment is unable to measure AV control actions when a hazardous event is detected during AV operations. These planned control actions can also pose risks during AV operations without real-world testing.
This thesis proposes a real-time risk assessment for AV operations, comprising a Real-time Risk Assessment Framework (ReRAF) within the AV and a Real-time Risk Assessment for Cooperative mode (ReRAC) within the infrastructure. The realtime risk assessment operates in a continuous and recursive loop for safety improvement.
In ReRAF, a novel Dynamic Acquired Risk Assessment (DARA) algorithm is designed, developed and verified to provide a Predicted Risk Number (PRN) for the AV to assess the risk of its ability to mitigate hazards through its control actions. The PRN is derived using the scenarios' risk tag figures and the AV control tag figures. The risk tag figures are achieved by object detection, scene segmentation and probabilistic modelling, while control tag figures are derived from the AV’s parametric controls. The resulting PRN is a quantitative outcome of ReRAF in an objective end-to-end approach, without any human intervention within the AV. The DARA algorithm was tested in real-world AV operations with unregulated traffic scenarios that consist of vehicles and pedestrians. The accumulative PRN results over time can be used to identify potential hotspots and improve AV’s path planning. The DARA algorithm demonstrated the ability to use a single PRN to represent the real-time risk assessment of an AV by measuring the risk mitigation of the AV control parameters based on the detected risk from its camera.
For ReRAC, a novel Spatial-Temporal Risk Estimation Ensemble Technique (STREET) algorithm is designed, developed and verified to provide remote advanced risk warnings to the AV. STREET compute the environment’s risk tag figure and provides hazard identifications and warnings from the infrastructure viewpoint to the AV. Risk tag figures are obtained by first performing a risk zoning of the environment, followed by probabilistic modelling to convert the scene into a risk matrix. Object detection is then used to map the detected object onto the risk matrix to provide risk tag figures for the scene. ReRAC can also derive the time to collide, while hazard identifications and warnings are obtained by detecting pedestrians and/or vehicles in proximity within an intersection or road section of the scene. STREET provides an objective end-to-end approach without human intervention and was tested in unregulated traffic scenarios that provide advanced AV warnings using cooperative mode. STREET results demonstrated the ability to perform real-time conversion from qualitative image to quantitative risk tag figures from the infrastructure’s environment scene and time to collision to act as AV’s preemptive warning purposes. In addition, the STREET algorithm also illustrated the ability to detect hazard identifications and warnings with roads and intersections, such as vehicle-to-vehicle and vehicle-to-pedestrian hazards, as well as pedestrian warnings and vehicle warnings for intersections. These hazard identifications and warnings are potential risks when the AV arrives at the infrastructure location. They can also detect potential accidents and road congestions if the detection persists.
The combination of ReRAC and ReRAF provides complete coverage of safety enhancements for AV operations in real time, within and beyond the AV. With ReRAC operating as remote advanced warnings, the AV's resultant actions can be safer as it moves towards the ReRAC location. In parallel, the ReRAF continuously monitors and assesses the AV's real-time risk assessment and acts as a trigger if further improvements for safer actions are required for subsequent routes
’I think they should be involved in some capacity.’ An interview study about perceptions of children’s possibilities and abilities for democratic participation and influence
Get PDFThis dissertation uses Wall’s (2010) concept of childism and Braun and Clarke’s (2022) reflexive thematic analysis to analyse the perceptions of students previously registered on a university course about children’s rights. The focus for the study is the students’ perceptions of children’s possibilities and abilities for democratic participation an influence in the private and public sphere.
Data were gathered through semi structured in-depth interviews with 13 participants, held and recorded through Zoom. All of the participants expressed the view that children should be ‘involved in some capacity’ democratically, but their suggestions for influence and participation ranged from emphasising activism and minor decisions related to the family and the school (the private sphere) to advocacy for a low voting age (the public sphere). Some, such as Mika, suggested that education needs to improve so children can make informed democratic decisions, or that more experience and maturity are needed for children to become involved in formal major democratic decisions. Issues of protection and the adultification of children were stressed by some participants as arguments against children’s involvement. Other perceptions underscored that children should not be deemed incompetent, that the experiences of all people are of relevance in a democracy, and that children should not be excluded from various arenas based on their age. Instead, it was argued that children should be able to represent themselves just like other groups in society. Many participants saw adults and their view of children as an obstacle to children’s genuine democratic participation and influence. Here national and cultural aspects were thought to be of relevance, with some countries being described as more supportive than others. The taken for granted view of children as adults in becoming wasquestioned by some participants but supported by others.
Wall’s (2010) concept of childism shines a light on normative adult views on democratic participation and calls for a reconstruction that empowers children and their perspectives. In an era where the rights and voices of children are of relevance, but democracy is declining (especially among youth), this dissertation argues that more attention should be given to the issue of children’s democratic participation
Immersive technologies for renewable energy systems: Enhancing user engagement and understanding
Get PDFRenewable energy, particularly solar energy, is a rapidly expanding field that is poised to address pressing global energy challenges. As energy demands rise, preparing future generations to leverage solar energy becomes important. However, traditional teaching methods in solar energy system design using simulations often offer limited practical interaction, poor integration of theoretical and practical learning and low learner engagement and motivation.
Virtual reality (VR) has emerged as a transformative technology for training and learning across various sectors due to its ability to replicate real-world scenarios and enhance user experience through immersive and interactive environments. This thesis investigates the application of VR in designing solar energy systems using computer based simulations, aiming to improve conventional methods by harnessing the “feeling of presence” unique to VR, which has been shown to promote high learner engagement. Therefore, the primary contributions of this research include:
• The development of innovative VR visualisations tailored for solar energy system design, enhancing the comprehension and interaction with complex solar configurations.
• A robust framework for evaluating VR applications in educational settings, focusing on engagement, enjoyment and technology acceptance.
• Guidance for developers and designers to optimise VR applications that aim to promote sustainability, thereby enriching the user experience and educational outcomes.
Methodologically, my thesis used a mixed-methods approach to evaluate the effectiveness of these VR environments. I incorporated qualitative data from surveys alongside quantitative physiological data gathered from wearable sensors and eye-tracking technology to assess user engagement and cognitive load. My findings show a strong user preference for integrating VR into solar energy system design, with significant improvements in understanding solar energy systems attributed to VR’s interactive and visual capabilities. Participants reported higher levels of engagement and interest when using VR compared to traditional learning methods.
In conclusion, my thesis demonstrates the potential of VR as a pivotal educational tool in the renewable energy sector, offering an immersive alternative that could replace conventional teaching methods and substantially enhance learner engagement and comprehension
Experimental characterisation and forming analysis of pure unidirectional stitched glass fibre non-crimp fabric
Get PDFThis research focuses on the experimental characterisation and forming analysis of pureunidirectional non-crimp fabric (pure-UDNCF). This fabric consists of low-stiffness polyamide stitches with a tricot-chain stitching pattern where the chain stitches run along the front of the fabric, perpendicular to the glass tow direction. Notably, there are no stabilising tows oriented transverse to the main tow direction in this fabric, a common feature in many ‘quasi-UDNCF’, this allows extension of the stitch in the transverse direction under certain loading conditions. The lack of stabilising tows introduces a possible low-energy deformation mode to the pure-UDNCF, which is absent in biaxial fabrics and primarily in quasi-UDNCF. Thus, compared to biaxial fabrics, the inplane deformation modes of pure-UDNCF are complex and dominated by transverse extension and shear. Therefore, characterisation tests typically developed to characterise woven fabrics are unsuitable for pure-UDNCF, because more than one low-energy deformation may occur, and the deformations can be coupled during deformation experiments. This was evident following the initial evaluation of pure-UDNCF with two principal shear tests, namely the Uniaxial Bias Extension test (UBE test is a tensile test performed on a rectangular-shaped fabric with the warp and weft rows oriented ±45° to the direction of applied tensile force) and the Picture Frame (PF) test (all four sides are clamped orthogonal to the tows). The results revealed a significant difference in shear stiffness, with the PF test being significantly higher than the UBE test. This unique observation encourages further investigation to determine the cause of the difference. The intent is to understand whether the different behaviour of the two tests is real, or a problem related to using the tests for this fabric.
The experimental error of the PF test was investigated using two modifications: the pre-displaced PF test and the G-clamped PF test. The pre-displaced PF test was used to investigate the misalignment error and involved two pre-displacements (4mm and 6mm). The existing tight clamps (nut and bolts) were replaced with G-clamps of two different pressures (high and low) to determine how the clamping condition affected the measured shear force. The resulting combined (high and low) G-clamped PF test curve was close to the 6mm pre-displaced PF test curve, and both curves were significantly reduced than the initial PF test results. Furthermore, the friction in the bearing of the PF rig was eliminated to improve the accuracy of the PF test, however, even after these modifications, the standard UBE test results showed less axial force than the PF test curve. Moreover, in addition to shear, an in-plane bending contribution was identified at high shear angles in the PF test. As a result, the axial force measured in the PF test of the pure-UDNCFs was considered to be a combination of shear and in-plane bending. In the UBE test, the in-plane contribution was not significant, however, stretching in the stitch direction was observed and assumed to help minimise the contribution from in-plane bending of the tows. Therefore, during the UBE test, was considered to be a coupling between shear stiffness-tensile strain in the stitch direction, and between tensile stiffness in the stitch direction-shear strain. Experiments were further performed to isolate the contribution of each deformation mode i.e., shear, in-plane bending and tensile strain in the stitch direction. New characterisation tests namely cruciform bias extension (CBE) test, parallelogram shear-stretch (PSS) test and simple shear (SS) test have been designed to explore a wider deformation space i.e., different combinations of shear and tensile strain in the stitch direction. In addition to these new shear tests, the PF test was performed with pure-UDNCF samples at different stitch pre-stretched levels to provide insights into unexplored areas of the (shear angle) - (tensile stretch) parameter space. The plotted results in the (shear angle) - (tensile stretch) parameter space revealed that the pure-UDNCF was sensitive to pre-stretching in the stitch direction during sample preparation. A new method for determining the amount of pre-stretch in each test was introduced, and the results were adjusted to be more realistic and accurate.
Except for the tensile test performed in the stitch direction, all the tests generated more than one contribution from the three low-stiffness deformation modes to the axial force i.e., shear, in-plane bending and tensile strain in the stitch direction. An iterative approach was developed to isolate the three stiffnesses of the pure-UDNCF by considering the combined experimental results of the PSS, SS, tensile, PF test, and pre-stretched PF tests. This has led to a new method of determining inplane bending stiffness using experimental data. The separate contributions from shear, in-plane bending and tensile strain in the stitch direction, provided good predictions of the measured axial force of each of the experiments. In addition to the shear tests, a cantilever bending test was performed on pure-UDNCF to measure the out-of-plane bending stiffness of the fabric. Furthermore, a new experiment was developed to measure the in-plane bending of pure-UDNCF qualitatively. This test could be further developed to quantify in-plane bending stiffness and is flagged as future work. Appropriate data obtained by these experimental methods can be used as input parameters to constitutive models to predict the material behaviour.
Following the shear and bending characterisation tests, hemispherical forming experiments were performed. Two fixation methods, i.e., acetone/epoxy solution and adhesive spray, were used to maintain the desired shape and facilitate the handling of formed specimens during post-analysis. Fabrics with different tow-stitch orientations were used to form the mono and bilayer hemispherical specimens. A combination of a 3D laser scanner (Escan H) and 3D modelling software (Autodesk 3ds Max) was successfully used to determine the shear angles and stretching in the stitch direction at selected locations on the formed hemispheres. Post-analysis of the formed bilayer hemispheres revealed that both fixation procedures provided long-term stability; however, the acetone/epoxy method caused fewer defects than the adhesive spray method due to facilitating inter-ply sliding and reducing the internal stresses between the two plies. The adhesive spray method worked well in modifying the fabric forming behaviour, offering improved control over fibre orientation, and helping to reduce gaps or inconsistencies in the fabric. Therefore, the fast and simple adhesive spray method can optimise the forming behaviour and be used locally to modify the behaviour in specific locations. These experimental forming results provide a solid foundation for validating numerical models that predict the forming behaviour of pure-UDNCF
The needs of mothers with an addiction who have had children removed from their care
Get PDFThis thesis brings together three key papers that each contribute to the understanding of the needs of mothers with an addiction who have children removed from their care.
The first paper focuses on child related risk information and recording of such in parents’ electronic records. From 736 services users, 62.8% were parents with 38.3% of those having children aged 16 years and under. 78.4% of female service users were mother yet 54.7% of male service users were fathers. Of the 913 offspring, 32.1% were either in or had been in local authority care, the majority were in kinship care - 17.6% of offspring sample and 54.9% of the offspring in local authority care. Seven (0.8%) offspring were deceased, a two-fold increase in mortality rate compared to the general population. In the records of 53 parents (11.5%; 68 children) there was a discrepancy between the electronic records and staff knowledge.
The second paper focuses on parents and gender differences in child removal. Mothers in the Alcohol and Drug Recovery Service were more likely to have children removed than fathers (56.6 vs. 17.7%, p<0. 001), had more children removed than fathers (2 vs. 1, p<0.001) and were more likely to have a series of individual child removals (22.5 vs. 4.3%, p=0.014). Female gender, younger age, drug use, mental health issues and a history of suicide attempts were associated with child removal from parents in the service. Mothers who had children in their care were less likely to have made an attempt on their lives than mothers who had children removed or women who were not mothers.
Paper three is a qualitative analysis of 12 mother’s lived experiences of child removal and contact with services – before, during and after child removal using Interpretative Phenomenological Analysis (IPA). Four themes were identified – ‘safe/unsafe’, ‘changing identity’, ‘loss’, and ‘no way to win’. Services that can develop a sense of safety in their client through continuity in workers, clarity and consistency about boundaries and communication with other services and supporting mothers to feel respected and validated as a person and as a mother, regardless of whether their child/ren are removed, are more likely to engage their clients and achieve better outcomes.
The findings of these three papers resulted in three key messages: (1) the prevalence of parenthood and child removal is high in Alcohol and Drug Recovery Services, particularly of female parents, (2) mothers with substance use issues who have children removed have unmet support needs before, during and after removal, but it is impossible to understand these needs without understanding them in the context of their children’s needs and the needs of the family unit, and (3) services need to consider how to make services feel safer for mothers and to improve connections and relationships between mothers and clinicians which could lead to better engagement and outcomes for mothers and their children.
The findings of these studies add to the knowledge and understanding of the needs of this group of mothers. Recommendations are made for services working with these mothers included:
• All service users in Alcohol and Drug Recovery Services need to be asked whether they have children, where those children live, and what level of contact they have with their own and any other children. This needs to be repeated regularly as circumstances can change.
• Services need to be aware of the role of gender in parenting and child removal, especially in younger mothers with mental health issues.
• Clinicians need to be aware of the link between child removal and suicide and provide increased support immediately after removal and for a period of time afterwards.
• Clinicians need to understand the concept of disenfranchised grief, how it applies to child removal and offer support for this loss and the subsequent grief response.
• Mothers suggested changes that services need to make them feel safe, such as consistency in staffing, clarity about roles, boundaries and information sharing, especially with child welfare services, and for staff to be persistent and provide outreach.
• Services need to consider and develop ‘soft’ skills such as compassion and authenticity in their staff teams.
Recommendations for researchers included:
• Further research into the high mortality rate in offspring, including the causes of these deaths and whether this is associated with parental substance use.
• Strengthen our knowledge about these constellations of parental risk factors to allow for the identification of mothers at risk of the removal of children and to aid service development and provision of support.
• There is a need for a better understanding of evidence-based treatment for women and mothers with substance use issues, as gender factors may have been overlooked in previous research.
• There is a need for co-production research with this group of mothers who are keen to have their voices heard
Exploring participant utilisation of a novel digital support service for people with COPD using a mixed methods study
Get PDFBackground
Chronic obstructive pulmonary disease (COPD) is a significant health concern thought to affect over 4 million people in the UK. Although not curable, effective management and treatment can minimise symptom and healthcare burden. Effective self-management - empowering patients to manage their own condition, recognise changes, and seek early intervention when needed – is a key component of value-based COPD care. There is however a mismatch between guideline-based recommendation and care resources, often exacerbated by socioeconomic disparities and quality care gaps. Digital solutions offer the opportunity to overcome service inefficiencies, widen access and increase uptake of strategies known to improve healthcare outcomes in COPD. A range of digital health solutions to support COPD management have been developed and evaluated over the last 10 years, but firm evidence of their sustained usage and utility has not yet been acquired.
COPD is a major healthcare challenge in Scotland, with notable variation in health outcomes and access to guideline-based care across the country. To address this, the Innovate UK-funded DYNAMIC project was created in 2018. The project aimed to co-develop and implement a digital intervention for people with COPD, with effectiveness testing of a digital infrastructure and associated service model to support effective delivery of supported COPD management and evidence-based care. The innovation team, in collaboration with a digital agency (StormID), co-designed and developed the COPD digital support service intervention, with the overarching goal to improve patient outcomes for those with COPD, whilst also integrating remote management and data capture.
The COPD digital support service consists of a patient-facing web application (app), where users can submit daily patient reported outcomes (PRO), access self-management info and medication information, link in Fitbit wearables and home non-invasive ventilation devices, and use patientclinician asynchronous messaging for non-urgent advice or queries. A linked support site gives access to further self-management resources. An associated clinician dashboard displays PRO responses and physiological data, and allows management of messaging and collated health record data.
Approach
The RECEIVER trial is the implementation and effectiveness observational cohort study that evaluated the use of the COPD digital support service alongside routine care. Its planned 1-year recruitment phase commenced in September 2019, with follow-up data censored in August 2021. Patients with COPD who had access to smartphone, tablet or computer and had had a severe COPD exacerbation requiring admission to hospital, and/or had chronic hypercapnic respiratory failure on home non-invasive ventilation were eligible for the study. The primary outcome of the trial was participant usage of the support service, measured through the submission of daily PROs. Secondary outcomes included clinical events, PRO and quality of life measures.
This research for this thesis sat within the RECEIVER trial and aimed to explore utilisation of the digital support service by people with COPD using an explanatory mixed methods study design. Study participant usage data and clinical outcomes were collated and analysed. Participant admission and occupied bed days (OBD) were compared to a matched contemporary control cohort created from a deidentified dataset of patients with COPD in NHS GGC.
The results from the quantitative evaluation that warranted further exploration were identified. Qualitative semi-structured interviews were designed and conducted in a sub-cohort of study participants who were best placed to give added insight into the findings seen. Interview transcripts were coded, and thematic analysis was undertaken to develop themes reflecting the use of the service from participant’s perspectives. This sequential combining of quantitative outcome data with qualitative semi-structured interview themes provided additional perspectives and deepened the understanding of the results obtained.
Findings
81 people with high-risk COPD were recruited to the RECEIVER study. Quantitative results showed consistent utilisation of the service, with participants submitting an average of 4 sets of PROs per week over the course of the study. There were notable reductions in hospital admissions and OBDs following enrolment, along with improvements in time to readmission and survival rates among RECEIVER participants compared to the matched contemporary controls.
To better understand the reasons behind the sustained usage of the service by some participants, semi-structured interviews were conducted with a sub-cohort of those who continued to engage with the platform. 14 interviews were performed and data from these analysed. Four themes were developed; conditions for success, added colour and detail, background care, and a means to help. These themes revealed key factors that appeared to contribute to successful use of the app and highlighted the perceived benefits experienced by participants from use of the COPD digital support service components.
The combining of qualitative and quantitative results allowed conclusions to be drawn about the practical drivers behind the persisting usage seen within the trial and identified aspects and priorities to incorporate into future service iterations and other project developments. This integration also broadened the insight into motivations behind usage and mechanisms that may have led to the improved outcomes seen. Aspects of the service that warranted further evaluation were revealed, including potential value gained from analysis of messaging patterns and physiological data.
Evaluation data from the scale-up of the COPD digital service provision during the COVID-19 pandemic (out with the RECEIVER trial) is also explored. Eligibility was extended to include any patient with COPD resident in NHS GGC. Results from the evaluation confirmed sustained utilisation and reductions in clinical events for patient users, mirroring the results from the RECEIVER trial. User feedback from this scale-up cohort group also captured similar sentiments to those recorded amongst RECEIVER trial participants.
Conclusion
This mixed-methods RECEIVER trial evaluation and complementary data from the subsequent service scale-up confirms sustained patient utilisation, perceived participant benefits and positive impact on healthcare utilisation from development and deployment of a co-designed COPD digital service. The outcomes from this research project have contributed to the adoption and evaluation of the service in other organisations, the publication of a supportive NICE early value assessment and to the extension of the digital tools including development, deployment and evaluation of artificial intelligence-based risk prediction models and transformation of the COPD diagnostic pathway. This research project and its evaluations also provide exemplar insights for implementation-effectiveness evaluations of additional digital technologies for COPD and other long-term conditions
Interactions of the Arabidopsis UVR8 photoreceptor with transcription factors
No full textAbstract not currently available
Genomic insights into Trypanosoma brucei and Leishmania major: compartmentalised DNA replication, modified base detection by Nanopore sequencing and nucleotide composition analysis
Get PDFThe genomes of the early-diverging eukaryotic parasites Trypanosoma brucei and Leishmania major are unusual in both their organisation and dynamics. The majority of transcribed genes in both vector-transmitted parasites are organised in polycistronic transcription units (PTUs), and the boundaries between these units act as transcription start and termination sites. The genome of T. brucei is further shaped by the parasite’s immune evasion strategy – antigenic variation. One variant surface glycoprotein (VSG) is expressed on the parasite’s surface at any given time, despite the trypanosome harbouring >2500 of VSG (pseudo)genes in its genome. This monoallelic gene expression is achieved whereby only one VSG is expressed at a time from a dedicated telomere-proximal site – the bloodstream-form expression site (BES), of which the parasite has ~15. The remainder of VSG genes are located in transcriptionally silent arrays in the subtelomeric compartments of the larger, so-called megabase chromosomes of the parasites, as well as the smaller mini- and intermediate chromosomes.
Analysis of DNA replication patterns using marker frequency analysis coupled with sequencing (MFAseq) in T. brucei showed that in the core of the larger chromosomes some PTU boundaries, as well as the annotated centromeric regions, co-localise with early S phase DNA replication initiation; this pattern was consistent between two T. brucei strains, TREU 927 and Lister 427, as well as between mammalian stage bloodstream-form (BSF) cells and insect-stage procyclic cells (PCF). Curiously, the active BES was also an early-replicating region, but only in the BSF cells; the origin – telomeric or upstream – of this replication could not be determined. In addition, complete, genome-wide analysis of DNA replication dynamics was not possible at the time due to incomplete genome assembly. Until recently, the genome of this parasite remained poorly assembled outside of the megabase chromosome cores, despite its small < 50 Mb size. In 2018, a number of major improvements in the genome assembly of T. brucei were achieved using PacBio long-read assembly, assisted by Hi-C DNA interaction data, but the chromosome ‘core’ sequences remained separate from VSG-containing subtelomeric sequences and BES. In addition, none of the centromeric regions, which co-localise with the earliest S phase DNA replication sites, have been fully resolved. Moreover, MFA-seq mapping to mini- and intermediate chromosomes was compromised by much of their content comprising 177 bp repeats.
In chapter 3 we discuss de novo long-read assembly of the genome of T. brucei brucei Lister 427 using Nanopore sequencing to improve contiguity along chromosome compartments (core, subtelomeres, BES), as well as improve repetitive region and sub-megabase chromosome assembly. This was motivated by a wish to provide a complete understanding of DNA replication, by expanding MFAseq mapping to the subtelomere compartments of the megabase chromosomes, across the core and subtelomere boundaries, across the entirety of the telomeric BESs, and within the submegabase chromosomes. Long-read assembly resulted in at least 1 bridging of previously separate genome sequences in 10/11 megabase chromosomes, overall improved contiguity, as well as assembly of full-length centromeric, 50bp, 177bp and 70bp repeats. Additionally, smaller, sub-megabase chromosomes of this parasite with the characteristic 177bp repeat region were at least partially assembled; the assembly of these smaller chromosomes showed that they contain more genes than previously thought. The improved genome assembly allowed mapping of MFAseq across the various genome compartments, and showed that megabase chromosome subtelomeres, surprisingly, contain no detectable early-replicating regions outside of centromeric repeats. We also show that 177bp repeats act as sites of DNA replication initiation in the submegabase chromosomes and that they are found in centromeric regions of the megabase chromosomes, revealing these repeats to be widespread, sequence conserved origins of replication. In addition, we demonstrate that early replication if the active BES in BSF cells initiates from the telomere. Finally, we show that, in addition to genome compartmentalisation being evident in gene content, organisation, transcription and DNA replication in T. brucei, genome stability is also compartmentalised, with the subtelomeric regions of megabase chromosomes showing pronounced genomic instability compared to the cores and, indeed, sub-megabase chromosomes.
Kinetoplastid parasite genomes also harbour an unusual DNA modification – a thymidine modification termed base J. It is thought to be generated in a twostep process – first, a thymidine is modified to 5-hydroxymethyluracil (5hmU), followed by glycosylation. Previous work has indicated that this modification is present in the T. brucei genome at very low levels, and that it is primarily detected at repetitive DNA, as well as PTU boundaries. Only mammalian stage parasites appear to harbour easily detectable levels of this base, as in the insect vector stage only very low or no base J has been previously detected. While both base J and 5hmU have previously been mapped genome-wide in T. brucei, the existing datasets have not been re-examined in light of improved genome assemblies, nor have they been evaluated together. In addition to offering longread DNA sequencing, the two main long-read sequencing technologies at the time of writing – Nanopore and PacBio – also possess the ability to detect modified bases in DNA and RNA. In chapter 4, we provide more comprehensive analysis of base J and 5hmU ChIPseq datasets in T. brucei, along with newly generated modified base detection data using Nanopore sequencing and Tombo software. We find that Nanopore-generated data recapitulates many base J ChIPseq enrichment patterns, specifically, at polycistronic unit boundaries, in repetitive regions and around coding sequence, while also offering strandspecific and base-resolution data. To our surprise, insect and mammalian stage parasites show similar levels and patterns of DNA modification based on Nanopore data, arguing that the two lifecycle stages may, in fact, have similar DNA modification distribution patterns.
In our analysis of modified DNA distribution in Chapter 4, it became clear that broader canonical base distribution genome-wide has not been fully described in T. brucei. DNA strand asymmetries in prokaryotes and eukaryotes often arise as a consequence of directional processes, such as transcription and DNA replication, as these processes are asymmetrical with regards to the two DNA strands. This often leads to overabundances of certain nucleotides on one DNA strand relative to the other, termed nucleotide skews. In many eukaryotic and prokaryotic genomes, analysis of nucleotide skews can even be used to detect origins in DNA replication. In chapter 5 we present efforts in describing the nature, and elucidating the potential contributors of nucleotide skews in T. brucei, and comparing these skews to those observed in L. major, as well as a broad range of trypanosomatid genomes. We found that T. brucei and L. major display clear and distinct skews associated with transcription direction, but similar skews associated with DNA replication, meaning differential processes are responsible for the distinct nature of transcription-associated skews. Additionally, through the inclusion of analysis in other trypanosomatids, we show that nucleotide composition and skew differences observed in T. brucei and L. major can be explained through their evolutionary divergence
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