Characterisation of factors that regulate homologous recombination and antigenic variation in Trypanosoma brucei

Trypanosoma brucei is an evolutionarily divergent eukaryotic parasite of mammals in sub-Saharan Africa and is transmitted by the tsetse fly vector. To evade the mammalian immune response, T. brucei utilises antigenic variation, which involves switches in the Variant Surface Glycoprotein (VSG) expressed on the cell surface. Such reactions can occur at very high rates (~10-3 switches/cell/generation) and occur primarily by the recombination of VSG genes, selected from an enormous silent archive, into specialised expression sites. It has been previously shown that such VSG switching is a form of homologous recombination, as mutation of RAD51 and a related gene, RAD51-3, impairs the process. BRCA2 has emerged as a significant regulatory factor during RAD51-catalysed recombination. In humans, BRCA2 contains eight BRC repeats, six of which have been shown to bind RAD51. Similar repeats are present in BRCA2 from other organisms, though normally in smaller numbers. This thesis describes a T. brucei BRCA2 homologue that appears exceptional in that it contains up to 12 BRC repeats. Furthermore, the sequence degeneracy that is observed between the BRC repeats in most organisms is absent in T. brucei, with all but the C-terminal proximal repeat being identical. It was hypothesised that this unusual BRCA2 organisation is due to the high levels of RAD51-directed recombination needed during antigenic variation. To examine the function of the putative T. brucei BRCA2 homologue, mutants were generated and found to display impaired growth, sensitivity to induced DNA damage, impairment in the ability to form sub-nuclear RAD51 foci, a reduced ability to recombine DNA constructs into their genome and a reduction in frequency of VSG switching, all of which are consistent with roles for BRCA2 in DNA repair and recombination. Furthermore, genome instability in the mutants was observed through the loss of silent VSG gene copies and substantial reductions in the size of the mega-base chromosomes. Interestingly, other chromosome classes (the so-called mini- and intermediate-chromosomes) appear not to be susceptible to such instability. A potentially novel function for BRCA2 was identified through DNA content analysis of the T. brucei BRCA2 mutants. Mutation of BRCA2 was shown to result in an accumulation of cells with aberrant DNA content that is most readily explained by an increased number of cells that undergo cytokinesis without having completed nuclear division, phenotypes that are not observed in other T. brucei recombination mutants, such as RAD51. This result suggests that BRCA2 has a role in the regulation of cell division, with mutation causing impaired replication of T. brucei nuclear DNA, but without a cell cycle stall, leading to the accumulation of chromosomal aberrations. In order to investigate the potential role of T. brucei BRCA2 in DNA replication and the unusual BRC repeat organisation phenotypes further, various truncations of BRCA2 were expressed in a mutant background. Cell lines expressing BRCA2 with only 1 BRC repeat displayed reduced efficiency in recombination, DNA repair and RAD51 foci formation, indicating that the large BRC repeat expansion in T. brucei BRCA2 plays a critical role in the proteins function. Expression of a BRCA2 variant encompassing only the region of the protein, C-terminal to the BRC repeats appeared able to function, at least partially, in regulating cell cycle progression. Moreover, this DNA replication role appears not to be provided by conserved DNA binding motifs present within the C terminus of BRCA2 since a fusion of T. brucei BRCA2 and the parasites homologue of the replication protein A 70 kDa subunit was impaired in cell division, but was proficient in repair of DNA damage. Taken together, these data infer that T. brucei BRCA2 possesses a function that is distinct from BRCA2’s role as a regulator of RAD51, and acts in DNA replication or cell division. In addition to the above research on BRCA2, I sought to examine the factors that interact with RAD51 in T. brucei. This work demonstrated that it is possible to add an epitope tag for tandem affinity purification (TAP) to the N-terminus of RAD51 in both the bloodstream and procyclic stages of T. brucei without disrupting its function. Preliminary data suggest that TAP is potentially a feasible way of examining RAD51 interacting factors

A Comparative Analysis of Protein and Peroxidase Blood Enhancement Reagents Following Laundering and their Impact on DNA Recovery

Blood is a commonly encountered biological fluid in criminal investigations concerning a violent incident, and visual traces of the fluid on a suspect’s clothing can be diminished through laundering. This study aims to analyze the effects of laundering and the application of commercially available blood enhancement reagents commonly used to improve visualization of dilute bloodstains and their impact on DNA recovery. Enhancement reagents Hungarian Red, Coomassie Blue, Amido Black, luminol, Bluestar® Forensic Magnum, and aqueous Leuco Crystal Violet (LCV) were used to enhance human blood on cotton, polyester, denim, and wool following laundering. DNA was extracted from these samples using a QIAamp® DNA Investigator Mini Kit and quantified using a NanoDrop™ OneC UV-Vis spectrophotometer. This study revealed the peroxidase based reagents to produce the greatest sensitivity on the natural fabrics, reacting positively down to a blood dilution of 1:1000. The protein reagents produced greater sensitivity on the synthetic fabrics, reacting positively down to a blood dilution of 1:10. Peroxidase stains relying on chemiluminescent properties rather than colorimetric results produced positive results on the dark colored fabrics as sufficient color contrast was not achieved with the protein stains. The resulting yields of extracted DNA suggest that quantifiable amounts of DNA originating from bloodstains persist despite laundering and enhancement. Additionally, measurements indicated that the application of some blood enhancement reagents, particularly Amido Black, may affect DNA recovery

Using group improvisation and imaginative listening to nurture creative autonomy in A-level music students: teaching composition for examination purposes in England

This thesis examines the effects of group improvisation and imaginative listening on teaching and learning in the composing component of A-level Music in one school in England. Problems with teaching composition in schools have long been acknowledged, with the same issues raised across studies spanning several years: how to teach it, and how to interpret assessment criteria. In that context, this action research project developed in response to a perceived association between students’ behaviours in composition and listening, with cautious, teacher-reliant composers also struggling to listen constructively to music that presented new aesthetic challenges. As studies with A-level Music students are rare and there is no research connecting listening, improvising, and composing in this context, a wide range of literature, educational and otherwise, was consulted. This scholarship underpins two central studies in this project, undertaken in the academic year 2020-21. The first study aimed to enable students to participate in group improvisation, addressing preconceptions about genre, skills, and expectations. Thematic analysis of data revealed a correlation between reduced self-consciousness and improved expression of observations and interaction when improvising, the latter supporting a view that positive self-constructs and creativity are linked. The second study used group improvisation alongside imaginative listening tasks as part of a developing composition curriculum. Characterful playing in improvisation, curiosity in listening, and a healthy dimension of perfectionism in composition were found to be connected by the attribute of risk-taking. Significant links emerged between behaviours in improvising, composing, and listening, indicating possible ways of indirectly nurturing creative autonomy and positive creative self-concept. This research informs a potential A-level composition curriculum that interweaves group improvising, listening, and composing. Rather than focussing on skills development, product assessment, or devising a model of the compositional process, this thesis recommends ways to nurture “unteachable” inner attributes such as intuition, self-trust, and aesthetic awareness, essential qualities in critical and autonomous composers

The Ability of the Landing Error Scoring System to Detect Changes in Landing Mechanics: A Critically Appraised Topic

Clinical Question: Is there evidence to suggest that the Landing Error Scoring System (LESS) is able to detect functional changes in landing mechanics in healthy individuals after participation in an injury prevention program (IPP)? Clinical Bottom Line: In a healthy, physically active population, there is strong evidence to support the use of the LESS as an outcome measure for changes in landing mechanics after the implementation of IPPs. Clinicians should consider the LESS as an evaluative tool for measuring the efficacy of IPPs in clinical practice

Improved sensitivity for detection of pathogenic variants in familial <i>NF2-</i>related schwannomatosis

Purpose To determine the impact of additional genetic screening techniques on the rate of detection of pathogenic variants leading to familial NF2-related schwannomatosis.Methods We conducted genetic screening of a cohort of 168 second-generation individuals meeting the clinical criteria for NF2-related schwannomatosis. In addition to the current clinical screening techniques, targeted next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification analysis, we applied additional genetic screening techniques, including karyotype and RNA analysis. For characterisation of a complex structural variant, we also performed long-read sequencing analysis.Results Additional genetic analysis resulted in increased sensitivity of detection of pathogenic variants from 87% to 95% in our second-generation NF2-related schwannomatosis cohort. A number of pathogenic variants identified through extended analysis had been previously observed after NGS analysis but had been overlooked or classified as variants of uncertain significance.Conclusion Our study indicates there is added value in performing additional genetic analysis for detection of pathogenic variants that are difficult to identify with current clinical genetic screening methods. In particular, RNA analysis is valuable for accurate classification of non-canonical splicing variants. Karyotype analysis and whole genome sequencing analysis are of particular value for identification of large and/or complex structural variants, with additional advantages in the use of long-read sequencing techniques

Trypanosoma brucei BRCA2 acts in a life cycle-specific genome stability process and dictates BRC repeat number-dependent RAD51 subnuclear dynamics

Trypanosoma brucei survives in mammals through antigenic variation, which is driven by RAD51-directed homologous recombination of Variant Surface Glycoproteins (VSG) genes, most of which reside in a subtelomeric repository of &gt;1000 silent genes. A key regulator of RAD51 is BRCA2, which in T. brucei contains a dramatic expansion of a motif that mediates interaction with RAD51, termed the BRC repeats. BRCA2 mutants were made in both tsetse fly-derived and mammal-derived T. brucei, and we show that BRCA2 loss has less impact on the health of the former. In addition, we find that genome instability, a hallmark of BRCA2 loss in other organisms, is only seen in mammal-derived T. brucei. By generating cells expressing BRCA2 variants with altered BRC repeat numbers, we show that the BRC repeat expansion is crucial for RAD51 subnuclear dynamics after DNA damage. Finally, we document surprisingly limited co-localization of BRCA2 and RAD51 in the T. brucei nucleus, and we show that BRCA2 mutants display aberrant cell division, revealing a function distinct from BRC-mediated RAD51 interaction. We propose that BRCA2 acts to maintain the huge VSG repository of T. brucei, and this function has necessitated the evolution of extensive RAD51 interaction via the BRC repeats, allowing re-localization of the recombinase to general genome damage when needed

COVID-19 and the role of Voluntary, Community, and Social Enterprises in northern England in responding to the needs of marginalised communities: a qualitative focus group study

Background: The Voluntary Community and Social Enterprise sector has a crucial role in supporting the health and wellbeing of people who are marginalised or who have multiple complex needs. We aimed to understand perceptions of those working in the sector and examine the short-term, medium-term, and long-term effects of COVID-19 on Voluntary Community and Social Enterprise organisations in northern England as they respond to the needs of marginalised communities. This research formed one component of a regional multiagency Health Inequalities Impact Assessment. Methods: We conducted qualitative focus groups with staff and volunteers from five organisations between March and July, 2021, via a video conferencing platform. Eight of nine focus groups were audio-recorded and transcribed verbatim. One focus group was not recorded due to concerns raised over anonymity and safeguarding, but non-ascribed fieldnotes were taken. Focus group transcripts were analysed using framework analysis. Findings: One organisation supported children and young people; two organisations supported vulnerable women, young people, and families; one organisation supported refugees and asylum seekers, and one organisation supported disadvantaged individuals to improve their mental and physical health and wellbeing. Three central themes were identified: the exacerbation of pre-existing inequalities, adversity, and challenges for vulnerable and marginalised populations; the cost of being flexible, innovative, and agile for Voluntary Community and Social Enterprise staff and volunteers; and the voluntary sector as a lifeline (organisational pride and resilience). Interpretation: The considerable expertise, capacity, and resilience of Voluntary Community and Social Enterprise organisations and the crucial role they have in supporting marginalised communities has been clearly shown in their response to the COVID-19 pandemic. The Voluntary Community and Social Enterprise sector therefore has an essential role in the post-COVID levelling-up agenda. The implications of these findings for service provision are that the Voluntary Community and Social Enterprise sector must be recognised as an integral partner within any effectively functioning local health system and, as such, adequately resourced to safeguard sustainability and to ensure that attempts to involve the sector in addressing the social determinants of health are not jeopardised. Funding: National Institute for Health and Care Research (Applied Research Collaboration North East and North Cumbria (grant reference NIHR200173) and Public Health England. SSo is supported by a Health Education England and National Institute for Health and Care Research Integrated Clinical Academic Lecturer award (reference CA-CL-2018-04-ST2-010) and Research Capability Funding, National Health Service North of England Care System Support. VJM is funded by the National Institute for Health and Care Research School for Public Health Research (grant reference PD-SPH-2015)

“I’ll meet you at our bench”: adaptation, innovation and resilience among VCSE organisations who supported marginalised and minoritised communities during the Covid-19 pandemic in Northern England – a qualitative focus group study

Background: The Covid-19 pandemic has exacerbated pre-existing inequalities and increased adversity and challenges for vulnerable and marginalised communities worldwide. In the UK, the Voluntary Community and Social Enterprise (VCSE) sector play a vital role in supporting the health and wellbeing of people who are marginalised or experiencing multiple complex needs. However, only a small number of studies have focused on the impact that Covid-19 had on the VCSE sector. Methods: As part of a Health Inequalities Impact Assessment (HIIA), we conducted qualitative focus groups with staff and volunteers from five organisations to examine short, medium and longer-term impacts of Covid-19 upon the VCSE sector in Northern England. Nine online focus groups were conducted between March and July 2021. Findings: Focus group transcripts were analysed using Framework Analysis and yielded three central themes: (1) exacerbation of pre-existing inequalities, adversity and challenges for vulnerable and marginalised populations; (2) the ‘price’ of being flexible, innovative and agile for VCSE staff and volunteers; and (3) the voluntary sector as a ‘lifeline’ - organisational pride and resilience. Conclusions: While the voluntary sector ‘adapted at pace’ to provide support during Covid-19 and in its continued aftermath, this resilience has potentially come at the cost of workforce and volunteer wellbeing, compounded by political obstacles and chronic shortage in funding and support. The VCSE sector has a vital role to play in the post-lockdown ‘levelling up’ agenda. The expertise, capacity and resilience of VCSE organisations, and their ability to respond to Covid-19, should be celebrated, recognised and supported adequately to maintain its resilience. To not do so threatens the sector’s sustainability and risks jeopardising attempts to involve the sector in addressing the social determinants of health

Essential features of responsible governance of agricultural biotechnology

Agricultural biotechnology continues to generate considerable controversy. We argue that to address this controversy, serious changes to governance are needed. The new wave of genomic tools and products (e.g., CRISPR, gene drives, RNAi, synthetic biology, and genetically modified [GM] insects and fish), provide a particularly useful opportunity to reflect on and revise agricultural biotechnology governance. In response, we present five essential features to advance more socially responsible forms of governance. In presenting these, we hope to stimulate further debate and action towards improved forms of governance, particularly as these new genomic tools and products continue to emerge