Parental care and cooperation in the burying beetle Nicrophorus vespilloides

In species that provide parental care, individuals must choose how to split their resources between caring for their current offspring and investing in their own reproductive potential. These decisions are made based on factors that shift the balance of costs and benefits associated with allocating resources to current or future reproduction. For parents providing uniparental care such factors relate to the value of the current brood and the likelihood of future reproduction. Females and males that cooperate to provide biparental care, must also consider factors that may influence the contribution of their partner. In this thesis, I explore what affects the level of care parents provide for their offspring and how females and males that provide biparental care balance their relative contribution in the burying beetle Nicrophorus vespilloides. I focus on four factors: previous reproductive allocation, nutritional state, social environment, and synchrony in the onset of care. First, I found that females provided the same level of care to a subsequent brood regardless of previous reproductive allocation and resource access, which suggests that neither affected future ability to provide care. Next, I found that females adjusted their level of care in response to both their own nutritional state and that of their partner and that these decisions were independent of their partner’s contribution, while males only responded to the contribution of their partner. Then, I found that parents provided a similar level of care regardless of the presence of female or male intruders. Finally, I found that males provided more care when the female and male started providing care asynchronously in comparison to when they started synchronously while females provided a similar level of care regardless

The Story of Ethiopian Governance: From a centralized public service monopoly to ‘big man’ politics

This report discusses how the EPRDF, from operating on the logic of a centralized political market, degenerated into an oligopolistic monopoly and later into a neo-patrimonial state that operates on patronage and sustaining disorder while maintaining a simulacrum of an institutionalized state. The report is divided into four parts. The first part discusses the genesis of the EPRDF and its highly centralized public service monopoly in government. The second part discusses the beginning of the end of the EPRDF’s centralized public service monopoly. The third part discusses the complete transformation of the state into what is called a typical African ‘big man’ politics. The fourth and final part is the conclusion

Development of high-throughput metabarcoding approaches to explore genetic diversity and anthelmintic resistance in sheep gastrointestinal nematodes

Sheep farming in the UK, integral to its cultural and economic fabric, contends with significant challenges from gastrointestinal nematodes (GINs) which impair livestock productivity and increase mortality, leading to substantial economic losses. The emergence of anthelmintic resistance, compounded by the impacts of climate change, has necessitated integrated parasite management (IPM) strategies and the adoption of advanced DNA technologies such as next-generation sequencing (NGS) for diagnosis and targeted treatment. In this project, rDNA ITS-2 and mt-ND4 genetic markers were employed to analyse GINs across various locations, farms, and treatment groups. The ITS-2 analysis, also known as the Nemabiome, provides robust identification and relative abundance of GIN species when combined with faecal egg count, while mt-ND4 markers offer insights into the origins, gene flow, and infection patterns of individual species. By leveraging these technologies, this thesis aims to enhance the management and understanding of GINs, contributing to the sustainability and productivity of UK sheep farming. A significant portion of the work focused on refining these techniques. Despite the effectiveness of Nemabiome sequencing in detecting all clade V nematodes, identifying lesser-known GIN species remains challenging due to reference sequence gaps, particularly in environments with cross-infections from GINs infecting diverse grazing animals. To address this, the reference library was expanded using various GIN sample sets, listing anticipated GIN species, extracting their ITS-2 rDNA sequences from NCBI GenBank, and enhancing the library through iterative BLAST searches and phylogenetic analyses. Concurrently, mt-ND-4 markers were developed for T. circumcincta and H. contortus, the two most prevalent GIN species in the UK. These mitochondrial markers, typically species-specific, were adapted into a primer multiplex to process both species simultaneously, significantly increasing efficiency and reducing costs. Reference libraries were developed for both species, along with the post-sequencing analysis pipelines, and the multiplex was tested on various laboratory strains, verifying its accuracy and reliability. To implement both these techniques in the field, a DNA biobank was established, collecting over 250 field samples from across Scotland and England to enable comprehensive comparisons across various locations, treatments, and farms. This biobank was further enhanced by the inclusion of nine characterised laboratory strains of H. contortus and seven of T. circumcincta to assist in the comparison of resistance mechanisms and genetic diversity. Nemabiome analysis identified seven GIN species within the UK, with T. circumcincta being the most prevalent, followed by H. contortus and Trichostrongylus vitrinus. Together, these three species accounted for approximately 88% of infections. Significant spatial differences in GIN distribution were observed between areas, with T. circumcincta being prevalent in Scotland and H. contortus in England, likely influenced by climatic conditions. Despite its generally lower presence, H. contortus was consistently found on some Scottish farms, suggesting an influence of local environmental or management factors. It also showed a higher prevalence and diversity in alpacas compared to sheep or other hosts, raising questions about the role of alpacas as a maintenance host. The effectiveness of levamisole, ivermectin, and their combined anthelmintic treatments was assessed through faecal egg count reduction tests (FECRT) across 18 farms in England. Treatments were more effective against some species than others: Resistance was indicated in T. circumcincta, while Trichostrongylus axei and Trichostrongylus colubriformis displayed mixed results. Notably, the combination treatment proved less effective than individual drugs on some farms, which could suggest antagonistic interactions between these drugs. In the mt-ND4 analysis, 60 H. contortus and 35 T. circumcincta amplicon sequence variants (ASVs) were identified, with a single ASV representing over one-third and over 62% of the total reads, respectively. Phylogenetic analysis suggested a common origin for H. contortus, but potentially two distinct origins for T. circumcincta. H. contortus showed a general trend towards balancing selection or population contraction across both areas, while T. circumcincta showed population expansion or purifying selection in Scotland and balancing selection or contraction in England. There was significant gene flow with minimal genetic differentiation between Scottish and English populations across both species. Post-treatment analysis revealed a reduction in genetic diversity for H. contortus but the opposite for T. circumcincta. Ivermectin-treated groups displayed higher H. contortus diversity compared to those treated with levamisole, with the combination yielding moderate diversity levels, supporting the idea of possible antagonistic interactions. No difference was observed between treatment groups for T. circumcincta. Overall, the study underscores regional and inter-species variations, alongside the challenges of emerging anthelmintic resistance, especially in T. circumcincta. It highlights the critical need for continuous monitoring and targeted management strategies to effectively control GIN prevalence and manage resistance within UK sheep farming. Additionally, the findings lay a foundation for utilising more diverse and specific genetic markers to further investigate genetic diversity and resistance mechanisms

Cooperation between two repressive epigenetic systems, DNA methylation and PRC2, in pluripotency and differentiation

The epigenome is established during embryogenesis by the acquisition of cell type-specific DNA methylation and histone modification patterns. Curiously, disruption of DNA methyltransferases or Polycomb Repressive Complex 2 (PRC2) components, which establish repressive DNA methylation and H3K27me3 modifications, respectively, does not affect the proliferation and self-renewal of pluripotent mouse embryonic stem cells (ESCs), despite resulting in differentiation defects and lethality during embryonic development. These modifications are deposited in a mutually exclusive manner, with H3K27me3 diffusing in a hypomethylated background and a limited increase in DNA methylation observed in PRC2 mutants. Therefore, a partial compensation mechanism between these two antagonistic modifications might exist to maintain the pluripotent state but becomes lost upon differentiation. In this thesis, I aimed to test this hypothesis by simultaneously disrupting DNA methylation and PRC2 in distinct pluripotent states and embryoid bodies. I inactivated Ezh2, a catalytic component of PRC2, in a cell line where DNA methylation levels can be controlled by doxycycline-inducible suppression of the endogenous maintenance DNA methyltransferase Dnmt1. I systematically compared the outcomes in ESCs cultured under the ground state naïve (2i/LIF) and metastable naïve (Serum/LIF) conditions, focussing on cellular morphology, stem cell identity and transcriptional changes. I identified that simultaneous disruption of DNA methylation and H3K27me3 had a minor effect on transcriptional regulation and stem cell identity in 2i/LIF conditions but led to a major destabilisation of transcriptional networks in Serum/LIF. This led to spontaneous differentiation, as evidenced by morphological changes, loss of pluripotency transcription factor expression and activation of lineage specification genes. Interestingly, the DNA methylation- and H3K27me3-deficient Serum/LIF cells continued to proliferate as a mixed population where differentiating cells co-existed alongside those that retained stem cell identity. This suggests that co-depletion of DNA methylation and H3K27me3 might be sensitising the primed subpopulation of Serum/LIF cells by amplifying stochastically expressed lineage priming factors. In contrast, 2i-induced signalling was sufficient to maintain the ground state naïve pluripotency independently of deficiencies in DNA methylation and H3K27me3. I also demonstrated that repression by DNA methylation and PRC2 has an antagonistic role in pluripotency and differentiation by co-depleting these pathways in embryoid bodies. In addition to morphological changes, the DNA methylation- and H3K27me3-deficient embryoid bodies showed an impaired ability to extinguish pluripotency networks but could induce gene expression programmes associated with early embryonic, endodermal and germline lineages. In summary, my findings suggest that while the epigenome might shape cell fate and lineage specification choices, the underlying transcriptional state plays a major role in maintaining stem cell identity. Additionally, I demonstrated that DNA methylation and PRC2 can partially compensate for each other, as spontaneous differentiation is only observed in double mutants cultured in Serum/LIF. Lastly, my observations highlight the dual requirement for DNA methylation and PRC2 in metastable naïve pluripotency maintenance and establishment of the appropriate lineage specification programmes during differentiation

A novel FLI1+ hybrid cell state in melanoma residual disease

Melanoma, formed from the malignant transformation of the pigment producing melanocytes, is the most aggressive form of skin cancer. In the UK, melanoma is the 5th most common cancer and its incidence has more than doubled since the early 1990s. Despite recent advances in treatment options, patients with metastatic melanoma typically have a poor prognosis, due to low response rate to immune therapies (e.g., checkpoint inhibitors) and high frequency of resistance to targeted therapies (e.g., BRAF inhibitors). Therefore, it is a necessity to develop more effective and durable therapeutic options for patients with metastatic melanoma. Melanoma is comprised of diverse cell populations of highly plastic transcriptional cell states which are considered key drivers of therapy resistance and disease progression. Previously, we showed via fate mapping that the cells which survive during disease regression, known as residual disease, directly contribute to tumour relapse (Travnickova et al., 2022). Thus, emphasising the importance of identifying and functionally interrogating the transcriptional cell states that arise and persist during disease regression and recurrence. To interrogate this heterogeneity and plasticity in melanoma, I have used an adult zebrafish model of cutaneous melanoma, in which I can follow tumour regression and recurrence, just as we see in patients as a result of therapy resistance. The origin of my project came from single cell RNA-sequencing of zebrafish melanomas completed by Travnickova et al., (2019), which showed a tumour subpopulation in residual disease unexpectedly expressing fli1. FLI1, an ETS transcription factor, is a key transcriptional regulator in development and homeostasis, and is predominately expressed in hematopoietic and endothelial cells. However, the functional significance of aberrant FLI1 expression in melanoma was unknown. To investigate this fli1+ tumour cell state, I first generated a Tg(fli1:GFP, crestin:mCherry) zebrafish line on the mutant melanoma background. Using IHC-IF, I was able to visualise fli1+ tumour cells in primary tumours, in persister cells at the residual disease site and in relapsed melanomas. Importantly, I demonstrated that this cell state is relevant to human disease, as I also detected FLI1+ melanoma cells in both patient biopsies and scRNA-sequencing data. Next, I used the Tg(fli1:GFP, crestin:mCherry) zebrafish melanoma model to quantify the fli1+ melanoma subpopulation in primary and regressed tumours using flow cytometry. Excitingly, these data showed that the fli1+ cell state is specifically enriched in residual disease, relative to the progressing tumour. Importantly, preliminary results from ongoing in vitro experiments generating BRAF inhibitor (Vemurafenib) resistant patient cell lines, indicate that FLI1 is also upregulated in response to prolonged Vemurafenib treatment. This provides an important link to the clinic, suggesting the enrichment observed in our genetic model of residual disease, is also conserved in response to targeted therapy. To further understand the dynamics of the fli1+ cell state, I successfully developed a tamoxifen inducible dual marker lineage tracing strategy, which enables a fluorescent ‘switch’ of the fli1+ melanoma cells, allowing tracking of this cell state through disease stages. Using this system in combination with IHC-IF and RNAscopeTM, to assess fli1 expression in fluorescently ‘switched’ cells, I demonstrated that the fli1+ tumour cells persist beyond residual disease and contribute to tumour relapse. In addition, this strategy revealed the plasticity of the fli1+ cell state, showing that fli1+ melanoma cells in residual disease can alter their transcriptional identity and turn off fli1 expression during tumour relapse. Next, to better understand the cellular identity of the fli1+ cell state, I isolated fli1+ tumour cells from primary and regressed zebrafish melanomas and performed transcriptomic profiling. Crucially, differential gene expression analysis showed that fli1 expression is more than simply a single marker gene, rather representative of a transcriptionally distinct tumour cell state. Furthermore, pathway analysis revealed that the fli1+ tumour cells are hybrid in nature, maintaining melanoma gene expression, while also being enriched for mesenchymal gene signatures. Moreover, a large proportion of the differentially expressed genes of the fli1+ cell state are both targets of Fli1 and expressed in the neural crest lineage during early development, suggesting that Fli1 may be driving a developmental mesenchymal programme, which could prove critical for tumour cell survival during regression. Therefore, to determine whether Fli1 is sufficient to drive the mesenchymal programme in vivo, and assess the impact this has on response to treatment and tumour relapse, I generated an inducible fli1 over-expression transgenic zebrafish line. Early validation experiments in embryos are promising and indicate this line will be effective in driving fli1 over-expression in melanoma tumours in adult zebrafish. Together, this work identifying and characterising this novel fli1+ cell state will better inform our understanding of tumour heterogeneity and plasticity in melanoma residual disease and how to combat therapy resistance

Breeding for reduced methane emissions in livestock

This project examined the potential reductions in livestock methane emissions through breeding, and the policy levers that could motivate these changes. We explored the technologies that detect and measure methane, manage data and are used in the breeding process and examined their potential availability in Scotland in 2030 and 2045. We also identified the relevant policy levers and behaviour changes and considered what Government, the post-farm market, pre-farm gate actors and farmers can do differently to encourage methane reductions through breeding

High precision tools for intraoperative brain cancer therapy

Glioblastoma remains to be the most aggressive brain malignancy to affect adults. The disease has a median survival of only 15 months, owing to tumour recurrence within 2 cm of the primary tumour site. Despite the poor prognosis, standard treatment still relies heavily on surgical resection of the tumours, followed by chemotherapy with temozolomide and radiotherapy. This treatment is hindered by the vast heterogeneity within and between glioblastoma subtypes. In this work, the author has proposed the use of photodynamic therapy as an adjuvant to surgical resection. Photodynamic therapy utilises light to induce a reaction between a photosensitiser and molecular oxygen in order to initiate cell death. This technique centres on three main components; the photosensitiser, light and oxygen. Each of these, in themselves, are non-toxic and cell-death is only elicited when all three are combined. While there are promising candidates for the treatment of glioblastoma through photodynamic therapy, these photosensitisers are often bulky molecules which lack specific targeting groups. This results in side- and off-target effects as well as patient-dependent differences in dosage in the target tissues at the time of treatment. Therefore, a library of photosensitisers based on the nitrobenzoselenadiazole (SeNBD) scaffold was designed. By taking advantage of the low molecular weight of SeNBD, this photosensitiser was readily conjugated to other small molecules, with the overarching aim of targeting glioblastoma metabolism. While differential uptake of metabolites was not effective enough to discriminate between healthy and cancer tissue, a further level of control was added through the design of an activatable photosensitiser. This was achieved by disrupting the electron density within the scaffold, thus quenching the absorbance of the photosensitiser. The nature of these quenching groups can be altered to tune the stimulus which uncages the photosensitiser. As a result, a library of orthogonal and environmentally sensitive photosensitisers based on SeNBD has been created, and this approach applied to other photosensitive scaffolds (i.e. 2-thioxocoumarin, thionaphthalimide, Nile Blue and methylene blue). This highlights the modularity of the approach and the wider implication on developing activatable photodynamic therapy agents. Combining these two strategies paves the way to the development of some of the first enzyme activatable, metabolically targeted photosensitisers. As a model, a cathepsin B sensitive pro-fluorophore was synthesised based on the ONBD scaffold. Transcriptomic data from glioblastoma stem cells (GCGR-E17, GCGR-E31, GCGR-E57) and healthy foetal controls (GCGR-NS12ST_A, GCGR-NS17ST_A, GCGR-NS9FB_B) suggest that this approach can provide an effective route to selective ablation of glioblastoma cells without significant damage to surrounding healthy tissues

Addressing microarchitectural implications of serverless functions

Serverless computing has emerged as a widely-used paradigm for running services in the cloud. In this model, developers organize applications as a set of functions invoked on-demand in response to events, such as HTTP requests. Developers are charged for CPU time and memory footprint during function execution, incentivising them to reduce runtime and memory consumption. Furthermore, to avoid long start-up delays, cloud providers keep recently-triggered instances idle (or warm) for some time, anticipating future invocations. Consequently, a server may host thousands of warm instances of various functions, their executions interleaved based on incoming invocations. This thesis investigates the workload characteristics of serverless and observes that: (1) there is high interleaving among warm instances on a given server; (2) individual warm functions are invoked relatively infrequently, often at intervals of seconds or minutes; and (3) many function invocations complete within milliseconds. This interleaved execution of rarely invoked functions leads to thrashing of each function's microarchitectural state between invocations. Meanwhile, the short execution time of functions impedes the amortization of warming up on-chip microarchitectural state. As a result, when a given memory-resident function is re-invoked, it commonly finds its on-chip microarchitectural state completely cold due to thrashing by other functions---a phenomenon we term lukewarm execution. Our analysis reveals that the cold microarchitectural state severely affects CPU performance, with the main source of degradation being the core front-end, comprising instruction delivery, branch identification via the BTB, and conditional branch prediction. Based on our analysis, we propose two mechanisms to address performance degradation due to lukewarm invocations. The first technique is Jukebox, a record-and-replay instruction prefetcher specifically designed to mitigate the high cost of off-chip instruction misses. We demonstrate that Jukebox's simplistic design effectively eliminates more than 95% of long-latency off-chip instruction misses. The second technique is Ignite, which builds on Jukebox to offer a comprehensive solution for restoring front-end microarchitectural state, including instructions, BTB, and branch predictor state, via unified metadata. Ignite records an invocation's control flow graph in compressed format and uses that to restore the state of the front-end structures the next time the function is invoked. Ignite significantly reduces instruction misses, BTB misses, and branch mispredictions, resulting in an average performance improvement of 43%. In summary, this thesis demonstrates that serverless systems present distinct workload characteristics that fail to match traditional CPU designs, severely impacting performance. Two simple techniques can overcome these bottlenecks by preserving the microarchitectural state across function invocations

The non-canonical role of the outer kinetochore protein KNL-1 in axonal development

The nervous system is composed of specialized cells; glia and neurons, that form an interconnected network to relay information. Proper transmission of information relies on the two main compartments within neurons, dendrites, that receive information, and axons, that relay the information through specialized domains called synapses. Axon development is a multi-step process that involves, axon outgrowth, guidance, termination and synaptogenesis. In every step of axonal development rearrangements in the microtubule and actin cytoskeleton are essential to mediate the morphological changes that the axon undergoes. The molecular mechanism governing cytoskeletal regulation during axon development is still not fully characterized. Recent findings have highlighted a novel, non-canonical role for the outer kinetochore protein network, KMN (Knl-1, Mis12, Ndc80), in neuronal development. KMN, primarily recognized for its role in tethering chromosomes to spindle microtubules during chromosome segregation in cell division, has emerged as a potential key cytoskeletal regulator in neurons. This work investigates the noncanonical neuronal role of the outer kinetochore signalling and scaffolding protein KNL-1, in brain organization and axon development. In my thesis I show that KNL-1 is essential for axon organization and termination in the nervous system of C. elegans. In the first part I show that KNL-1 is required for the organization of the C. elegans nerve ring axons and ganglia organization in the brain. Specifically, loss of KNL-1 affects the correct placement and fasciculation of the axons within the nerve. Structure-function analysis of KNL-1 showed that this function requires both the signalling and microtubule binding domains of KNL-1. The second part of my work reveals an essential role for KNL-1 in axon termination, a process whereby the axonal growth cone is destabilized and stops its growth upon reaching its target. The effect of KNL-1 in axon termination, requires reorganization of F-actin at the axonal tip and is regulated by microtubule dynamics. In the final part, I have used Correlative Light-Electron Microscopy and a GFPTrap of KNL-1 in C. elegans embryonic neurons to identify the neuronal structures and proteins that KNL-1 associates within the axon. KNL-1 associates with endo-lysosomal structures in the cell body and synaptic vesicles in the axon. Mass spectrometry analysis revealed a synaptic protein as a potential interactor-candidate of KNL-1. This work showed a new potential link of KNL-1’s neuronal activity with synaptic organization and function. Overall, this study provides insights into the mechanism by which the outer kinetochore component KNL-1 functions in brain development, identifies a novel role for this protein in axon termination and reveals neuronal interactors of KNL-1 highlighting a potential role of the protein in synapses

The ‘layered time’ of xenophobia and racial capitalism: comparing the everyday challenges and identities of African migrant women in Johannesburg and London

The literatures on xenophobia and its violence in post-apartheid South Africa and post-Brexit UK, while each providing a deep and important contextual analysis, lacks a broader examination of the everyday manifestations of anti-foreigner discrimination for different groups. Particularly, they lack the perspectives of African migrant women. Using a relationally comparative approach, this interdisciplinary study examines both the conceptual and context-specific literature on xenophobia and its relationship to racism in South Africa and the UK, drawing on primary qualitative, ethnographic and creative fieldwork with Congolese and Zimbabwean women in Johannesburg and London using both an intersectional and temporal lens. Comparing contexts, concepts and lived experiences, this study embeds African migrant women’s experiences of discrimination over time within scholarship, policy and practice. Theoretically, it contributes towards an expanded conceptual understanding of xenophobia in three ways. Firstly, by examining its relationship to racism in wider literature and specifically within the Congolese and Zimbabwean migrant literatures in both contexts, arguing for it to be conceptually understood as embedded within racism and as a form of structural discrimination. It then goes onto argue for xenophobia and racism to be grounded within a global and contextually specific understanding of racial capitalism. Secondly, by applying a framework of ‘layered time’ to the lived experience of xenophobia, to reveal the longer-term psychological and ontological effects of this multi-faceted form of discrimination on women migrants’ livelihoods and wellbeing. Thirdly, by analysing barriers to access for African migrant women in everyday areas of education, work, healthcare and housing through an intersectional lens. This analysis reveals that to attempt an accurate portrayal of women’s lives and to inform legal and rights-based responses, intersectional research must include not only how lives and livelihoods are affected by their multiple overlapping categories of structural identity, but also by individual choice, background and social networks. Calling for a transnational, temporal and racially capitalist analysis of xenophobia, this study emphasises the importance of exploring the mundane ‘everyday’ as well as sporadic eruptions of violence. It warns against the tendency to homogenise the lived experiences of African migrant women, highlighting the need to recognise their human complexities