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Investigating the effect of PAX6 haploinsufficiency on neuronal projections using human cerebral assembloids
No full textInter- and intra-hemispheric communication in the cerebral cortex relies on axons. PAX6 is a crucial transcription factor for axon development, and its haploinsufficiency leads to defects in inter-hemispheric structures like the corpus callosum. This structure forms from SATB2-expressing cells, with Pax6 influencing their specification in mice.
We investigated the impact of PAX6 haploinsufficiency on human iPS cell-derived cerebral assembloids. Using a 3D-printed device, we established a method to generate these assembloids, optimised the RatDISCO protocol for assembloid imaging using 3D light sheet microscopy, and used AAV-hSyn-EGFP to visualise projections.
In our comparison, PAX6⁺/⁻ organoids showed a transient increase in SATB2- expressing cells at day 90, returning to control levels by day 120, indicating a role for PAX6 in age-dependent SATB2 specification. Tracing projections revealed only local extensions, with preliminary data indicating no morphological differences between genotypes. These findings suggested that PAX6 has a role in SATB2 specification, but a partial loss of PAX6 likely did not affect the morphology of neuronal projections
The role of the cGAS-STING pathway in TIF-IA dependent induction of the senescence associated secretory phenotype
No full textCellular senescence is a stress-induced program characterized by irreversible cell cycle arrest and the secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP). While acute SASP promotes tissue repair and tumor suppression, its chronic activation contributes to many age-related diseases including cancer. NF-ₖB is recognised as a key transcriptional driver of the SASP. However, the pathways that activate NF-B following exposure to senescence triggers remains elusive. The abstract is too long so I would suggest just a couple of sentences of background highlighting the importance of the SASP and the lack of understanding of its initiation.
Previous work from the STARK Lab demonstrated that the PolI complex component, TIF-IA, accumulates in response to senescence triggers and that this is essential for NF-ₖB activation and the SASP. However, the molecular mechanism linking TIF-IA accumulation to SASP induction remain unclear. The cGAS–STING DNA-sensing pathway has been implicated in senescence effects on NF-B signalling. Therefore, I hypothesized that cGAS-STING may act as an intermediary. To explore this possibility, I used a model of therapy-induced senescence in which cells are treated with the DNA damaging agent, etoposide. Firstly, I compared A549 (STING-low) and HCT116 (STING-high) cells. Using qPCR western blot analysis and immunocytochemistry, I found that both cell lines showed comparable transcription of SASP factors and TIF-IA accumulation following etoposide exposure, despite the differential baseline expression of STING. Furthermore, siRNA depletion of TIF-IA significantly abrogated etoposide-mediated SASP factor transcription in both cell lines. To further evaluate STING’s role in SASP regulation, I performed STING siRNA knockdown in HCT116 cells. Contrary to my finding in the STING low A549 cells, qPCR indicated that depletion of STING in HCT116 cells caused a significant reduction in SASP factor expression in response to etoposide. Together, these results suggested that, at least in HCT116 cells, both TIF-IA and STING are required for the SASP. Therefore, I next set out to understand the relationship between these two pathways. I found that depletion of TIF-IA significantly reduced mRNA and protein levels of STING both basally and in response to etoposide, while the opposite was observed upon TIF-IA overexpression. In contrast, STING depletion had no effect on TIF-IA accumulation in response to etoposide. These data suggest STING may be downstream of TIF-IA in the pathway to the SASP. In support of this suggestion, I found that depletion of STING significantly reduced the increase in SASP factor transcription observed after TIF-IA overexpression. These data identify a new pre-senescence pathway upstream of SASP activation that has significant relevance to age-related diseases, including cancer
Early warning systems and dryland communities in the Horn of Africa: A desk review of actors, challenges, and opportunities
No full textThis report is part of a series drafted to respond to the challenge: “How can we bridge the disconnects between food security, climate, and natural hazards early warning systems and the anticipatory actions that pastoral and agro-pastoral communities in the Horn of Africa can take to overcome recurring shocks and threats to their lives and livelihoods?
Reframing the Nation: Army Media, Memory, and Militarised Legitimacy in Sudan’s War
No full textSince the start of the war in Sudan in 2023, the Sudan Armed Forces (SAF) has used Arabic-language media to portray itself as Sudan’s only legitimate national institution—non-Islamist, inclusive, and the last safeguard against fragmentation. This report explores how that projection is constructed, and how it draws on selective historical memory and co-opted revolutionary rhetoric to consolidate military legitimacy. This report assesses the contribution of the SAF’s Facebook pages – including its official page, and the specific page for the affiliated al-Quwwat al-Musallaha newspaper – to its propaganda campaign during its current conflict with the Rapid Support Forces (RSF). It focuses in particular on the ways in which the military uses its social media presence to appeal to a broad range of social categories in Sudan, whether through its calls for mobilisation, its narrative of political developments, or its reports from the battlefield. It pays detailed attention to the way the army has sought to present itself as the guarantor of the Sudan’s national interests, and the ways in which it has used history to legitimise itself. This report shows that SAF propaganda rebrands the military not by abandoning the past, but by curating it – mobilising nostalgia, nationalism, and selective silence to present itself as Sudan’s last remaining centre of gravity
On the Peace Negotiations Between Russia and Ukraine: Prioritising the Human Dimension
No full textThis report highlights the importance of raising the “human dimension” in the talks aimed at ending the Russo-Ukrainian War. It identifies 31 key proposals on the human dimension and places them in the context of the fragmented global order, situating them in five thematic areas: safety and security; justice and accountability; participation and inclusion; socioeconomic recovery; and culture and identity. The report develops the argument that the multilayered and polycentric nature of this order, for all its tendencies to breakdown and violence, does provide avenues for civil society interventions in negotiations, which are crucial to mitigating harm
Dynamic ubiquitylation in Plasmodium transmission: from writers to targets
No full textUbiquitylation is a key post-translational modification, controlling protein turnover, localisation,
activity, and additional regulatory functions. Despite the implication of ubiquitin regulation in
Plasmodium blood stages, including in the resistance to key anti-malarials, there has been
little research into the function of ubiquitin in the transmission stages. By employing a ubiquitin
capture system, we identified over 1300 proteins associated with ubiquitin signalling, and
>600 ubiquitylation sites in ~200 proteins during Plasmodium transmission. We found that
ubiquitylation was involved in most key processes in early parasite development stages in
the mosquito, including DNA replication, axoneme biogenesis, and translational control. To
further investigate the proteins that mediate these signalling events we employed genetic
engineering to knock out eight E3 ligases and assess their function during transmission.
We found that six of these E3s contributed to fitness during transmission, of which four had
critical functions. Further investigation revealed one RING-finger E3 (RING1) to be essential
for proper DNA replication and or nuclear segmentation in oocyst stages, while a U-box E3 (UBox1)
is essential for sporozoite re-infection of the naive host. A second U-box (U-Box2) and
RING-E3 (RING2) display functions earlier in development, where both impact the efficiency
of ookinete production. We found RING2 regulates inner membrane complex (IMC) integrity,
and its deletion results in bulbous ookinetes with a low potential for oocyst production. UBox2
on the other hand appears to be essential for transmission progression, and its deletion
results in a widespread stress response which is ultimately lethal for the parasite. Finally, we
investigated an identified target of ubiquitylation, ZNF1. We found that ZNF1 is essential for
transmission, including for the fertility of both male and female gametocytes. ZNF1 deletion
results in widespread dysregulation of the transcriptome and proteome of gametocytes,
including the precocious translation of some translationally repressed transcripts. Together
this demonstrates the key role of ubiquitylation in multiple stages of parasite transmission,
increasing our understanding of the regulation involved in Plasmodium propagation at the
host-vector interface
The pursuit of status: Russia’s engagement with the Western-led peacebuilding processes in Bosnia and Herzegovina and Kosovo
No full textThis thesis explores how status concerns shape state behaviour in international processes by focusing on Russia’s engagement with the Western-led peacebuilding efforts in Bosnia and Herzegovina (BiH) and between Serbia and Kosovo under Vladimir Putin’s leadership (2000-2024). It seeks to understand how Russia’s preoccupation with its great power status influenced its involvement in these post-conflict processes. Using an analytical framework based on social identity theory (SIT) and status inconsistency approaches, I argue that these international processes provided Russia with opportunities to engage with its significant Others – the United States and Europe – and to seek recognition of its great power status.
My research suggests that two key factors shape Russia’s behaviour: first, its self-perception as a status overachiever or underachiever at the systemic level, and second, its perception of the legitimacy and permeability of the Western-led initiatives in the Western Balkans. The first factor influences whether the Russian administration views symbolic equality (membership) in the great power club as sufficient or whether it seeks to enhance Russia’s positional ranking within the club. The second factor influences which of the identity management strategies outlined by SIT Russia adopts in its status-seeking efforts within the Western-led international peacebuilding initiatives: social mobility (follow), social competition (challenge), or social creativity (change by expanding or reframing).
Methodologically, the thesis employs a case study research design supported by process tracing that relies on document analysis. It makes the following contributions: First, it furthers the status literature by bridging the gap between SIT and status inconsistency approaches to delineate the conditions under which great powers perceive status as a ‘club good’ versus a ‘positional good’. This theoretical advancement is the primary contribution of the thesis. Second, it provides a framework that explains how great powers’ status concerns guide their regional policies and approaches towards international processes, expanding the empirical scope of status literature. Third, empirically, it offers an in-depth account of Russian foreign policy towards BiH, Serbia, and Kosovo, as well as the international peacebuilding efforts for these countries
Exploring the presence of mental fatigue in elite orienteering training and competition
Get PDFLaboratory-based experiments have found that engaging in mentally demanding tasks can induce mental fatigue (MF), which is known to impair endurance and decision-making performance in sports. However, these experiments often lack ecological validity, and their generalisability is limited by individual differences in the perception of MF. These differences can be influenced by factors such as training level, environment, and occupation. Consequently, the transferability of findings from laboratory-based intervention to real-world sports settings may be limited. To address these limitations and improve our understanding of MF in applied sports contexts, this thesis explores how orienteers perceive and respond to MF in ecologically valid environments. This thesis comprises three studies investigating the presence and impact of MF in orienteering.
Study 1 employed a three-round online Delphi approach with 24 international orienteering athletes and practitioners to explore sport-specific perspectives on MF. The consensus reached in this study showed that international orienteering experts recognise the occurrence of MF during both orienteering training and competition, while also reinforcing differences in how MF is perceived between these contexts. However, there was no consensus that MF experienced in competition could be replicated in orienteering training. Furthermore, no consensus was reached on the existing definition of MF accurately reflects the MF experienced in orienteering. Consequently, a sport-specific definition of MF was developed to increase specificity and clarity in future research and support more accurate discussions of MF within the context of orienteering.
Study 2 analysed 43 orienteering races from 16 national-level orienteers and found a moderate increase in perceived MF (ES = 0.84 [0.49 , 1.19], mean difference: 19.7 [12.5 , 27.2]) following an orienteering competition. The increased MF ratings sustained up to 48 hours, with the perceived MF ratings remaining higher than pre-competition values to a small extent (ES = 0.54 [0.08 to 1.15], mean difference: 10.4 [-1.8 , 22.7]). These findings imply that orienteering competition can acutely induce perceived MF, with effects that may extend beyond the immediate post-competition period and influence recovery.
Study 3 examined the changes in perceived MF and other psychological responses among eleven national junior orienteers during a 4-day orienteering training camp. The combined analysis of 42 pre- and post-orienteering training responses found a moderate increase in perceived MF (ES = 1.06 [0.66 , 1.45], mean difference: 22.5 [15.6 , 29.5]). A moderate increase in perceived MF was observed following each training session, with a cumulative effect resulting in the highest MF ratings on the final day of the training camp. Importantly, perceived MF remained moderately elevated 48 hours after the termination of the orienteering training camp compared to the initial pre-camp ratings (ES = 0.86 [-0.07 , 1.75], mean difference 17.3 [-1.7 , 36.3]), suggesting that simulated orienteering training can also induce sustained MF.
Importantly, studies 2 and 3 revealed significant individual variability in perceived MF and related psychological responses such as physical fatigue, stress, tiredness, and mood states. These findings highlight that orienteers experience MF differently. The model comparison consistently supported the use of random intercept models that accounted for individual variability, emphasising the importance of considering variability between participants in statistical analyses. Furthermore, the differences in the magnitude and pattern of changes across outcome variables support the interpretation that MF is a different fatigue construct. Therefore, MF should be monitored and managed separately from other psychological responses.
In summary, this thesis provides evidence that both national and junior national-level orienteers experience MF during orienteering training and competition. This thesis advances the conceptual understanding of MF in orienteering by providing an orienteering-specific definition of MF and reinforces the importance of ecologically valid research designs that reflect the real-world experiences of athletes. Future research should investigate the acute and cumulative impact of MF on orienteering performance and recovery, as well as develop individualised strategies to monitor and manage MF alongside other psychological responses. This will be essential for optimising performance, recovery and fatigue management in orienteering
Protein-RNA interactions during stress responses in Saccharomyces cerevisiae
Get PDFThe survival and adaptability of living organisms depend on their ability to adjust to
environmental changes. In the yeast Saccharomyces cerevisiae, glucose serves as
the preferential carbon and energy source. When glucose becomes scarce, the yeast
undergoes a rapid metabolic reprogramming, known as the diauxic shift, to sustain
growth and survival. This adaptation to glucose starvation is biphasic, involving both
transcriptional changes at the DNA level and post-transcriptional changes at the RNA
level. Transcriptional changes occur within minutes following stress, characterized by
the downregulation of genes associated with glucose metabolism and the upregulation
of stress response genes and genes involved in utilizing alternative carbon sources.
In contrast, immediate post-transcriptional changes commence within seconds of
stress, mainly influencing mRNA stability, localization, and translation, and thus
immediately impacting gene expression. RNA-binding proteins (RBPs) play a crucial
role in modulating these RNA-related processes, thereby determining the fate of the
cell following stress.
Previous studies have identified significant reorganization of the RNA-protein
interactome in yeast following glucose removal and heat shock 4. Specifically, a suite
of RBPs exhibited substantial changes in their RNA binding after stress.
Unsurprisingly, most of these proteins have known roles in RNA metabolism.
However, among the most affected RBPs were a few previously uncharacterized
proteins, including Pin4 and Rtc3. In this work, we used our improved UV crosslinking
method called reCRAC to examine Pin4-RNA and Rtc3-RNA interactions, under both
glucose-rich and stress conditions to identify their function in RNA metabolism. Our
findings reveal that Pin4 plays an integral role in post-transcriptional regulation and,
unexpectedly, that it is also a key protein for the transcriptional adaptation to glucose
starvation. Additionally, we identified Rtc3 as a protein that binds to a specific site in
the large ribosomal subunit, potentially influencing ribosome dynamics following
stress
Using synthetic biology to understand DNA methylation maintenance in cancer
Get PDFLoss of DNA methylation is common to the vast majority of human cancers and is considered a hallmark of cancer epigenomes. Current models propose that improper maintenance of DNA methylation could underpin this hypomethylation. However, these models are based on evidence derived from static snapshots of cancer methylomes. In addition, DNA methylation represents an important therapeutic target in cancer treatment. DNA hypomethylating agents (HMAs) are evidenced to promote antitumour immunity, however, existing HMAs have limited applications due to poor pharmacometrics and cytotoxicity. Thus, advancing our understanding of DNA hypomethylation in cancer, alongside identifying more effective drugs to target DNA methylation, is crucial for enhancing therapeutic strategies in cancer treatment.
To gain insight into the dynamics of DNA methylation maintenance in colorectal cancer cells, I developed a methylation-sensitive reporter system. This piggyBac transposon-based system allows stable integration of a methylated eGFP transgene (eGFPme) at distinct locations across the genome. I show that methylation of this reporter construct represses its expression in colorectal cancer cells. Reporter integrations were identified across the whole genome, enabling tracking of genome-wide DNA methylation maintenance. To test whether HCT116 cells are able to faithfully propagate DNA methylation, cells containing premethylated reporters were maintained in culture for up to 8 weeks, equivalent to ~75 cell divisions. The majority of methylated reporters remained repressed and maintained their methylated status over this time course. These findings demonstrate that methylation patterns remain consistently preserved at synthetic reporters in HCT116 colorectal cancer cells. My results highlight the functionality of the DNA methylation maintenance machinery in colorectal cancer cells, despite the presence of extensive hypomethylation.
Having demonstrated that DNA methylation is maintained in colorectal cancer cells, I next investigated the contribution of different components of the DNA methylation maintenance machinery to methylation maintenance. To do this, I assessed reporter activity in knock-out (KO) and degron cell lines. DNMT1 is the primary maintenance methyltransferase, responsible for maintaining DNA methylation following replication. As expected, reporter repression and methylation were not maintained in HCT116 cells lacking DNMT1 function. It has been previously reported that cooperation between DNMT1 and the de novo methyltransferase DNMT3B is required to maintain DNA methylation in mouse embryonic stem cells. However, this has not previously been tested in human cancer cells. To investigate the contribution of DNMT3B to DNA methylation maintenance, I assessed reporter activity in DNMT3BKO cells. No significant differences in reporter expression or methylation were detected in DNMT3BKO cells compared to HCT116. My results indicate that DNMT3B does not have a detectable contribution, in this experimental system, to methylation maintenance in human HCT116 cells. Next, I investigated the contribution of UHRF1 to DNA methylation maintenance. UHRF1 is an emerging therapeutic target of interest due to its essential role in DNA methylation maintenance, and its overexpression in some cancers. I integrated the eGFPme reporter into DNMT1 and UHRF1 degron cell lines. This degron system enables rapid depletion of the degron-tagged proteins, and I monitored reporter activity in response to protein depletion. Following DNMT1 or UHRF1 depletion, rapid upregulation of the reporter gene was detected. These results support that targeting UHRF1 is an efficient method to deplete DNA methylation colorectal cancer cells. Furthermore, these findings demonstrate that the reporter is activated in response to acute loss of DNA methylation.
Finally, I sought to test whether the reporter system developed in this study could be utilised as a drug screening platform, to investigate compounds that modify DNA methylation. My previous results provided evidence for therapeutic targeting of DNMT1 and UHRF1. Therefore, I screened several novel inhibitors of DNMT1 and UHRF1. Removal of DNA methylation maintenance using the DNMT1 inhibitor GSK-3484862 resulted in a ~60% reduction of genome-wide methylation and rapid reporter activation. Importantly, I showed that GSK-3484862 treatment led to the activation of HERV-1 elements and IFN-1 signalling, which is thought to be important for the immunostimulatory applications of hypomethylating agents in cancer treatment. Together, my findings provide proof-of-concept for the use of this reporter system in large-scale compound library screen, to identify novel hypomethylating agents.
The methylation-sensitive reporter system developed in this study was utilised to provide novel insights into the mechanisms underlying DNA methylation maintenance in cancer. This reporter system has future applications as a screening platform, to identify genetic factors involved in DNA methylation, and novel hypomethylating compounds with therapeutic potential
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