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The role of neutrophils in metastatic non-small cell lung cancer
No full textLung cancer is a leading cause of cancer deaths globally. Metastasis, the process by which cancer spreads from its site of origin to a more distant site, accounts for approximately 90% of cancer deaths. Lung cancer is broadly split into non-small cell lung cancer (NSCLC), which represents around 85% of lung cancer cases, and small cell lung cancer (SCLC). When cancer cells have spread from the lung to the pleural fluid around the lung (malignant pleural effusion), this is considered metastatic disease.
Neutrophils are a heterogenous population and have been shown to be polarised to anti-tumour or pro-tumour roles depending on their tumour microenvironment. T cells are known to be important effector cells in cancer. Neutrophils and T cells are both found in malignant pleural effusions, but little is known about their interaction in this context. The objective of this project was to gain a better understanding of how neutrophils interact with T cells in the NSCLC pleural fluid metastatic niche.
Using pleural fluid from NSCLC patients to model the metastatic niche, I have shown when healthy donor T cells are cultured in the presence of NSCLC pleural fluid, there is an uplift in the percentage of CD4+ and CD8+ T cells that divide. However, when healthy donor neutrophils are added to this co-culture model, this pleural fluid uplift in the percentage of CD4+ and CD8+ T cells that divide is significantly suppressed. Further to this I have observed that the neutrophil induced suppression of T cell proliferation is a result of reduced entry into proliferation as opposed to inhibiting the ability of T cells to continue to divide once they have started to divide. This suppression is contact dependent. I have also identified that the neutrophil induced T cell suppression also occurs in the presence of pleural fluid from pneumonia patients, suggesting a common mechanism induced by both the infective and metastatic niche. Additionally, at 72 hours post stimulation, neutrophils cause a suppression in T cell activation as measured by reduced CD25 and CD71 and increased CD62L T cell expression when T cells are cultured with neutrophils and NSCLC or pneumonia pleural fluid compared to when cultured with pleural fluid alone. Taken together, this data suggests neutrophils are reducing T cell activation and causing a block to T cell entry into proliferation, suggestive of the neutrophils having an effect early in the T cell activation process. This is supported by the failure of T cells to induce expression of the early activation markers CD69 and phosphorylated ribosomal protein S6 (pRPS6) in the presence of neutrophils and NSCLC pleural fluid compared to pleural fluid alone 4 hours following stimulation. I have established that the suppressive neutrophil induced effect in the presence of NSCLC pleural fluid is dependent on active signalling, as the neutrophils were unable to cause suppression when they were fixed prior to co-culture, and is likely to involve a protein factor, as the suppressive effect was lost when the NSCLC pleural fluid was heat treated.
Proteomic analysis revealed the majority of the 358 proteins identified were expressed to similar degrees in both NSCLC and pneumonia pleural fluid. Metabolomic analysis of pleural fluid showed both methionine and tryptophan essential amino acids were present in both types of pleural fluid although at higher levels in NSCLC compared to pneumonia. NanoString transcript data of CD8+ T cells cultured with NSCLC pleural fluid alone or with NSCLC pleural fluid and neutrophils then sorted into CD69+ or CD69- T cells showed some of the genes differentially expressed between CD69+ (“activated”) and CD69- (“non-activated”) T cells were perturbed by the addition of neutrophils in the co-culture. Interestingly SMAD3 was the only gene that was upregulated in both CD69+ and CD69- T cells in the presence of neutrophils compared to without neutrophils. Despite this, neither SMAD3 nor transforming growth factor beta (TGFβ) inhibitors reversed the neutrophil induced T cell suppression shown in the context of NSCLC pleural fluid.
In summary, I have established a robust phenotype of neutrophils causing suppression of T cell activation and entry into proliferation in the context of NSCLC pleural fluid. I have shown this to be the case for both CD4+ and CD8+ T cells and across multiple NSCLC pleural fluids and pneumonia pleural fluids. I have used proteomic, metabolomic and transcriptomic data to try to understand how the suppressive effect may be manifesting. Further work is required to identify a target and functionally prove reversal of the suppressive effect. Establishing mechanisms by which neutrophils cause a suppressive effect on T cells may help provide therapeutic targets to optimise the overall anti-tumour response in NSCLC treatments
An empirical investigation of alignment in dialogue: situational and cognitive factors
Full text linkIn dialogue, interlocutors build similar representations of what they are talking about. That is, they align on representations that are relevant to the conversation. We define aligned representations simply as representations held by at least two interlocutors, and which are similar to each other. Alignment at the level of representations leads to similarities in interlocutors’ behaviour: aligned interlocutors are likely to use the same words to refer to the same objects or events (i.e., they entrain on their lexical choices). We define entrainment as between-interlocutors repetitions of words during dialogue, and we understand it as a consequence of the alignment of lexical representations. Alignment is pervasive: speakers entrain with their interlocutors in written and spoken, online and in-person interactions, when talking with both human and conversational agents (such as bots and robots). At the same time, speakers can use entrainment for strategic purposes: to aid comprehension or to regulate social distances and enhance rapport. In this sense, alignment must be a mechanism that speakers can use flexibly. On top of that, interlocutors keep track of alignment and communicate about whether they believe they are aligned or not, through commentaries on alignment. We define a commentary on alignment as any behaviour that indicates an interlocutor’s belief about whether the interlocutors are aligned or not. Interlocutors can comment on alignment in several ways: by using backchannels (e.g., mh-mm, yeah, okay), by requesting repairs when needed (e.g., what was that? or who are we talking about?), or by repeating and sometimes elaborating on what their interlocutor said.
This thesis is about the mechanisms that allow interlocutors to align and to communicate whether they are aligned in situated interactions, with other people and with conversational agents. The first part of the thesis focuses on entrainment in highly controlled interactions, where participants believed they were playing a reference game with a remote human interlocutor or with a virtual agent, under normal or high cognitive load. In Experiments 1-2 we presented interlocutors as high or low in social status and asked participants to rate the social status of the interlocutors either before or after playing the reference game. We found that participants entrained more with interlocutors presented as high in social status than low in social status, but only when they rated the social status before playing the game. In Experiments 3-4 we presented interlocutors as highly or poorly competent virtual agents, and we manipulated participants’ cognitive load using a dual-task paradigm. We found that participants entrained more with virtual agents when presented as poorly than highly competent, but only when participants could fully focus on the reference game. These results suggest that speakers can use alignment strategically when they can focus on the interaction, and when there are salient properties of the interaction that trigger communicative and social intentions, but that they can also rely on simpler – and more automatic - mechanisms when such intentions are less salient and when they are distracted.
The second part of the thesis (Chapters 5-6) focuses on alignment and use of commentaries in more naturalistic interactions, and whether they are affected by the topic of the conversation and the interlocutors involved. In Experiment 5, participants ranked some items from the most to the least useful for people stranded in the desert, and, after discussing the items with a partner, they ranked the items once again. The order was presented as factual in one group (i.e., unknown to participants, but defined by the British Army) or contestable in the other group (i.e., unknown to the participants, who were made aware that there was no right or wrong order). In Experiment 6, participants performed the same task but discussed with a social robot, whose appearance was either human- or machine-like. Alignment was measured as the increase in similarity between rankings after the discussion, compared to the similarity in rankings before the discussion. In both experiments, participants aligned with their interlocutor, but there was no effect of how the ranking was framed or of how the robot appeared. However, participants used more commentaries when discussing the items in the ranking presented as contestable compared to the ranking presented as factual. Additionally, participants’ use of commentaries was similar in human-human and human-robot interactions.
Overall, these results confirm that alignment – and its commentaries – are pervasive in interactions with human and conversational agents, but that they can be adapted flexibly to context. Such ubiquity and flexibility may be allowed by the existence of multiple mechanisms and which mechanism is used may be triggered by specific situational properties of the interaction. These properties include the communicative needs of the interlocutors, their nature, the social dynamics embedded in the interaction, whether or not the speakers can dedicate full attention to them, and the topic of conversation
The design and synthesis of peptides for fluorescence imaging applications
No full textTesting new function - does this work?? Fluorescence microscopy is a tool
routinely utilised to address biological questions. Direct visualisation of features of interest, confirmation of protein identity or detecting the presence of a posttranslational modification enable researchers to study differences between healthy and pathological phenotypes. As the questions get more complex, more advanced imaging techniques are required to address the limit of resolution, probe specificity to target and fluorescent background. This thesis explores two advancements to the field of fluorescence microscopy: the development of a novel imaging platform
and the introduction of a new imaging modality.
The study covers the design and construction of the Full Spectrum Fluorescence Lifetime Imaging Microscope (FS-FLIM): a new imaging platform. The FS-FLIM maximises the information collected in a fluorescence imaging experiment. It is capable of 3-in-1 imaging, collecting fluorescence intensity and lifetime data at 512 wavelengths simultaneously. The instrument is compatible with a wide range of samples. The spectral region observed can be matched to the emitters present in the sample. Moreover, a wide range of lifetimes (from sub-nanosecond to tens of nanoseconds) can be recorded using the FS-FLIM. The performance of the
constructed instrument is validated through solution lifetime measurements of several fluorescent dyes, and its application in environment sensing is described using a model system.
Next, the thesis focuses on developing fluorescent peptides for imaging applications. Although peptides can be labelled with a range of fluorophores, this study mostly uses a selenium-derivatised nitrobenzoxadiazole dye - SeNBD, a recent exciting addition to the imaging toolbox. The fluorogenic properties of the new dye are characterised in this study, and the incorporation of the dye on-column using standard solid state peptide synthesis methods is described. The switch-on character, as well as the small size of the dye, are leveraged throughout the work, with short peptide sequences and internally-labelled peptide sequences developed as a new generation of peptide probes.
The specificity of peptides enables their application as therapeutic agents
for otherwise undruggable proteins. One such example is α-synuclein, a protein associated with Parkinson’s disease (PD). It is commonly thought that aggregation of α-synuclein and formation of cytotoxic inclusions is involved in PD progression. An α-helical bacteria-derived peptide, phenol soluble modulin α3 (PSMα), was previously shown to bind to small oligomeric α-synuclein species. Here, the peptide sequence was derivatised with several labels to explore its applications in imaging. Pilot experiments to detect α-synuclein species in vitro were conducted to confirm the binding to the protein-of-interest. A biotin-labelled peptide analogue was used to perform immunohistochemistry staining on patient tissue for in situ detection. Lastly, first attempts at fluorescence lifetime imaging of α-synuclein on the FS-FLIM instrument were made.
To further illustrate the capabilities of SeNBD dye specifically, and its potential
for super-resolution imaging, a range of sequences targeting the PDZ domain
(a common structural motif of anchoring and signalling proteins) were then
considered. The chosen sequences were only a few amino acids long, however they
demonstrated retained transient binding to the protein-of-interest upon labelling
with SeNBD. Moreover, an internally-labelled sequence was also synthesised and successfully used to target PDZ domains. The transient nature of the peptideprotein binding was utilised in a Point Accumulation in Nanoscale Topography (PAINT) experiment, where the on-off binding enabled precise localisation of the dye molecules and super-resolution image reconstruction. The PDZ contained in post-synaptic density proteins of a brain-derived sample was successfully imaged, and nanoclusters of the protein super-resolved, corroborating literature findings. Furthermore, an extension to the approach using a coiled-coil interacting peptide pair: 101A and 101B, was investigated as an alternative approach for proteiniv peptide interaction pairs for super-resolution microscopy. Whilst the delivery of
the synthesised targeting sequence (101B) proved challenging, expressing 101B attached to the target protein in cellulo, and subsequent staining with fluorescent 101A sequence produced promising results and enabled direct visualisation and super-resolution imaging of TOM-20 and LAMP-1 in fixed HEK cells.
Overall, the work presented herein showcased a range of fluorescent peptides that could be used across versatile fluorescence imaging platforms. The small novel dye, SeNBD, enabled super-resolution imaging using peptide sequences containing less than 10 amino acids. The small size of the peptides decreased the linkage error and ensured the fluorescent signal was localised at target of interest. The environment sensing properties of the dye were explored in preliminary experiments to observe α-synuclein on a purpose-built novel microscope, the FS-FLIM. It is hoped the techniques developed here could further progress research in the field
of neurodegeneration and beyond
The progressive 1920s: government in American states and cities, 1923-1929
Full text linkIn the first two decades of the twentieth century, progressive American politicians sought to improve their society and to make it fairer. Addressing a broad range of societal issues, they had in common a conviction that government must serve the interests of ordinary people rather than of privileged elites. Contrary to the view of many historians, progressive government continued through the following decade. This study of twelve states and twelve cities demonstrates that their governments were consciously progressive in the 1920s. They enhanced the quality of public services, introducing improvements to systems of education and training, public health, working conditions, residential care and criminal justice. And they expanded the scope of government into areas that foreshadow themes of the New Deal: major public works, parks and conservation, and social welfare.
As the range of public services grew and their quality improved, so their costs soared. Despite their progressive convictions, states and cities failed to introduce fair systems of taxation to fund their expanding programmes. Their failure to tax wealth, in the form of personal incomes and corporate profits, saw the burden of additional tax fall most heavily on people who had little to spare. It also meant the states and cities did not acquire the financial means to sustain their progressive policies. Throughout the 1920s, they looked for financial assistance to the federal government, with its power to tax incomes and profits across the nation, and their calls for aid increased in the troubled circumstances of the Great Depression. From 1933, Congress would respond with record levels of financial aid supporting New Deal initiatives. Radical as it was in its use of federal funds, the New Deal was also a force for continuity, serving to re-energise the progressive government of the 1920s
Policy development for diet and climate
No full textThe Climate Change Committee’s 2023 Report to the Scottish Parliament called for stronger action on food system emissions. Policy interventions need to address the environmental impacts of food production and consumption while ensuring dietary improvements and economic sustainability.
This report assesses Scotland’s diet and climate policy landscape, identifying areas for policy development and providing recommendations to support the Scottish Government’s climate, public health, and food security goals going forward.
The study combined desk-based research, stakeholder engagement and categorisation using a PESTLE (Political, Economic, Social, Technological, Legal, and Environmental) framework
Understanding the ‘China Factor’ in the Russia-Ukraine War
No full textThis paper examines the evolution of China-Ukraine relations from 1991 to April 2025, focusing on the strategic, economic, and geopolitical implications of China’s role in the context of Russia’s full-scale invasion of Ukraine. Traditionally centered on trade, agriculture, and strategic industries, China’s engagement with Ukraine is part of a broader effort to reshape the international order and expand Beijing’s global influence. Since 2022, China has pursued a dual-track policy—maintaining close ties with Moscow while seeking to preserve relations with Europe—leading to increasing skepticism in Kyiv.
Ukraine’s early hopes for China’s constructive role in conflict resolution have faded amid evidence of economic and military support for Russia. Beijing’s diplomatic positioning, especially in multilateral forums, seeks to avoid direct criticism while appealing to the Global South. This reflects China’s competing interests: countering US dominance, encouraging European strategic autonomy, and bolstering its leadership claim among developing nations.
The report argues that China’s ambiguous stance complicates Ukraine’s foreign policy and post-war planning. Key recommendations include reducing dependence on Chinese investment, strengthening European partnerships, and ensuring European leadership in any future peacekeeping or reconstruction efforts. Understanding the “China factor” is essential for shaping a stable and balanced security environment in Ukraine and Europe
Exploring genetic alterations and protein marker expression in cancer: investigating ARID1A and SMARCA4 knock-outs in colorectal cancer and evaluating histological markers for targeted therapy in ovarian cancer
Full text linkThis dissertation presents two distinct yet connected research projects focused on the
alteration and evaluation of DNA and protein expression in common cancers, both of
which are subjects of ongoing research into their mechanisms and potential therapeutic
advancements. Colorectal cancer, one of the most prevalent and deadly cancers
globally involves metastasis and therapy resistance driven largely by tumour plasticity,
which is affected by changes in the expression of epigenetic modifying proteins.
Ovarian cancer, on the other hand, is common among women and exhibits various
histotypes that differ in their origin, tumour microenvironment, and genetic and
epigenetic expressions. Both cancers stand to benefit from further study to facilitate the
development of new treatments.
The first project focuses on producing SWI/SNF CRISPR/Cas9 knockouts in a mouse
colon organoid line (Apcfl/fl;KrasLSL-G12D/+;Trp53fl/fl). Knockouts were validated
using qPCR and Western blot analysis. Incomplete knockouts were produced,
indicating the need for further evaluation using DNA sequencing. Additional knockout
experiments are recommended, utilising the existing protocol with different clones
picked from the lentiviral transduction pool.
The second project involved the use of TMAs (tumour microarrays) taken from patients
with different ovarian cancer histotypes. These TMAs were sectioned and stained for
membrane proteins that are potential targets for antibody-drug conjugate (ADC)
therapy. The TMAs were then scored by two independent assessors for the expression
of these targets, providing a foundation for future studies exploring the suitability of ADC
treatments in different patient demographics and ovarian cancer histotypes, as well as
the role of target expression levels.
Though these two projects investigate different types of cancer, together they provide a
broad foundation in research methods and the diverse skills required for cancer
research. Both projects contribute valuable groundwork for future studies on the
mechanisms and treatment of colorectal and ovarian cancers
Towards open-source optimisation for bespoke cell-free protein synthesis systems
No full textCell-free protein synthesis (CFPS) enables transcription and translation outside the con-
fines of a cell. CFPS systems are based on either crude cell lysates, which contain most
of the soluble cellular machinery, or the PURE system, a minimal system reconstituted from
purified components. CFPS has been essential in molecular biology discovery, biosensing,
and bioproduction and is emerging as a useful tool in acquisition of large datasets via high-
throughput expression e.g. de novo protein design prototyping. It has the potential to play
a significant role in future biotechnology including in industrial biologics manufacturing and as the transcription translation (TXTL) chassis on which to base bottom-up synthetic cells capable of self-replication. However CFPS reactions exhibit lower titers than their in vivo
equivalents and still use expensive substrates limiting their current utility meaning for CFPS
to fully realise its potential, further system engineering is required to improve protein yields and enable the use of cost-effective substrates. Design of Experiments (DoE) is a powerful statistical method for efficiently designing and analysing candidate screens and optimisation experiments, where it can produce insights with relatively few experimental trials. However, its accessibility is hindered by the need for specialised knowledge and the cost of proprietary software.
This thesis presents work addressing these challenges. DoEasy, a free, open-source DoE
platform was developed and its efficacy proven via the optimisation of deterministic model. E. coli Lysate CFPS was established and the platform was applied to modulating key buffer concentrations of lysate reactions expressing different GFP-tagged proteins relevant to medicine and biotechnology. The PURE system was implemented and advances towards fine-grained optimisation of its components were made through the development of a multi-pot PURE configuration. The substrates of a de novo synthetic metabolism system were optimised resulting in a 60.2% improvement in yield. The platform was demonstrated in multiple
CFPS optimisations and democratises advanced statistical tools, making them accessible to a broader scientific community and enabling CFPS to reach its full potential
Exploring an online professional learning community for curriculum reform supported by school-university collaboration
No full textCurriculum reforms usually encounter conflicts between theory and practice, especially in foreign language education in China where policy borrowing is frequent. This longitudinal study focuses on a six-month online professional learning community (PLC) involving policymakers, primary school English teachers, and academics in China. The PLC is distinctive as an attempt to connect stakeholders in curriculum reforms, bridge the policy-theory-practice gap, and achieve sustainable resolutions. Through observations of PLC workshops and semi-structured interviews, this study elaborates on members’ trajectories of engagement in such PLC and how such PLC develops accordingly. Three key findings suggest the nature of the PLC:
1. Construction of professional learning (PL): The PLC was initially designed in a top-down way. However, self-adaptation enabled bottom-up developments. Top-down and bottom-up features mutually shaped each other, whilst jointly shaping self-adaptation. PL was constructed through this cyclical process in the PLC.
2. Affordances and constraints for sustainability: Three interrelated factors shaped the PLC sustainability: ownership (of the PLC), authenticity (of interactions in the PLC), and confidence and trust (in the PLC process and outcomes) jointly formed a context for sustainability.
3. Members’ points of view: Members consider the PLC as an attempt to connect policy, theory, and practice. Through dynamic identity construction (role switching), the PLC reshapes PL. The PLC has the potential to achieve sustainability, but there are challenges in transforming short-term gains into long-term ones.
The study contributes to understanding teacher-researcher-policymaker collaboration and fostering sustainable relationships against the background of curriculum reforms and other educational changes, which are relevant for researchers, teacher educators, policymakers, and school leaders
Philosophical paddling: reviving the aesthetic core of canoe journeying in nature and outdoor education
Full text linkIn this thesis, I build on the work of Scottish philosopher Ronald W. Hepburn to develop a pluralistic model of aesthetic education in the context of educational canoe journeys. I begin by integrating concepts and arguments from philosophical aesthetics into canoe journeying by exploring emergent metaphors of journeying drawn on in outdoor education literature. While acknowledging that there is limited conceptual analysis in the discourses of aesthetic education and outdoor education journeys, I integrate research to lay the groundwork for building new insights into this identified knowledge-practice gap.
I demonstrate that opportunities for reconsidering aesthetic education in nature and canoe journeys are plentiful. By adopting a philosophical viewpoint and recognising the significant impact of Ronald W. Hepburn on the aesthetics of nature, I develop an original application of his analyses into the context of educational journeying. Furthermore, I discuss how the continued narrowing of aesthetic education to the study of art or the philosophy of art is problematic in the context of the natural environment, as educators lack the sensible sounding language to describe the meaning of their own and their students’ experiences when journeying. Building on Hepburn’s legacy, I draw upon three further aesthetic philosophers and their models of aesthetics: Allen Carlson – Scientific Cognitivism; Yuriko Saito – Everyday Aesthetics; and Arnold Berleant – Experiential Aesthetics. Each model provides a unique set of aesthetic concepts, which, I argue, are especially beneficial for educators in their efforts to facilitate and guide aesthetic education experiences.
To develop the reader's awareness regarding the practical application of the concepts discussed in this thesis, I employ two novel approaches within its structure and style. Influenced by Mary Midgley, with her argument claiming that philosophy is like plumbing, I have sketched original illustrations of the philosophical concepts and their relationships to educational journeying for each model. Again, inspired by Midgley – and with the assistance of stylistic insights from Nussbaum – I undertake canoe journeys with each philosopher in fictive philosophical narratives. Hepburn, Carlson, Saito and Berleant become imagined canoe companions to enable me to address an essential question that Midgley posed: “Who is thinking when you’re thinking?”.
Although some aspects of these models are not entirely aligned, I argue that there are considerable conceptual tools within them that educators can utilise to cultivate aesthetic sensibilities and maximise aesthetic rewards for both themselves and their student paddlers. To better understand and practice educational journeys, I show the reader how aesthetic concepts and arguments can provide fertile ground for invigorating and growing opportunities for conceptual and practical work in this area. I conclude that a pluralistic model of aesthetic education in nature and journeying can facilitate better world-making by maximising aesthetic rewards in and after the journeys
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