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Synthesis of photocleavable supramolecular rotaxanes towards the controlled binding of nucleic acid three-way junctions
Get PDFThis work aimed to develop a supramolecular system which is responsive to an external trigger for the controlled formation of nucleic acid three-way junctions. Metallo-helicates capable of junction formation were modified with photocleavable capping groups and encapsulated in cucurbit[10]uril rings to form supramolecular rotaxanes. Removal of the capping groups by irradiation for the release of the metallo-helicate to trigger junction formation was investigated.
In Chapter 2, the structure of an imidazole-based nickel helicate is modified to include a photocleavable protecting group. Synthesis of a supramolecular rotaxane is then attempted via two different pathways. The photocleavage and junction binding of the nickel complexes are investigated by UV-visible spectroscopy, mass spectrometry, and polyacrylamide gel electrophoresis in Chapter 3.
Alternative designs for supramolecular rotaxanes are detailed in Chapters 4 and 5 where the terminal imidazole rings of the helicates investigated in Chapters 2 and 3 are exchanged for 4- and 5-hydroxypyridines. The formation of supramolecular rotaxanes from the 4- and 5-hydroxypyridine based helicates with photocleavable capping groups is then investigated. Chapter 5 also includes the synthesis of a 3- hydroxypyridine based metal helicate designed to permit the control of junction binding activity without encapsulation in the cucurbit[10]uril macrocycle.
The removal of the capping groups from the hydroxypyiridne-based complexes in response to irradiation is then explored by UV-visible spectroscopy and mass spectrometry in Chapter 6. The formation of nucleic acid three-way junctions by the 5- hydroxypyridine based complexes is then investigated by polyacrylamide gel electrophoresis
Investigating the impact of single bouts of exercise on the number and function of peripheral blood immune cells
Get PDFSingle bouts of exercise elicit changes to the number of immune cells within the bloodstream of humans. The initial transient increase in cell types including haemopoietic and progenitor cells (HSPCs) may offer relevance in the context of peripheral blood stem cell donations, which are used to collect HSPCs to treat various haematological disorders. The composition of the collected HSPC-rich immune graft is equally important, with higher numbers of cytolytic natural killer (NK) cells predicting better health outcomes in patients, and strikingly, these cells are mobilised up to 10fold during exercise. Mobilisation of immune cells during exercise forms part of a dynamic response in recovery, that may drive immune remodelling when repeated over time. The mechanisms underpinning this are not well understood, but recent work has started to examine bioenergetic responses within the total peripheral blood mononuclear cell (PBMC) fraction. PBMCs are a highly heterogeneous population of T and B lymphocytes and monocytes, each with various subsets with distinct bioenergetic profiles. Given that single bouts of exercise evoke marked compositional changes to PBMCs, providing single cell resolution of immunometabolic changes is crucial to facilitate understanding of the acute immune response to exercise.
Chapter 2 investigated differences in HSPC and cytolytic NK cell concentrations during and after continuous and interval-based cycling bouts utilising high intensity interval exercise (HIIE). We demonstrated that 2 x 2-minute bouts of HIIE (90-95% HRmax ) were sufficient to increase peripheral blood HSPC concentrations above rest, but not in response to 30 minutes of continuous cycling at moderate intensity. Furthermore, peripheral blood was enriched with higher numbers of cytolytic NK cells following bouts of high and low volume HIIE, compared to moderate intensity cycling. These data indicate that a very low volume of cycling at a high intensity can potentiate peripheral blood HSPC concentrations and provides a rationale for exploring whether intermittent cycling during PBSC collections can increase the yield of HSPCs for transplant.
Chapter 3 developed methods using extracellular flux analysis to examine the metabolic phenotype and real time responses to ex vivo activation of enriched naïve Helper and Cytotoxic T cells vs. PBMCs. Conditions were optimised for nutrient assay conditions (i.e., glucose and glutamine concentrations) and blood collection procedures (anticoagulant and blood ‘sitting’ time) to provide a robust experimental model for chapter 4 of this thesis.
Chapter 4 utilised an innovative human study design and methodology to investigate the impact of prolonged moderate intensity cycling on the energy metabolism of naïve T cells. This study provided detailed analysis at the single cell level for both naïve CD4+ and CD8 + T cell subsets. This chapter showed that a 2-hour session of cycling at a moderate intensity (define by a power output that reaches 95% of the lactate threshold1) did not cause any changes in the metabolic profiles of naïve CD4 + and CD8 + T cells, or peripheral blood mononuclear cells (PBMCs), immediately after or 2 hours into recovery, compared to rest. This included comprehensive measures of mitochondrial respiration, glycolytic flux, and ATP synthesis rates. Furthermore, there were no differences in the bioenergetic responses to ex vivo activation of PBMCs or enriched naïve T cells based on sampling before, immediately after or 2 hours following cycling completion. These data indicate that the metabolism of PBMCs and naïve T cells are unaltered within 2 hours of prolonged moderate intensity cycling.
Overall, this thesis presents novel data on acute changes in circulating immune cell number and function within peripheral blood
Development of autologous macrophage-encapsulated gellan fluid gel for the prevention of blindness from ocular surface scarring
Get PDFBackground: Corneal scarring is the fourth leading cause of preventable visual loss. Following infection /injury, the cornea undergoes a repair mechanism involving a strong inflammatory response, repair and degradation mechanisms. This results in an alteration of its molecular components and structure, which could potentially lead to scar formation, and ultimately cause blindness. Corneal transplantation is the standard treatment for corneal scarring, which has donors’ shortage and a rejection risk. Thus, the development of a novel treatment is significantly required for maintaining the intact corneal structure. Our group using a novel gellan fluid gel to deliver a single anti-scarring molecule in a sustained manner into the effected cornea showed promising findings. Thus, we proposed using gellan fluid gel to allow sustained release of multiple molecules instead of one to limit inflammation and promote corneal tissue remodelling while minimising fibrosis, which would lead to regenerative healing of the cornea and untimely maintenance of vision. Macrophages are critical players during wound healing repones, meditating infection elimination, damaged cell clearance, resolution of inflammation, and tissue remodelling. Thus, we proposed that administration of a balance of M1 and M2 macrophages at the right time on the ocular surface using gellan fluid gel as an effective delivery system may lead to regenerative corneal wound healing and eventual preservation of sight. The present project aimed to develop autologous macrophage-encapsulated gellan fluid gel for delivering bioactive molecules that aid re-epithelialisation, while minimising fibrotic signalling cascades to aid scarless healing of the ocular surface and the preservation of sight.
Method: Human monocyte derived macrophages with pro-inflammatory (M1), anti-inflammatory (M2), and pro-healing (M2) properties were generated using GM-CSF, LPS+IFN-, and M-CSF, IL-4+IL-13, respectively. The viability of M1/M2 in gellan fluid gel and its effect on macrophage phenotype at 4h were assessed by fluorescent microscopy (FM), and Flow cytometry and qPCR, respectively. To test macrophage polarisation states, M1/M2 were cultured with opposing cytokines, i.e., M1 with IL-4/13, and analysed by RNAseq and Luminex.
Result: The gellan fluid gel maintained both M1/M2 survival and polarisation states. However, the polarisation states of our generated subset of M1/M2 macrophages are not maintained in response to sequential stimulation with positing stimuli.
In conclusion: these results show that gellan fluid gel is biocompatible and can be used to deliver autologous macrophage therapy into the cornea. The instability of the M1/M2 polarisation states necessitated caution when applying them to the cornea with an unstable microenvironment
Transcription start site usage in cancer heterogeneity
Get PDFResolving intra tumour heterogeneity (ITH) remains an extensive therapeutic challenge in cancer research. Despite an emphasis on the importance of the transcriptional landscape in understanding cancer cell behaviours, the role of the core promoter remains widely ignored. Recent studies have shown the presence of alternative transcription start sites (TSS) and found a distinctive YC TSS enrichment in cancers that correlated with global phenotypes. However, tumour dynamics are underpinned by a complex network of sub clonal diversity therefore, the true biological significance of TSS usage remains elusive. Here we further characterise the phenotypic landscape of YC enriched cancers on both an inter and intra level. Through successful undertaking of single cell CAGE sequencing, we identify ITH of TSS usage even at a single cell level. We find differential YC enrichment associated with cellular physiological states that facilitate a growth phenotype. Next, we identify cell cycle phase associated TSS shifting and find YC enrichment associated with G0 and G1 phases. Scrutinisation of genes utilising YC enrichment showed subsets critically involved in directing the metabolic state of the cell. Overall, our findings highlight an important TSS association to cancer cell growth dynamics that act in a cell cycle dependent manner, even at a single cell resolution. Finally, attempts to characterise the spatiotemporal mapping of cycling phases through imaging emphasised the dynamic landscape of cancer behaviours. The work presented here further highlights the importance of TSS usage investigations that are masked in conventional gene expression analysis
Developments to established dose-finding methodologies for application in trials with complex and innovative designs
Get PDFThe results and decisions made in early phase clinical trials play an important role in the drug development process. Failure to make correct or accurate decisions may result in further unwarranted clinical research and may also cause potential future harm to patients.
This thesis explores methodologies used in dose-finding trials, specifically looking at the implementation of these methods and how they can be extended to answer different questions and assist with decision-making.
Firstly we detail our experiences implementing a novel methodology for a dose-finding trial where monotonic ordering is not possible. We then present an extension to a seamless adaptive phase I/II trial design which aims to conduct dose-finding and replace the need for a randomised phase II trial.
We also present two extensions to dose transition pathways, a visualisation tool to aid designing and decision making in early phase trials. The first looks at how they can be applied to more complex time-to-event methodologies and the second looks at how the same concept can be applied to single-arm phase II trials.
Throughout this thesis we include exemplar trials and showcase our methods being motivated or implemented into clinical trials conducted by the Cancer Research UK Clinical Trials Unit at the University of Birmingham
A linguistic and discursive analysis of the humour in Arabic novels written in vernacular: examples of e-Arabic genre
Get PDFThe primary objective of this research is to analyse the linguistic aspects of humour and satire in a new literary genre, known as e-Arabic literature. The research focuses on the emergence of this distinctive literary genre, emerging from Information Technology (IT) use in what is recognised as Computer-Mediated Communication (CMC) and how it has been impacting Arab culture in general and literary scene, in particular. Satire and humour are one specificity of this genre, and this research aims to situate this new genre as one of the subfields of humour in Arabic literature. This study makes a contribution to theories about humour and adds e-Arabic literature, as a new genre which belongs to the global area of CMC and highlight a new variety in Arabic, known as e-Arabic. Furthermore, this study clarifies and analyses the development of the satirical writing style and the use of humour as means by which Arab writers cultivate an awareness of social and political issues within their cultures. In addition, it examines the significance and function of rhetorical questions, interrogative phrases, and punctuation marks in constructing a humorous and satirical writing style that is both original and creative.
The current study seeks to investigate the utilisation of satire in e-Arabic literature, as a genre that uses a hybrid language and mixes in styles to create humorous effects, adding to Arabic literature a new variety. Moreover, this research reveals the underlying factors that contribute to the extensive popularity of political satire in e-Arabic literature and its impact on societal transformation. The predominant Arabic literature in this study originates from authors hailing from Saudi Arabia, Egypt, and Syria. The data for this research is collected from novels that exemplify the e-Arabic genre. These novels are written in several Arabic regional dialects, such as Egyptian, Najdi, Hijazi and Syrian. Due to the great dissemination of Egyptian media, the Egyptian dialect has emerged as the most prevalent used vernacular. The data collected revealed that Arabic novels written in vernacular language have ample evidence of the utilisation of satirical and humorous discourse. A thorough understanding of comedy’s intended purpose serves as the driving force behind this. The humour in each piece is associated with a criticism of religious institutions, politics, and political systems. The writers of these novels utilise many linguistic features, including lexical aspects, and purposefully depart from traditional norms in order to educate readers. The data demonstrates the excessive use of linguistic tactics based on juxtaposition, contradiction, and implausibility as linguistic devices to generate humour and construct a satirical framework for criticism
‘Natural piety’ and sentiment: children, landscape and religion in the paintings of William Collins, R.A. (1787-1847)
Get PDFVery little has been written about the work of the painter William Collins, R.A. (1787-1847) despite the fact that his contemporaries considered him to be one of the leading artists of his time. Most of his paintings are unknown to modern scholarship, with the result that an assessment of his significance for nineteenth-century British art has never been properly undertaken. Whilst a number of his contemporaries, such as David Wilkie, William Mulready, Charles Lock Eastlake, William Dyce and John Rogers Herbert have been the subject of detailed examination, and their contributions recognised, Collins has not.
This thesis fills that gap in art historical scholarship of the period, first by identifying as many of Collins’s paintings as possible, and then by undertaking a close reading and detailed visual analysis of them. It establishes the links between his painting and the social, literary and religious cross-currents of his time, and demonstrates that Collins was actively involved in the fields of genre, landscape and coastal painting, and that in all these fields he gave children an unusual agency. Between 1838 and 1843, during the first phase of the Oxford Movement, he produced a series of paintings which reflected major Tractarian pre-occupations. During that same period, towards the end of his life, he painted a number of biblical subjects which demonstrate that he was at the forefront of attempts to establish a distinct Protestant approach to religious art. Many features found in his approach were adopted a few years later by members of the Pre-Raphaelite Brotherhood who knew the Collins family well.
I conclude that the currently understood histories of nineteenth-century British art need re-evaluation, so as to include Collins’s significant contributions to landscape painting, the portrayal of children, paintings of the coast, and religious art of the period. I also make the case for regarding Collins as a Pre-Raphaelite precursor
An investigation into invaginated membrane system development, dynamics, and actin regulation throughout megakaryopoiesis
Get PDFThrombocytopenia is a condition defined by low blood platelet count, often linked to defects in platelet synthesis. Megakaryocytes are the precursors to platelets that undergo endomitosis and grow to up to 64n ploidy in the bone marrow. In the later stages of megakaryocyte development, a reservoir of membrane mass known as the invaginated membrane system (IMS) accumulates within megakaryocyte cytoplasm. Importantly, structural defects in the IMS can be seen in a range of conditions characterised by thrombocytopenia (, and ), linking defects in IMS maturation to a low platelet count. Furthermore, many thrombocytopenic conditions are caused by mutations in proteins linked to F-actin regulation , , , ), whilst chemical inhibition of actin cytoskeleton during megakaryopoiesis leads to aberrated or halted IMS development, highlighting actin cytoskeleton as an essential component for functional IMS maturation.
During development, the IMS transverses through several distinctive morphological stages. Whilst IMS development has been broadly categorised: , , and , there is a lack of definition surrounding IMS morphologies, or how IMS transitions through those morphologies during development. This study uses a range of microscopy techniques and bespoke image analysis pipelines to measure 4D IMS morphology in large populations of megakaryocytes. Here, we categorise megakaryocytes into 7 different stages of development: , , , , , , and using qualitative morphometric data, and probe F-actin regulation of IMS maturation. Our data shows that:
• Megakaryocytes transition through polarisation of the IMS prior to proplatelet formation.
• A network of underlying invaginated tubules which prelude IMS compression and expansion.
• K-means cluster analysis can be used to perform unlabelled classification of morphometric datasets, and define rates of IMS maturation.
Furthermore, we provide evidence for:
• An Arp2/3 regulated actin cytoskeleton during IMS maturation
• An F-actin regulated polarisation and expulsion of the IMS
Loved to death: an Anabaptist critique of Christendom Christology through the narrative themes of kenosis and death in the Gospel of Mark
Get PDFThis thesis expands the Anabaptist critique of Christendom Christology by examining the narrative motifs of kenosis and death in the Gospel of Mark.
Within their respective literary documents, modern Anabaptism, early Anabaptism, and Christendom Christology all present opposing Christological views related to God’s ability to change, the constitution of humanity, and the definition of death. One view exemplified in the Tome of Pope Leo and the Council of Chalcedon promotes the belief that God is necessarily immutable, humans are body/soul composites, and death is the separation of body and soul (a dualist view). An alternative view within Christendom and Anabaptist literature holds that God is kenotic, humans do not have souls, and death is the decomposition of a corpse (a physicalist view).
Historically, Anabaptism has critiqued Christendom by implementing a Christocentric narrative approach to developing theology. Working from within the Anabaptist tradition, this thesis applies a narrative critical examination of the Gospel of Mark to determine whether a dualist or physicalist view is most faithful to the story the author of Mark presents to his audience. To aid in this analysis, I adopt the strategic approach of narrative critic Peter Bolt, who suggests that knowledge of the first-century audience’s cultural mind provides helpful insight into the practice of narrative criticism. Using Bolt’s approach, I suggest that certain rhetorical, socio-religious, and linguistic knowledge of the first-century audience’s cultural minds proves useful in constructing a robust narrative analysis of Mark’s anthropology, thanatology, and eschatology.
I begin my narrative criticism by examining the narrative motif of death in Mark’s Gospel. From this analysis, I conclude that the author of Mark intends for his audience to embrace a physicalist understanding of death. Next, I investigate the theme of kenosis within Mark’s Gospel. From this analysis, I conclude that the author of Mark seeks to persuade his audience that Jesus was a kenotic messiah. Finally, I examine pericopes in Mark’s Gospel that combine the themes of kenosis and death. From these texts, I conclude that the author of Mark intended to convince his audience that Jesus was a kenotic mortal messiah.
In response to my conclusion that Mark desired his audience to view Jesus as a kenotic mortal messiah, I argue that the Anabaptist stream of Christology that has embraced the dualist view found in Christendom Christology should be rejected. In its place, I attempt to construction an Anabaptist Christology that embraces Mark’s portrayal of Jesus as a kenotic mortal messiah. I contend that this definition of Jesus has significant implications for how Anabaptists think about God, Jesus’ death, and human death
Demystifying emotion-processing: autism, alexithymia, and the underlying psychological mechanisms
Get PDFDespite extensive research, the mechanisms underpinning successful emotion recognition remain unclear. Constructionist, template-matching, and signal detection theories illuminate several emotion-related psychological processes that may be involved – namely the conceptualisation, experience, visual representation, and production of emotion – however, this requires empirical verification. Therefore, across the six empirical chapters described here, I developed and applied several novel experimental paradigms to assess the way in which individuals conceptualise, experience, visualise, produce and recognise emotion, and created new mathematically plausible, mechanistic models that shed light on the processes involved in emotion recognition. In doing so, I identified several candidate mechanisms that may underpin the emotion recognition difficulties seen in a range of clinical conditions, including autism spectrum disorder, and I (1) determined whether there are differences between autistic and non- autistic individuals in these emotion-related psychological processes, and (2) ascertained whether differences therein underpin emotion recognition challenges for autistic people.
Ten years ago, it was theorised that the emotion-related difficulties of autistic individuals do not stem from autism per se, but rather alexithymia – a subclinical condition highly prevalent in the autistic population characterised by difficulties identifying and describing emotions. Since its inception, this theory has gained empirical support, with multiple studies documenting that alexithymia, and not autism, is associated with emotion- processing differences. However, to date, this evidence has largely been confined to the domain of emotion recognition. As such, it is unclear whether there are differences between autistic and non-autistic individuals in the conceptualisation, experience, visual representation, and production of emotion, after controlling for alexithymia. Here, I resolved this ambiguity, discerning the explanatory scope of the “alexithymia hypothesis”: there were no differences between autistic and non-autistic individuals in the understanding or differentiation of emotion concepts (Chapter 6), the precision or differentiation of emotional experiences (Chapter 6), and the speed (Chapter 3) or differentiation of visual emotion representations (Chapter 5), after controlling for alexithymia. Nevertheless, there were differences between groups with respect to the precision of visual representations (Chapter 5), the production of emotional facial expressions (Chapter 7), and recognition of specific emotions (Chapter 2), even after accounting for this confound.
Despite suggestions that autistic individuals adopt alternative strategies to recognise the emotions of others, very few studies have examined mechanistic differences in emotion recognition between autistic and non-autistic people. Therefore, here I aimed to compare the processes involved in emotion recognition for these groups. Across multiple empirical chapters, I identified that there are similarities and differences in the processes implicated in emotion recognition for autistic and non-autistic people (Chapters 4, 5, 6, and 7), with autistic individuals relying on fewer emotion-related psychological processes. By elucidating several candidate mechanisms underpinning superior emotion recognition, my doctoral work paves the way for future supportive interventions to help both autistic and non-autistic individuals to accurately interpret other people’s emotions, thus ultimately fostering more successful and fluid social interactions
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