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Ontology through the lenses of mathematics: mathematical intermediates in Plato (Meno, Phaedo, Republic)
Get PDFAristotle’s report in Metaphysics A.6, 987b14-18, says that, apart from the Forms and the sensible things, Plato posited a third kind of entity: the objects of mathematics or mathematicals (τὰ μαθηματικά). This latter entity would share properties with the former two, but not fully belonging to either of them, belonging to a place in between Forms and sensible particulars. Hence its name: intermediates. This report generated an important controversy for ancient philosophy scholars, since nowhere in Plato’s dialogues it is explicitly stated that he advocated for this third kind of entity. Most efforts to make sense of this report have been based on an exegesis of Plato’s works, looking for hints that would indicate that he advocated for objects of mathematics, or, conversely, to dismiss Aristotle’s report as spurious. My proposal here is to freshen this discussion by focusing on Greek mathematics and the way it worked. Indeed, most of the literature that deals with intermediates tends to focus on Plato’s ontology, but lacks a more detailed account of the mathematical side of things. Moreover, Plato’s Academy was one of the main places in Antiquity to pursue the study of mathematics. My contention is that, by looking at Plato’s dialogues through the lenses of mathematics (i.e. by having a clear account of how it worked and what makes it different from the mathematics we nowadays use), we would be able to find new insights in Plato’s mathematical passages in the middle dialogues. The upshot of this analysis is that Plato not only posited these mathematicals, but he also incorporated this notion of third kind of entity into his account of the soul in the Phaedo and the ideal city in the Republic
The role of the IL-6/STAT3 pathway in invasive lobular carcinoma
No full textInvasive lobular carcinoma (ILC) is the second most common subtype of breast cancer (BC)
after invasive ductal carcinoma (IDC) and accounts for 15% of BC cases. ILC is
characterised by loss of E-cadherin, slow and discohesive invasive growth, with a unique
pattern of metastatic sites which includes the gastrointestinal tract, peritoneum, and ovaries.
90% of ILC cases are estrogen receptor (ER) positive and are considered ideal candidates for
endocrine therapies (ET), including letrozole and tamoxifen. Initially, ILC patients respond
well to ET but eventually develop resistance. Retrospective analysis has shown poor
outcomes in ILC compared to IDC. Therefore, it is crucial to comprehend the mechanisms of
resistance to ETs to enhance the care and outcomes of ILC patients.
Interleukin-6 (IL-6) has been identified as a cancer-associated fibroblast (CAF) secreted
factor in human ILC cells driving the phosphorylation and translocation of Signal Transducer and Activator of Transcription 3 (STAT3) into the nucleus where it acts as a transcription factor. The IL-6/STAT3 signalling pathway has been associated with resistance to ET in BC cell lines and patients via multiple mechanisms including signalling from the tumour microenvironment (TME). We initially characterised this pathway in a mouse model of ILC, where significant expression of Il6 was found in mouse CAFs, with no measurable expression in the mouse epithelial tumour cells. CAF-conditioned media (CM) and recombinant IL-6 stimulation of mouse epithelial cells resulted in phosphorylation of STAT3 confirming the paracrine activation of STAT3. Additionally, the effect of IL-6 on mouse ILC cell proliferation, migration, and invasion was assessed in vitro, where we observed that both CAF CM and recombinant IL-6 resulted in a decreased proliferation rate of ILC tumour cells compared to the control. Recombinant IL-6 stimulation resulted in increased migration and invasion of ILC cells.
RNA sequencing analysis of human ILC cell lines SUM44PE, MDA-MB-134VI, and two
patient-derived xenografts, HCI-013 and HCI-018, following stimulation with recombinant
IL-6 showed downregulation of ER expression and suppression of generic and ILC-specific
ERα-dependent gene sets, as well as upregulating a subset of letrozole and tamoxifen
resistance genes. We generated a 60-gene IL-6 dependent gene signature and found a
significant negative correlation between ESR1 expression and expression of IL6 and the IL-6
dependent gene signature in ER+ ILC and IDC tumours in the TCGA dataset. To further
investigate and understand the clinical significance of IL-6/STAT3 pathway, we carried out
immunohistochemical (IHC) analysis on 246 primary operable ILC (Glasgow cohort) BC
patients for alpha-smooth muscle actin (αSMA), STAT3, pSTAT3, and the STAT3 target
gene, BCL3. The analysis highlighted the significant association of αSMA and cytoplasmic
STAT3 with disease-free survival as well as other clinicopathological features of the patients.
This project also aimed to investigate the effect of Letrozole treatment in ILC. IHC analysis
in 32 ILC patients (Edinburgh cohort) who were treated with letrozole, and their matched
primary tumour sections, for STAT3, pSTAT3, and BCL3 revealed a differential pattern of
expression of nuclear STAT3 and pSTAT3 upon letrozole treatment. NanoString GeoMax
Digital Spatial Proteome Profiling of the Edinburgh cohort revealed a distinct proteome in
stromal and tumour compartments. Letrozole treated patients showed a reduction in
proliferation, PI3K-AKT and MAPK pathway-related proteins in tumour cells and a subset of
immune cell markers in the stromal compartment compared to their baseline biopsies. These findings suggest that letrozole may not only inhibit tumour growth directly by reducing estrogen levels, but also alter the tumour microenvironment.
To conclude, this work provides valuable insights into the importance of tumour-stromal IL-6 signalling in BC and suggests that inhibiting IL-6/STAT3 signalling could represent a
promising therapeutic strategy for investigation in BC
Neural mechanisms of dance communication in honeybees
No full textThis thesis brings a new approach to the study of honeybee dance communication: evaluating computational models against observed behaviour to understand how dance communication could be implemented by the insect brain. After a foraging trip, a bee has internal knowledge of the flight vector to the food which it communicates by producing a stereotyped motor pattern known as the waggle dance. This creates mechanical cues that surrounding nestmates can assimilate to obtain their own flight vector. Independently of communication, the acquisition and utilisation of vectors for navigation is believed to occur in the central complex in the insect brain. Our key hypothesis is that this circuit is sufficient, with minor adaptations, to explain both the production and assimilation of dance information.
First, we propose how an anatomically grounded model of path integration for foraging, based on the central complex, could be adapted to produce the dance in the hive. We assume the existence of vector memory, where a snapshot of the bee's path integrator state at the food source can later be utilised by the steering circuitry to guide a return journey. We also impose a parameter to limit the angular velocity of the bee when waggling based on properties of natural dances. By simulating neural activity for foraging routes, we demonstrate that natural features emerge from the subsequent dancing behaviour produced by the circuit, including waggle and return phases. The simulated dances also exhibit patterns of angular scatter that align with those observed in real bees for different feeder distances. Our results suggest that performance of the dance could arise from a pre-existing neural circuit that underlies large-scale navigation and supports the idea that the dance is a miniature re-enactment of the foraging flight.
We then explore how this circuit could be used by the receivers of information: nestmates following the dance. We present a new dataset of nestmates as they follow a dance and uncover a previously unreported feature of their antennal positioning that correlates to their relative angle to the dancer. Knowing its own orientation to gravity on the vertical comb, this could allow followers to deduce the dancer's orientation, which indicates the direction of the food relative to the sun. Integrating the estimates of this direction during the waggle phase could then enable the follower to obtain a vector to the food. Based on recent evidence of antennal inputs and spatial encoding in the central complex of the fruit fly, we propose how the circuit could be adapted to use the antennal information to perform these computations and recover the signalled dance vector. Using the real positional data of the followers as input, the simulated circuit predicts that their recovered vectors would be appropriately centred on the signalled direction. It also predicts the distribution to be up to ±90° wide from this direction.
To follow up this result, we devise an experiment to compare these predictions with the real flight vectors expressed by bees. Inspired by the forced-detour paradigm in ants, we track the correction angle of bees navigating to a feeder after an imposed detour, as a measure of the estimated food location. Similar to the circuit's predictions, we observe a characteristic spread of vectors centred on the feeder. Our work thus indicates that the central complex could underlie the encoding, decoding and expression of spatial information in dance communication
Visualising prison violence: exploring the micro-dynamics of violence in men's prisons using video data
Get PDFThe present thesis presents an exploratory study in the field of prison violence. Until recently, most research on physical violence, both in and outside of prison, has been concerned with background and causal factors as well as social and structural contexts, leaving out the actual moments of violent action. A novel research trend, Visual Data Analysis (VDA), emerging from one of the major theories of violence of recent times (Collins, 2008), draws on photos and video footage to shift the focus towards interactions and behaviour observable in violent incidents. The VDA method allows for studying the micro-dynamics of physical violence by means of video-footage. Employing CCTV and mobile device-footage of violent incidents from YouTube as empirical material in combination with focus group interviews with current prison officers and ex-prisoners, the thesis contributes to the micro-sociology of violence, particularly prison violence, by examining the strengths and limitations of video data sampled online - as a general method as well as in the specific context of prison violence research - thus addressing a gap in the field of VDA research. At the same time, it tests the applicability of Collins’ theory of violence to the prison setting, i.e., to a non-public context to which it has not previously been applied.
By analysing interactional processes and expressions of emotion and discussing interpretations of these with individuals familiar with the prison setting, the thesis finds that audiences in prisoner-on-prisoner fights regulate both the severity and the course of the fights; where (co-prisoner) audiences exhibit enthusiasm through cheers and encouragements, fights are prolonged, a finding which echoes Collins’ (2008) observations of audience impact. The analysis further suggests that prison officers who exhibit anger or aggression through shouting, giving orders, or by invading prisoners' personal space provoke prisoners to exhibit similar behaviours or to use violence against the officers. Lastly, the study also shows that officers who exhibit fear through struggles to control bodily movements allow prisoners to take charge of the course of interaction, i.e., to gain or maintain emotional dominance, and to use violence, a dynamic resonating with Collins’ (2008) notion of how violent interactions are initiated. Based on the findings, the thesis is able to present implications of technical features for future video research drawing on online-sampled data, thus contributing to a furthering of the VDA method. The findings have also laid a foundation for (indicative) practical suggestions for prison officer training based on both Collins’ micro-dynamic approach and the VDA method. This highlights the potential of using video recordings of real-life incidents in the design of this type of training. The thesis concludes that Collins’ theory holds promising potential in furthering the understanding of the dynamics of (especially) escalation and de-escalation of potentially violent encounters – with or without the presence of audiences - between prisoners and between officers and prisoners, and thus for future development in the field of violence prevention and intervention in the prison context
The impact of soothing videos on the generation and content of prospective mental imagery
Get PDFIn recent years, there has been a growing interest in prospective mental imagery (PMI) and its effects on individuals. Research indicates that positive PMI cannot only improve but also stabilise the mood of those suffering from depression and other mental health conditions. It has been established that those who suffer from mood and anxiety disorders have difficulty producing a positive PMI and have a higher tendency to produce a negative PMI. However, few studies have explored the content of PMI and alternatives (non-clinical methods) to access positive PMI. As an expansion of Project Soothe's findings and recent literature connecting nature and mental imagery, this thesis explores the effect of soothing videos on the generation and content of Prospective Mental Imagery. The first study (Chapter 4) was a systematic literature review that investigated the content, and appraisals of mental imagery of both past and future mental imagery across mood and anxiety disorders. The findings indicate that mental imagery content surrounds specific topics, such as interpersonal relationships, and crossing one’s boundaries. The appraisals are subjective and context specific. Chapter 5 (Study 2) explored the subjective experiences and themes of PMI content,and the subjective experiences of using nature videos to produce PMI scenarios. In the first part of the study, the study found that PMI content can be positive (as a motivational tool) and negative (providing a feeling of not being there yet). Furthermore, common PMI content themes are typically related to interpersonal interactions (including spending time with family and friends) and accomplishing milestones, both personally (e.g. having a family) and professionally (e.g. reaching the next level on the career ladder).
Regarding the second part of the study, participants reported that it was easier and more vivid to use a visual cue to produce PMI. As an extension of Chapter 5, Chapter 6 (Study 3) explored the connection between episodic memory and PMI content. Chapter 6’sstudy concluded that there was no statistically significant relationship between the affective descriptions of PMI and EM. Furthermore, the study concluded that there were no statistically significant differences between the soothing categories and perceived level of soothe experienced by participants. Chapter 7 (Study 4) explored the effect of soothing videos displayed on different platforms (VR or Desktop) on the generation of PMI. Overall, the results demonstrated a higher PMI vividness when stimuli (both non-soothe and soothe) were presented via a desktop. In addition to, no statistically significant differences were found between the percentages of positive words when comparing the conditions or platforms. Furthermore, the results show that it was easier to produce non-cued PMI in VR, meanwhile, it was easier to produce visual cued PMI in desktop.
These pilot findings suggest that VR has the potential to assist in the production of non-cued PMI and contribute to our overall understanding of PMI content and its affective effect
High resolution multispectral estimation of sea surface salinity and temperature in coastal areas
Get PDFSea surface temperature (SST) and sea surface salinity (SSS) are essential climate variables
(ECVs) that serve as critical indicators of ocean health, influencing global climate dynamics
and ecological processes in coastal environments. Coastal regions, encompassing nearshore
waters and zones affected by riverine discharge, upwelling, and sediment transport, pose unique
monitoring challenges due to their dynamic nature and spatial heterogeneity. This thesis aims
to advance the accurate estimation of SST and SSS in coastal areas using high-resolution ocean
colour satellite imagery, addressing three core research questions:
(i) understanding the relationships between absorption and scattering coefficients of water
column constituents and SST and SSS,
(ii) how these relationships can be used together with machine learning to estimate SST and
SSS from ocean colour satellite imagery, and
(iii) whether image segmentation of satellite images can improve these estimation models
through the inclusion of spatial information.
Chapter 3 establishes foundational links through a comprehensive analysis of hyperspectral
radiance data from Patagonia, informing how inherent optical properties relate to SST and
SSS variations. Water-leaving radiance is linked to the inherent optical properties of the water
column, affected by constituent parts such as coloured dissolved organic matter, chlorophyll,
and total suspended matter. These constituents influence the backscatter, absorption, and
reflectance coefficients, thereby affecting the spectral signature of the radiance. Temporally and
spatially matched water data of SSS and SST with hyperspectral data at ground level is used
to train linear regression and neural network models to predict SST and SSS, achieving a root
mean square error (RMSE) of 0.2 PSU and 0.1 °C respectively. Principal Component Analysis
(PCA) and feature selection are also explored to understand the spectral bands contributing
most strongly to salinity and temperature values.
Building on this groundwork, Chapter 4 uses multispectral satellite data and employs machine
learning algorithms to mitigate nonlinearities and atmospheric effects, enhancing spatial and
temporal resolution to capture coastal variability accurately. A global neural network model
trained with in-situ and multispectral Sentinel-2 satellite data achieves an R2 of 0.83 for temperature
and 0.65 for salinity. In a case study of the Gulf of Mexico, the RMSE for temperature was 0.83°C for
test cases and 1.69°C for validation, outperforming previous methods in dynamic coastal
environments. Feature importance is analyzed using Shapley values, revealing key spectral
features influencing SST and SSS estimation. Near-infrared bands play a crucial role in
SST prediction, while blue/green colour bands are more influential for SSS. This approach addresses
the ”black box” nature of machine learning models, providing insights into the relative
importance of spectral bands.
Chapter 5 introduces an innovative methodology using unsupervised clustering to classify ocean
colour images by water types, integrating these classifications into SST and SSS estimation
models to improve predictive accuracy and facilitate ecological monitoring. Two methodologies
are explored: clustering based solely on spectral radiances and clustering applied directly to
satellite images. The former improves model generalisation by identifying similar water clusters
across different locations, reducing location dependency and enhancing regression model performance,
improving a global ocean colour SST regression model RMSE error by 10%. The latter
approach, incorporating spatial information, enables the identification of front boundaries and
gradient information, improving global SST models RMSE by 20% and SSS models by 30%
compared to initial ocean colour models. Beyond improving algorithm performance, optical
water classification can monitor and interpret changes to water optics, such as algal blooms
and sediment disturbances caused by climate change or anthropogenic activities. For example,
clusters have been used to show the impact of a category 4 hurricane landfall on the Mississippi
estuarine region.
This thesis demonstrates the potential of integrating high-resolution ocean colour satellite imagery,
machine learning, and unsupervised clustering to enhance the estimation of SST and SSS
in coastal areas, thereby contributing to a more comprehensive understanding of ocean health
and its implications for global climate and ecological processes
Investigating IFITM1 signalling and regulation in cancer
No full textInterferons (IFNs) are a family of pleiotropic cytokines with widespread
physiological roles. Despite their well-characterised antitumour applications,
prolonged IFN signalling induces gene programmes which favour therapy
resistance and tumour progression. Interferon induced transmembrane
proteins 1 and 3 (IFITM1 and IFITM3) are thought to be part of this IFN protumorigenic
programme. Specifically, IFITM1 is a core gene in an interferon
related DNA damage signature (IRDS). However, the mechanisms of IFITM
induced pro-tumour effects are not clear. Here, phenotypic studies of
CRISPR/Cas9 mediated IFITM1 Knockout cells showed an impaired capacity
to form colonies an increased radiation sensitivity in these cells. These
differences were related to an impaired regulation of the formation of focal cell
adhesions upon IFITM1 deletion. Importantly, the effect seeing in IFITM1
knockout cells was significantly more pronounced than that seen upon IFITM3
deletion. Liquid Chromatography-Mass Spectrometry (LC-MS) was performed
to obtain a comparative analysis of the proteome of Wild-Type SiHa and
IFITM1 K/0 cells. This analysis revealed a significant downregulation of
multiple genes related to cell adhesion and regulation of cytoskeleton
dynamics in IFITM1 K/0 cells compared to Wild-Type counterparts confirming
the role of IFITM1 in the regulation of cell adhesion. Both IFITM1 and IFITM3
seemed to contribute to cell adhesion in the data shown here and previous
work in our group revealed a tumour cell specific co-localisation of IFITM1 and
IFITM3 upon IFN stimulation. This suggests that IFITM1 and IFITM3 might
interact to promote tumour progression and that this localisation might be of
interest. Here previous findings are confirmed and expanded. By using a
combination of Wild-Type and IFITM3 knockout Flp recombinase mediated
integration (Flp-In) models constitutively expressing IFITM1 and a panel of
IFITM1 and/or 3 deleted SiHa and Glioma stem cells (GSCs) it was shown that
the levels of IFITM3 rather than IFN signalling are responsible for the
regulation of the perinuclear co-localisation of IFITM1 and IFITM3. Finally, a
structural functional analysis of IFITM1 was attempted using Flp-In SiHa cell
models expressing Wild-type or mutant forms of IFITM1. However, these
studies showed no significant differences between the mutant forms and
suggested that the expression balance between IFITMs might be important for
their regulation of pro-tumorigenic phenotypes. Overall, this work confirms
previous findings suggesting that IFITMs are key regulators of tumour
progression and resistance to therapy and points towards regulation of cell
adhesion as a key mechanism underlying these effects. The tumour specific
interaction of IFITMs around the perinucleus might be an important component
of IFITM signalling and could be a target for future therapeutic strategies
Investigating spelling change and Anglicization in Scots correspondence
Get PDFThis thesis aims to explore what extra-linguistic variables, if any, conditioned a Scottish
letter-writer’s spelling choice during a time of increased influence from the emerging
(southern) English standard. This is done by examining the effect of three predictor
variables on orthographic variant usage for three Older Scots (OSc) features in 16th–18thC
Scottish correspondence. Crucially, OSc never fully achieved forming a standard spelling
system in the way that English did. The 16th–18thC especially represents a unique time for
OSc during which, due to various events increasing Southern English influence, Scots
underwent anglicization. As a result, by the end of the period, emblematic Scots features
were eventually replaced by English spelling practices. These changes may represent
simply an orthographic change, but perhaps they are representative of changes to
pronunciation.
Scots orthographic change is a vastly under-researched field warranting further
explanation and this thesis aims to contribute to establishing a clearer picture of the
influences that determined variant selection during this period of anglicization. Using
data from the Helsinki Corpus of Scottish Correspondence (1540–1750, Meurman-Solin &
VARIENG 2017), I analyze the influence of three variables on variant selection: writer
gender, audience, and region. I explore the effect of these predictors within three case-studies,
each looking at an emblematic Scots feature. These features are Sibilant
Interchange (e.g., sal > shall), Q-W (e.g., quhilk > which), and etymological /x/ and /xt/
(e.g., nicht > night). The analyses are informed in part by statistical models, specifically
generalized linear models.
The findings of these studies are most insightful regarding writer gender, where women
were found to be ahead in the change to the incoming spelling variant. This is especially
interesting as it aligns more with the previous studies on English—where women were
more innovative in the selection of variants—than with those on Scots, which found
women to be more conservative. Ultimately, writer gender and region were found to be
better predictors of variant selection than audience gender
A practical approach to the sustainable management of gastrointestinal nematodes on extensive sheep farms
Get PDFExtensive farming systems play an important role in sheep production across Europe yet face many innate management and production challenges compounded by narrow economic margins which threaten their sustainability. Gastrointestinal nematodes (GINs) pose a significant challenge to the productivity, health and welfare of grazing ruminants worldwide, causing economic losses in excess of £40 million each year in the UK alone. Increasingly, anthelmintic resistance also challenges the ability to control these parasites, thus more sustainable strategies will need to be employed to prolong the longevity of these treatments.
Traditionally, extensive farming systems were not thought to have a significant GIN challenge compared with intensive lowland ones. However, there is a need to identify the scale and importance of GINs on extensive farms to inform the need for, and practicality of, sustainable control strategies on these farms. Therefore, the aims of this thesis were to focus on extensive sheep farms and (i) investigate the GIN and anthelmintic resistance challenges; (ii) determine practical evidence-based solutions to improve GIN control; (iii) assess the quantitative and qualitative impact of implementing the solutions, (iv) identifying the main challenges impacting GIN management on these farming systems. Finally, with accurate interpretation of test results being crucial, this thesis also aimed to (v) develop a decision support tool to help farmers understand the impact of faecal egg count (FEC) results, an accessible diagnostic test for roundworms and anthelmintic resistance.
A questionnaire of extensive sheep farmers across Scotland (n=34) was conducted to identify the perceived GIN challenge and anthelmintic resistance challenges on their farms. Nine extensive sheep study farms, located across Scotland, were then recruited for a 3-year study to identify the actual GIN and anthelmintic resistance challenge faced on these farms. Initially, parasitological data, including FECs, FEC reduction tests and GIN species identification through ITS-2 deep amplicon sequencing were employed to establish the GIN challenge and anthelmintic efficacy on each farm. The results showed a production-limiting challenge in lambs on most study farms, with a higher GIN challenge observed on improved pastures. In addition, over half of the surveyed extensive farmers perceived anthelmintic resistance to be a greater problem for intensive farmers, with only 20% of respondents reporting known anthelmintic resistance. However, all study farms had evidence of resistance to at least one group of anthelmintics. This demonstrated that despite a perception of low parasitism on extensive farms, there is a need to increasingly consider its impact and take a proactive approach to sustainable control.
A workshop comprising experts in extensive sheep production systems and veterinary parasitology was then conducted to identify practical opportunities to improve GIN management for each study farm. Two to three potential solutions were identified for each farm, informed by the parasitological and management data collected across the first sampling season. While solutions were uniquely tailored to individual production systems, four underpinning themes informed the development of each solution: treatment failure, low pre-treatment FECs, high FECs on improved grazing, and preserving susceptible GIN populations. This demonstrated that despite differences in farms’ scale, management, GIN challenge and anthelmintic resistance status, there may be opportunities to improve GIN management on extensive sheep farms.
The proposed solutions for each farm were subsequently implemented across two further production years, and the quantitative impact measured using parasitological (FECs, FEC reduction tests and species compositions) and production data (e.g. weight gain) where available. Generally, farmers successfully implemented solutions on their farm, which included increased FEC monitoring to guide treatment decisions, targeted anthelmintic treatments informed by species data and weight-based targeted selective treatment of lambs. Continued monitoring also facilitated the development of a larger evidence-base which could guide the further refinement of solutions going forward.
The qualitative impact of, and challenges for, the implementation of sustainable control solutions were then identified through semi-structured interviews with eight of the study farmers. Farmers identified challenges of resource availability, knowledge gaps, practical and structural production challenges, and planning for future resilience. Furthermore, farmers believed the project unanimously improved their understanding of GIN management, with some farmers changing their approach to parasite management as a result of their involvement in the project.
In response to identifying challenges with FEC results interpretation throughout this study, the final objective of this work was to develop a decision support tool to improve their interpretation using a simple visual of potential clinical impact. A preliminary results reporting template was developed in R Markdown and used with the nine study farmers for an initial 12-month period. A decision support tool was then developed in R Shiny and validated by the nine study farms and a further four focus groups with farmers, veterinary clinicians and animal health advisors. This tool has the potential to be used by farmers beyond the end of this work to continue, and build on, the solutions adopted in this thesis, and as a tool for the wider industry.
Ultimately, this thesis has provided a practical understanding of the GIN challenge facing extensive sheep farms and has shown that existing sustainable parasite control strategies can be successfully applied on these farms. However, it has also highlighted the often unique challenges that impact the ability of extensive faming systems to implement solutions, which will need to be considered in future control efforts
Biochemical and functional insights into maintenance DNA methylation by DNMT1 in context of chromatin
No full textPrecise regulation of DNA methylation patterns during cell division is critical for maintaining heritable epigenetic memory and vital for preserving lineage specific characteristics of differentiated cells. During our lifetime DNA methylation is principally maintained by DNMT1. DNMT1 preferentially recognizes the hemi methylated (HeMe) parent DNA strand formed as a result of semi-conservative DNA replication and uses it as a template to copy the methylation pattern on the daughter strand. DNMT1 is a large multidomain protein with regulatory N-terminal region comprised of RFTS, CXXC, and BAH domains, and catalytic C-terminal methyltransferase domain. DNA methylation is inherently linked and guided by the chromatin environment, in part by direct recognition of multiple histone post-translational modifications. H3 trimethylated at K9 (H3K9me3) and ubiquitinated at K14 and/or K18 and/or K23 (H3ubs) are directly recognized by the RFTS domain and H4 trimethylated at K20 (H4K20me3) is read by BAH1 domain of DNMT1. This multivalent interaction facilitates the precise recruitment and recognition of HeMe substrate in chromatin and fine-tunes DNMT1 enzymatic activity.
Biochemical and structural investigations conducted thus far have primarily relied on truncated versions of DNMT1 and short DNA fragments or modified linear histone peptide sequences. While these studies have provided valuable insights, they have obscured the physiological relevance of chromatin in regulating maintenance methylation. Therefore, this study is focussed on elucidating the role of various histone post-translational modifications and how they interplay in modulating DNMT1 activity within the fundamental chromatin unit, the nucleosomes. By examining DNMT1 function within the context of nucleosomes, I aim to shed light on the intricate regulatory mechanisms governing maintenance of methylation in chromatin.
Through a combination of binding and DNA methylation enzymatic assays, I have demonstrated that interaction and catalytic activity of DNMT1 are not entirely interlinked. I have shown that nucleosomes are better substrates for DNMT1-mediated maintenance methylation. These findings challenge previous suggestions that nucleosomes create a barrier and do not confer any advantage for DNMT1 activity. Our results indicate that the chromatin context provided by nucleosomes enhances DNMT1 function, underscoring the importance of studying DNA methylation within the physiological chromatin environment. Additionally, my data provides evidence that DNMT1 interaction with nucleosomes is not facilitated through the acidic patch. Interestingly, I found that unmodified H3 and H4 tails are not necessary for DNMT1 catalytic activation, contrary to previous assumptions. This highlights the complexity of DNMT1-nucleosome interactions and suggests that additional factors beyond histone tail and acidic patch contribute to DNMT1 activation within chromatin.
Further investigation suggests that DNMT1 preferentially recognizes H3ub2 and might have the ability to recognize the symmetry and stoichiometry of ubiquitin signature within the nucleosome. Moreover, my data indicate that DNMT1 may distinguish between different sites of ubiquitination on the H3 tail relative to the position of HeMe on DNA. These findings shed light on DNMT1's intricate interactions with ubiquitinated histones and provide valuable insights into its role in chromatin regulation. In addition to ubiquitination, I show that heterochromatin marks H3K9me3 and H4K20me3 alone and in tandem does not have an additive effect on DNMT1 catalytic activity in nucleosomal context.
Finally, I have investigated how the nature of DNA, including DNA length, sequence and hemi-methylation position within the nucleosome context can affect DNMT1 activity. I found a complex interplay between position of hemi-methylation and flanking sequence around methylation sites have influence on DNMT1 catalytic activity in context of nucleosome. I also show that H3 ubiquitination could alleviate poor DNMT1 activity due to unfavourable DNA sequences or position of HeMe. This suggests that H3 ubiquitination may play multiple roles in activating DNMT1 through mechanisms that are not yet fully understood.
In conclusion, my research attempts to provide a mechanistic understanding of DNMT1 regulation and the roles of different post-translational modifications and their interplay within a physiological nucleosomal context. By elucidating the functions and interactions of different players, I pave the way for further in-depth exploration into the unknown aspects of DNMT1 regulation, including its multivalent interactions and cross-talk with chromatin elements
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