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Effects of STAT3 gain of function on myeloid cells in human peripheral blood mononuclear cells
Get PDFPhD ThesisBackground: Monogenic germline mutations in signal transducer and activator of
transcription 3 (STAT3) can cause gain of function (GOF) characterised by hyperactivity of the
transcription factor and an increase in transcription of STAT3 target genes. Mutations can be
hereditary or sporadic and occur in the various functional domains of the STAT3 gene product.
The disease presents early in life with IPEX‐like, ALPS‐like, and STAT5 deficiency‐like
symptoms, multisystem autoimmunity and immune deficiency. The phenotype is best
described as STAT3 GOF induced immune dysregulation and has severe clinical implications.
To date there remains a paucity of research into this condition and an unmet clinical need
meaning this research could aid understanding of this very complex condition.Objective: To
assess how STAT3 GOF mutations affect gene expression, cell populations, cytokine
production and to identify the mechanism of hyperactivity.
Approaches: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood
obtained from STAT3 GOF patients and healthy control and profiled using single cell RNA
sequencing. In addition, the myeloid compartment of these donors were phenotyped using
Trucount and sequenced using mini‐bulk RNA sequencing to confirm observations from single
cell work. Furthermore, inducible pluripotent stem cell (IPSC) derived macrophages were
used to validate observations made during RNA sequencing experiments. Finally, primary
human dermal fibroblasts from STAT3 GOF patients and healthy controls were used to
identify the mechanism of hyperactivity of STAT3 GOF mutants.
Key findings: Single cell RNA sequencing of PBMCs identified 17 populations in total including
all major myeloid cells, DCs and T‐cells as well as identifying increased expression of glycolytic
genes in STAT3 GOF samples. Phenotyping of the myeloid compartment identified all
monocyte and dendritic cell (DC) populations but with some reductions in some DC
populations in STAT3 GOF. Mini‐bulk RNA sequencing of the myeloid compartment confirmed
an increase in the expression of genes related to glycolysis. Validation studies demonstrated
STAT3 GOF IPSC’s were able to generate mature functional macrophages that displayed
altered glycolytic characteristics as well as altered response to lipopolysaccharide (LPS)
challenge compared to controls. Mechanistic investigations revealed no altered protein
stability or phosphorylation kinetics with STAT3 GOF but a retention of STAT3 in the nucleus
was observed. Preliminary data suggests that STAT3 GOF mutations may not increase DNA
binding capacity but may alter acetylation kinetics.
Conclusion: STAT3 GOF mutations do not appear to affect development or maturation of
PBMC populations but may alter some DC population numbers possibly through
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sequestration. Moreover, metabolic investigations suggest that STAT3 GOF may perturb
glycolysis and LPS response in IPSC derived macrophages, but the mechanism remains
unknown. Finally, preliminary mechanistic data reveals a possible mode of action for STAT3
GOF via altered acetylation kinetics which may increase transcriptional activity of STAT3
several fold
Uptake of peatland ecosystem service knowledge for decision-making
Get PDFPh. D. Thesis.Severe and ongoing loss of biodiversity and changing climate present major threats to nature and
society. This requires urgent action at the science-policy interface. However, progress towards
achieving international biodiversity and climate targets is currently insufficient. To improve the
implementation of environmental governance processes, robust and up-to date evidence is
needed to inform decision-making, built upon effective knowledge exchange and strong multistakeholder networks. However, if evidence of the need for action is so strong, the question
remains – why is knowledge not taken up effectively and efficiently in decision making, and why
does a strong science-policy gap remain?
In order to evaluate science-policy communication and exchange this doctoral research drew on
data from two case studies around peatland ecosystem services in the UK and Germany and a
questionnaire with international policy makers. Peatlands were chosen due to their global
importance to combatting climate change and halting biodiversity loss. Data was collected using
a mixed-method approach and analysed via Bayesian network analysis, social network analysis
and analyses of interviews, questionnaire data and stakeholder workshop discussions. The
combination of quantitative and qualitative approaches enabled a triangulation of the results,
providing rigorous, in-depth insights into the science-policy process in each case study.
The results showed that in order to make informed decisions, evidence needs to successfully reach
the target audience, which was not always the case given the mismatches between the transfer
and perceived reception of knowledge in the case study networks. However, where there was
dialogue between the case study actors, this not only improved the likelihood that knowledge and
evidence was taken up, it also resulted in higher probabilities that decisions were made based on
the communication. If knowledge exchange occurred regularly (at least quarterly), the likelihood
for evidence-informed decision-making increased. Both bilateral and group exchange led to
decisions being made. Trust was found to be an important factor in stakeholder relationships, and
the critical time for trust building appeared to be in the years 1-5. This includes a deliberate choice
to engage with both homophilic (similar) and heterophilic (dissimilar) actors in the network, and
the creation of strong ties as well as weak ties between actors, including from pre-existing network
connections, from the outset and continuing throughout the project. At the beginning of the
project, this can be done by centrally located actors, however, the involvement of professional
knowledge brokers can help to deliberately manage and decentralise the network structure over
time, making it more stable and less vulnerable to break apart. If knowledge exchange interactions
improve awareness and hence address stakeholder needs, evidence can be tailored, delivered or
co-produced accordingly.
The results shed light into the broader processes of knowledge exchange in science-policy settings.
Results also seem to indicate that trust is more easily formed between homophilic actors (who
close in physical distance) who held central positions in the science-policy network. Ongoing
network management seem to be required to build stable networks, which enable efficient
knowledge exchange. This research may help multiple stakeholders to collaborate more
effectively around environmental knowledge in the future, therefore moving the world closer to
successfully and collectively solving environmental issues of our timeNewcastle Universit
Isolation and identification of actinomycetes from Fire Mountain, China, and the elicitation of antimicrobial production
Get PDFPhD ThesisThe increasing emergence of antimicrobial resistance alongside the decrease of novel compounds being discovered is an emerging worldwide health threat. The discovery of novel antibiotics, especially from natural products, is an important area of research for all. Natural products from secondary metabolites produced by actinomycetes are a recognised and established source of antimicrobial compounds, with many clinically relevant drugs on the market.
This thesis aimed to investigate several approaches to the discovery of novel antimicrobial compounds from putative streptomycetes, using classical methodology alongside elicitation techniques. In chapter 3, selective isolation and classical whole cell approaches were investigated to isolate putatively novel strains of Streptomyces spp. from a soil sample taken from Fire Mountain in China, which were subsequently tested for their ability to produce bioactive secondary metabolites. The results showed incidences of broad-spectrum activity from >7% of isolates tested. The known compounds streptothricin and wailupemyin were purified and identified from the Fire Mountain isolates, as well as a compound that inhibited the growth of multidrug resistant strains. In chapter 4, various chemical, biological, and co-culturing techniques were carried out to investigate how these methods could induce the production of antimicrobial compounds from non-active isolates. Ferrioxaime E, a siderophore commonly isolated from Streptomyces spp., was identified from DEM60169 and DEM60164. DEM60169 was also found to produce lipozalidinone A, a broad-spectrum antimicrobial compound, and gentamicins C1 and C1a. Additionally, the use of solid scaffolds was investigated to simulate the natural soil environment that the strains were isolated from. Co-culturing techniques were employed to stimulate the production of antimicrobial secondary metabolites. Six-hundred and twenty incidences of antimicrobial action were observed using this method, and twenty strain combinations transferred to liquid media to investigate further.
It is concluded that elicitation techniques should be utilised to further investigate the production of antimicrobial compounds from Streptomyces spp
A sociolinguistic study of dialect contact and change in Omani Arabic
Get PDFPhD ThesisThis study investigates change in the Nizwa dialect due to inter-dialectal contact caused by
population movements towards Muscat. Particularly, it examines the use of five linguistic features
of Nizwa Arabic belonging to different levels of the grammar. The phonological variables include
the labialization of the high vowel /i/ and the vowel syncope in CV.C word-onsets. The morphosyntactic variables are affricating the second-person feminine singular suffix, marking the future
with the prefix [Ɂa-] and adding /-ə/ clitics when composing yes/no questions.
Data was collected from 38 participants stratified by sex, age, age of arrival (AoA) and length of
residence in Muscat (Labov 2001; Siegel 2010). It was elicited using sociolinguistic interviews,
picture and map tasks and a judgment/transformation test.
Results confirm that the migrants’ dialect is indeed undergoing change with regard to labialization,
syncope, future marker and the yes/no question clitics. This is attributed to: (i) Improved
interlocutor comprehensibility in contact settings and (ii) Speakers’ desire to conform to the
prestigious norms. This study presents several interesting findings to the field. First, it shows that
older speakers (ages 25-50) are the highest adopters of the innovative features due to their
involvement in the linguistic marketplace which increases their awareness of and thus desire to
avoid stigmatized linguistic variants (Simmons 2003; Sankoff and Wagner 2006). Furthermore, a
younger AoA is associated with conservative local use which is interpreted to result from
maintaining local contacts in such speakers’ social networks (Milroy and Milroy 1985).
Additionally, a convergence to Nizwa dialect is attested in monitored speech styles. This dialect
maintenance emphasizes speakers’ desire to retain their Nizwa identity (Ivars 1994). This study
shows that speakers’ ideologies and identity affiliations can be strong correlates for predicting
migrants’ dialect divergence and convergence patterns (Ervin-Tripp 2002; Eckert 2003)
Donor liver optimisation and real-time pre-implant assessment
Get PDFPh. D. ThesisThe number of livers offered for cadaveric donation currently exceeds demand in the United Kingdom. However, the quality of these organs means that nearly a third are rejected for transplantation. In particular utilisation of fatty (steatotic) and organs from donors after circulatory death (DCD) are poor. The aims of this project were to develop novel cost effective ex-situ perfusion viability and reconditioning techniques and explore accurate methods of steatosis quantification.
Ex situ perfusion was introduced using our previous experience in kidney perfusion, taking advantage of the inherent safety benefits of hypothermic perfusion before progressing to normothermic perfusion. A novel perfusion setup was utilised for Hypothermic Oxygenated Perfusion of livers (HOPE), with a custom-designed circuit and adapting existing experience of ex-vivo kidney perfusion using Medtronic perfusion equipment.
A total of 10 livers were transplanted following HOPE, all with significant donor and recipient risk factors, with very promising results. All recipients are alive with a minimum of 20-months follow-up and none have developed clinically-significant ischaemic cholangiopathy. This compares favourably with a historical local cohort that would have met current inclusion criteria for perfusion. Only 1 recipient has had a retransplant, due to rejection.
A number of modalities for assessing livers during perfusion have been explored. We have demonstrated that lactate clearance in normothermic machine perfusion (NMP) cannot be safely relied upon, as livers that had sustained damage well beyond the scope of transplantation still cleared lactate. Bile production has proved to be of some importance in our series, but not all published evidence supports this. Extensive analysis of perfusate biomarkers has identified a potential scoring system that could differentiate between transplantable and untransplantable livers in our series, but needs further validation. We have described an objective method of analysing liver microcirculation, and have demonstrated that NMP improved the arterial microcirculation, but that with extended ischaemia the benefit of NMP is lost.
We have demonstrated in rodent cell lines that steatosis adversely affects viability in hypoxia. In addition we have developed a preliminary point-of-care steatosis assessment method, which could prove beneficial with further calibration and validation.
Utilising ex-situ perfusion to modulate and treat organs is an exciting potential to further improve marginal organs. We selected Pregnane-X Receptor (PXR) activation as our treatment target for this based on rodent experiments demonstrating its benefit in reducing ischaemia-reperfusion injury. These experiments demonstrate upregulation of the end-targets of PXR during human NMP, proving
its potential as a platform for drug delivery. We were unable to demonstrate significant benefits to the livers within the limited timeframe of perfusion, as it is likely that any potential advantages of PXR activation would only become apparent in a much longer time period.NHSBT Blood and Transplant Research Unit, Royal College of Surgeons, LiverNorth, Live Life Give Lif
Utilising O2 chemical potential difference to drive CO2 separation by ceramic-carbonate dual phase membranes
Get PDFPhD ThesisCarbon dioxide (CO2) separation using selective membranes is a promising technology for continuous CO2 capture. However, driving force for CO2 permeation is often limited, resulting large energy input to sustain CO2 permeation flux. In this work, ceramic-carbonate dual phase membranes were fabricated for high temperature CO2 separation at 600-850°C. These membranes consist of molten tertiary carbonate mixture (Li/Na/K, melting point 397°C) infiltrated into porous network of ceramic solid oxide support. When mixed ionic and electronic conductive La0.6Sr0.4Co0.2Fe0.8O3-δ (LSCF6428) is used for membrane support, O2 can co-permeate with CO2 electrochemically via the molten salt (CO32-) based on the principle of molten-carbonate fuel cells (MCFCs). Chemical potential gradient of O2 can be exploited to drive CO2 permeation. It overcomes the driving force limitation and potentially reduces the energy requirement for CO2 capture, even allowing CO2 to permeate against its own chemical potential gradient (uphill permeation).
This project aimed to investigate a novel approach on promoting CO2 permeation flux by enhancing the thermodynamic driving force of O2. Firstly, LSCF6428 membrane has been selected to investigate CO2-O2 co-permeation from a mechanistic viewpoint. Temperature dependence of CO2 flux in presence of O2 in feeding gas has been studied in order to find out the apparent activation energy for CO2 permeation. Several CO2 flux-driving force models have been established; each model was associated with a rate-determining scenario incorporating the driving force contribution from O2. Experimentally observed CO2 flux-driving force relationship revealed that the CO2-O2 co-permeation was likely limited by global interfacial reaction (CO2 + 1/2O2 + 2e- ↔ CO32-).
It was further proposed that permeate side O2 removal may enhance the overall driving force and promote uphill CO2 permeation flux. A system combining uphill CO2 permeation with downstream O2 removal has been developed, utilising LSCF6428 membrane to separate out CO2 and O2 from feeding gas (1:1:20 ratio of CO2, N2 and O2), and Cu-based oxygen carrier for O2 removal. The Cu successfully removed O2 on membrane permeate side from 400 ppm to 30ppm at 600°C and from 3000ppm to 200ppm at 800°C. Uphill CO2 flux at 800°C reached 7.62 × 10-4 mol·m-2·s-1 with O2 removal, a 30% enhancement to the flux without O2 removal. This result was consistent with the prediction from suitable flux-driving force model
A theory-based investigation of patient and healthcare professional behaviours linked to optimal removal of short-term urinary catheters
Get PDFPh. D. ThesisCatheter-associated urinary tract infections (CAUTIs) are among the most prevalent healthcare-acquired infections. Prolonged catheter duration increases CAUTI risk by 5% per day. The average duration of postoperative short-term urinary catheterisation in the UK remains higher than recommended standards. Implementation of early catheter removal guidance is likely influenced by patient and healthcare professional (HCP) factors. This research investigates behavioural determinants (barriers and enablers) of prolonged catheter duration using the theoretical domains framework (TDF).
Study 1 (literature review) highlighted that existing interventions and current guidance on catheter removal focus on either limiting the use of urinary catheters or on early catheter removal. A combination of strategies appears more effective than a singular strategy, but sustained improvement over time remains unclear. Current UK guidelines on the timing of catheter removal were non-specific. Two TDF-based interview studies explored barriers and enablers to prolonged catheter duration with hospital-based HCPs (doctors, nurses and ancillary staff) (Study 2) and with hospitalised/catheterised patients (Study 3).
For HCPs, several key barriers to catheter removal practice were identified, for example, low motivation to remove catheters promptly due to competing demands; poor knowledge of removal guidance and evidence-based practice. Key enablers for HCPs included being highly skilled at removing catheters; the use of handover notes, reminders and daily task lists; and having senior and specialised colleagues as role models. Patient barriers to engaging with HCPs were, for example, perceived inability to manage without a catheter/dependence on the catheter, environmental and resource factors such as the lack of accessible toilets and staff assistance with toileting, staff attitudes, and ward culture. Patient enablers were: motivation to engage to speed-up hospital discharge and social support from the family.
A range of modifiable barriers linked to HCP and patient-related behaviours were identified which may help explain the prolonged duration of short-term urinary catheters. A behaviour change intervention targeting these barriers whilst strengthening the enablers may reduce catheter duration and subsequently CAUTIs.National Institute for Health Research (NIHR
Three dimensional estimation of vegetation moisture content using dual-wavelength terrestrial laser scanning
Get PDFPhD ThesisLeaf Equivalent Water Thickness (EWT) is a water status metric widely used in vegetation health monitoring. Optical Remote Sensing (RS) data, spaceborne and airborne, can be used to estimate canopy EWT at landscape level, but cannot provide information about EWT vertical heterogeneity, or estimate EWT predawn. Dual-wavelength Terrestrial Laser Scanning (TLS) can overcome these limitations, as TLS intensity data, following radiometric corrections, can be used to estimate EWT in three dimensions (3D). In this study, a Normalized Difference Index (NDI) of 808 nm wavelength, utilized in the Leica P20 TLS instrument, and 1550 nm wavelength, employed in the Leica P40 and P50 TLS systems, was used to produce 3D EWT estimates at canopy level. Intensity correction models were developed, and NDI was found to be able to minimize the incidence angle and leaf internal structure effects.
Multiple data collection campaigns were carried out. An indoors dry-down experiment revealed a strong correlation between NDI and EWT at leaf level. At canopy level, 3D EWT estimates were generated with a relative error of 3 %. The method was transferred to a mixed-species broadleaf forest plot and 3D EWT estimates were generated with relative errors < 7 % across four different species. Next, EWT was estimated in six short-rotation willow plots during leaf senescence with relative errors < 8 %. Furthermore, a broadleaf mixed-species urban tree plot was scanned during and two months after a heatwave, and EWT temporal changes were successfully detected. Relative error in EWT estimates was 6 % across four tree species. The last step in this research was to study the effects of EWT vertical heterogeneity on forest plot reflectance. Two virtual forest plots were reconstructed in the Discrete Anisotropic Radiative Transfer (DART) model. 3D EWT estimates from TLS were utilized in the model and Sentinel-2A bands were simulated. The simulations revealed that the top four to five metres of canopy dominated the plot reflectance. The satellite sensor was not able to detect severe water stress that started in the lower canopy layers.
This study showed the potential of using dual-wavelength TLS to provide important insights into the EWT distribution within the canopy, by mapping the EWT at canopy level in 3D. EWT was found to vary vertically within the canopy, with EWT and Leaf Mass per Area (LMA) being highly correlated, suggesting that sun leaves were able to hold more moisture than shade leaves. The EWT vertical profiles varied between species, and trees reacted in different ways during drought conditions, losing moisture from different canopy layers. The proposed method can provide time series of the change in EWT at very high spatial and temporal resolutions, as TLS instruments are active sensors, independent of the solar illumination. It also has the potential to provide EWT estimates at the landscape level, if coupled with automatic tree
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segmentation and leaf-wood separation techniques, and thus filling the gaps in the time series produced from satellite data. In addition, the technique can potentially allow the characterisation of whole-tree leaf water status and total water content, by combining the EWT estimates with Leaf Area Index (LAI) measurements, providing new insights into forest health and tree physiology.Egyptian Ministry of Higher Educatio
Novel data association methods for online multiple human tracking
Get PDFPhD ThesisVideo-based multiple human tracking has played a crucial role in many applications
such as intelligent video surveillance, human behavior analysis, and
health-care systems. The detection based tracking framework has become
the dominant paradigm in this research eld, and the major task is to accurately
perform the data association between detections across the frames.
However, online multiple human tracking, which merely relies on the detections
given up to the present time for the data association, becomes more
challenging with noisy detections, missed detections, and occlusions. To
address these challenging problems, there are three novel data association
methods for online multiple human tracking are presented in this thesis,
which are online group-structured dictionary learning, enhanced detection
reliability and multi-level cooperative fusion.
The rst proposed method aims to address the noisy detections and
occlusions. In this method, sequential Monte Carlo probability hypothesis
density (SMC-PHD) ltering is the core element for accomplishing the
tracking task, where the measurements are produced by the detection based
tracking framework. To enhance the measurement model, a novel adaptive
gating strategy is developed to aid the classi cation of measurements. In
addition, online group-structured dictionary learning with a maximum voting
method is proposed to estimate robustly the target birth intensity. It
enables the new-born targets in the tracking process to be accurately initialized
from noisy sensor measurements. To improve the adaptability of the
group-structured dictionary to target appearance changes, the simultaneous
codeword optimization (SimCO) algorithm is employed for the dictionary
update.
The second proposed method relates to accurate measurement selection
of detections, which is further to re ne the noisy detections prior to the tracking
pipeline. In order to achieve more reliable measurements in the Gaussian
mixture (GM)-PHD ltering process, a global-to-local enhanced con dence
rescoring strategy is proposed by exploiting the classi cation power of a mask
region-convolutional neural network (R-CNN). Then, an improved pruning
algorithm namely soft-aggregated non-maximal suppression (Soft-ANMS) is
devised to further enhance the selection step. In addition, to avoid the misuse
of ambiguous measurements in the tracking process, person re-identi cation
(ReID) features driven by convolutional neural networks (CNNs) are integrated
to model the target appearances.
The third proposed method focuses on addressing the issues of missed
detections and occlusions. This method integrates two human detectors
with di erent characteristics (full-body and body-parts) in the GM-PHD
lter, and investigates their complementary bene ts for tracking multiple
targets. For each detector domain, a novel discriminative correlation matching
(DCM) model for integration in the feature-level fusion is proposed, and
together with spatio-temporal information is used to reduce the ambiguous
identity associations in the GM-PHD lter. Moreover, a robust fusion
center is proposed within the decision-level fusion to mitigate the sensitivity
of missed detections in the fusion process, thereby improving the fusion
performance and tracking consistency.
The e ectiveness of these proposed methods are investigated using the
MOTChallenge benchmark, which is a framework for the standardized evaluation
of multiple object tracking methods. Detailed evaluations on challenging
video datasets, as well as comparisons with recent state-of-the-art
techniques, con rm the improved multiple human tracking performance
Values and Decision-making within an Acute Medicine Service
Get PDFPhD ThesisTheoretical frameworks of values in healthcare tend to come in one of two kinds: monism
and pluralism. However, both value-monism and value-pluralism prove inconsistent and
unrealistic when applied to controversial cases. The lack of clarity about values in healthcare contributes to the contentious nature of key concepts in decision-making, such as sickness and futility.
The prominent decision-making models considered in this thesis are substituted decision-making, supported decision-making, shared decision-making, dual process theory, values-based practice and values-based medicine. Whilst each has its strengths, each model fails to provide a compelling account of the role of values in decision-making, on the grounds of arbitrariness, incompleteness or ambiguity.
A lack of clarity about the role of values in decision-making is particularly problematic in busy and pressured clinical environments. An Acute Medicine Service (AMS) is a part of a hospital which is dedicated to the early management of medical emergencies. The workload of an AMS entails a heavy decision burden concerning a broad range of clinical problems under time pressure.
In this ethnographic study, I was participant-observer within an AMS over a period of 16
months. This involved numerous informal interviews and 27 formal interviews with staff,
patients and relatives. A thematic analysis of empirical data, borrowing insights from the
tradition of philosophical hermeneutics, provides a plausible interpretation of the role of
values in decision-making within an AMS.
The data I present are organised around three main themes: restoring order, working together and what’s best. Within an AMS, a course of action is appraised according to three values: welfare, choice and effectiveness. These values are interdependent in a manner distinct from monism and pluralism. The implications of these findings for the philosophy of medicine and clinical decision-making are discussed, with some suggestions for future prescriptive models and further research