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Defining the cellular heterogeneity of healthy human skin using single cell technologies
Get PDFPhD ThesisSkin is a complex organ comprising many synergising cell types. It serves a vital role in the immune system as the first line of defence against pathogens. In homeostatic conditions, skin must be primed to respond to an overwhelming diversity of pathogens from the environment while simultaneously avoiding excessive immune responses and autoimmunity. Understanding of the intercellular heterogeneity that is required for this balance during steady state is crucial to understanding how these cells act during dysregulation, and could aid in the understanding of diseases such as eczema and psoriasis.
Healthy adult skin, obtained from reconstructive surgery, was enzymatically dissociated and profiled using single cell RNA sequencing and mass cytometry, and further validated using flow cytometry and immunohistochemistry. By sequencing 82,490 single cells from healthy adult human skin of three donors, it was possible to profile the different transcriptomic states of keratinocytes, melanocytes, vascular and lymphatic endothelium, pericytes, fibroblasts, schwann cells, lymphoid cells and antigen presenting cells. 1,959,717 single cells from four donors were analysed using a 37-marker mass cytometry panel, allowing for a comparison between the proteomes and transcriptomes of skin cells.
Interrogation of the stromal cells at high resolution revealed previously unreported heterogeneity, including the presence of specialised vascular endothelial structures in healthy skin, which appear to mirror the high endothelial venules found in lymphoid tissues. These structures may be critical to leukocyte infiltration during homeostasis and inflammatory conditions.
This study provides a powerful resource, as a repository of healthy skin single cell heterogeneity, for future research into deviations from health, including inflammation, infection and disease
Use of the androgen signalling pathway to identify ovarian cancer patients suitable for hormonal therapy
Get PDFPhD ThesisBackground
Despite confirmed AR expression in epithelial ovarian cancer (EOC), clinical response to anti-androgen treatment is poor. Stratification of susceptible individuals with a specific biomarker, such as the previously described Rab35, might enable more effective treatment strategies. Abiraterone, a steroid synthesis inhibitor, might be of therapeutic benefit in specific EOC subgroups.
Methods
Primary cell cultures (PCO) generated from ascites were used as a representative model for the heterogeneity of EOC. PCOs were examined for AR and Rab35 expression at mRNA and protein level and were stimulated with androgens to evaluate subsequent Rab35 expression.
CYP17 expression was measured in ovarian cancer cell lines and PCOs and the effect of abiraterone on proliferation was assessed in two ovarian cancer cell lines.
Results
The AR expression was widely different when examined with qRT-PCR, Western blotting and immunohistochemistry. No correlations were found between the modalities for either AR or Rab35 expression.
In contrast, AR and Rab35 expression showed a positive correlation at the protein and mRNA level. However, androgen treatment of PCOs showed >50% increase in Rab35 mRNA expression in only 40% of PCOs.
CYP17 expression was confirmed in all examined cell cultures and PCOs at both, the protein and mRNA level. Abiraterone treatment of the ovarian cancer cell lines led to significant inhibitory effects on proliferation. On protein level however, abiraterone exposure resulted in increased expression of AR and CYP17.
Conclusion
Although AR expression was confirmed in POCs, it remains unclear which technique would be most suitable to stratify for androgen expressing tumours.
Rab35 in PCOs appeared to be androgen-related and hence may not be a suitable biomarker in EOC for AR.
The inhibitory effect of abiraterone on proliferation that was observed in ovarian cultures is suggestive of a dual action of the compound. Response to abiraterone exposure in PCOs might help to determine potential treatment effects.Northern Gynaecology Oncology Centre (NGOC
Optimisation of protocols for ex vivo expansion of limbal stem cells and their enrichment
Get PDFPh. D. ThesisThe corneal epithelial cells are constantly replaced by the stem cells located at the limbus, the peripheral edge of the cornea, therefore known as limbal stem cells (LSCs). LSCs can be destroyed by numerous factors which results in the condition called limbal stem cell deficiency (LSCD).
Ex vivo expansion of LSCs is a well-established technique used successfully to cure patients with LSCD. Therapeutic use of LSCs must be performed in compliance with good manufacturing practice (GMP) as a quality assurance system. However, traditional culture media for ex vivo expansion of LSCs contains a number of ingredients derived from animal sources which may compromise its safety profile for human transplantation. The first aim of the study was to define new GMP grade medium for cultivation and maintenance of LSCs in vitro. Formulation of new GMP compliant media resulted in equal growth to non-GMP grade media.
Strick regulations for cell therapy promote centralization of culture units, therefore definition of reliable and practical transportation strategies is vitally important. The second aim of this study was to optimise the transport conditions for limbal biopsies (LBs) and cultured limbal epithelial cells (LECs). Transport of LBs at room temperature proved to be significantly superior to 4°C transport. We also showed that cultured LECs may be stored in serumfree media and transported up to 7 days at 23°C without any negative effect on cell number, viability, colony forming efficiency or gene expression profile.
Due to the absence of specific LSC markers, identification and isolation of putative LSCs is a complicated task. The third and final aim of this study was to identify novel cell surface markers for LSCs. We reported herein the identification of a new cell surface marker for LSCs (CD200) as well as a cell surface marker for proliferating progenitor cells (CD109)
Polar codes combined with physical layer security on impulsive noise channels
Get PDFPh. D. ThesisThe need for secure communications is becoming more and more impor-
tant in modern society as wired and wireless connectivity becomes more
ubiquitous. Currently, security is achieved by using well established
encryption techniques in the upper layers that rely on computational
complexity to ensure security. However, processing power is continu-
ally increasing and well-known encryption schemes are more likely to be
cracked. An alternative approach to achieving secure communication is
to exploit the properties of the communication channel. This is known as
physical layer security and is mathematically proven to be secure. Phys-
ical layer security is an active research area, with a significant amount
of literature covering many different aspects. However, one issue that
does not appear to have been investigated in the literature is the effect
on physical layer security when the noise in the communication channel
is impulsive. Impulsive noise adds large spikes to the transmitted signal
for very short durations that can significantly degrade the signal. The
main source of impulsive noise in wireless communications is electromag-
netic interference generated by machinery. Therefore, this project will
investigate the effect of impulsive noise on physical layer security.
To ensure a high level of performance, advanced error-correcting codes
are needed to correct the multiple errors due to this harsh channel. Turbo
and Low-Density Parity-Check (LDPC) codes are capacity-approaching
codes commonly used in current wireless communication standards, but
their complexity and latency can be quite high and can be a limiting fac-
tor when required very high data rates. An alternative error-correcting
code is the polar code, which can actually achieve the Shannon capacity
on any symmetric binary input discrete memoryless channel (B-DMC).
Furthermore, the complexity of polar codes is low and this makes them
an attractive error-correcting code for high data rate wireless commu-
nications. In this project, polar codes are combined with physical layer
security and the performance and security of the system is evaluated on
impulsive noise channels for the first time.
This project has three contributions:
Polar codes designed for impulsive noise channels using density evo-
lution are combined with physical layer security on a wire-tap chan-
nel experiencing impulsive noise.
The secrecy rate of polar codes is maximised. In the decoding of
polar codes, the frozen bits play an important part. The posi-
tions of the frozen bits has a significant impact on performance and
therefore, the selection of optimal frozen bits is presented to opti-
mise the performance while maintaining secure communications on
impulsive noise wire-tap channels.
Optimal puncturing patterns are investigated to obtain polar codes
with arbitrary block lengths and can be applied to different modu-
lation schemes, such as binary phase shift keying (BPSK) and M-
ary Quadrature Amplitude Modulation (QAM), that can be rate
compatible with practical communication systems. The punctured
polar codes are combined with physical layer security, allowing the
construction of a variety of different code rates while maintaining
good performance and security on impulsive noise wire-tap chan-
nels.
The results from this work have demonstrated that polar codes are ro-
bust to the effects of impulsive noise channel and can achieve secure
communications. The work also addresses the issue of security on im-
pulsive noise channels and has provided important insight into scenarios
where the main channel between authorised users has varying levels of
impulsiveness compared with the eavesdropper's channel. One of the
most interesting results from this thesis is the observation that polar
codes combined with physical layer security can achieve good perfor-
mance and security even when the main channel is more impulsive than
the eavesdropper's channel, which was unexpected. Therefore, this thesis
concludes that the low-complexity polar codes are an excellent candidate
for the error-correcting codes when combined with physical layer security
in more harsh impulsive wireless communication channels
Progressive collapse of damaged ship structures
Get PDFPh. D. ThesisThis research investigates the progressive collapse of stiffened panels in a ship’s structure under several damaged conditions. The main focus is on the behaviour of stiffened panels under three conditions: intact condition; damage represented by a circular, clear-cut-out hole; and damage represented by penetration simulations. The same damage conditions have also been applied to double bottom box girders. The results of these analyses are used to better understand the behaviour of damaged ship structures and develop a novel modification to a simplified method for predicting a ship’s ultimate strength.
The non-linear, finite element method is used in order to simulate the damaged condition and to estimate ultimate strength behaviour in both undamaged and damaged stiffened panels. The damaged conditions are divided into two categories: damage represented by a circular, clear-cut hole and damage represented by penetration with an indenter. The damaged scenario assumes the damage to be located in the middle of the stiffened panel. The diameter of the damaged area and diameter of indenter are controlled by a ratio between the diameter of damaged area (D) or diameter of indenter (Din) and the width of the stiffened panels (W) respectively. Pre-existing characteristics of the structure are considered as an average level in terms of both residual stress and geometric imperfection. An in-plane compression load is applied to the stiffened panel in order to generate the ultimate strength, which is affected by the damaged condition.
The results are used to extend an existing hull girder progressive collapse method, using a novel approach to adapt the load shortening curves. A knockdown factor is generated by using regression formulae from the finite element models and is applied to modify a load shortening curve for damaged ship structures. The modification curves are combined with moment curvature to find the ultimate strength of the damaged hull girder.
The method is verified with case study analyses of double bottom box girders. The same damaged conditions applied for the stiffened panels are used with the hull girder. The damaged area is located in the middle of the bottom part of the structure. The hogging condition is applied for the verification model. The validation results show excellent agreement between the finite element method and modified hull girder progressive collapse method, which can be used to predict the ultimate strength of a damaged ship structure.ON
Clinical presentation and genetic characterisation of mitochondrial disease in Kuwait
Get PDFPh. D. ThesisMitochondrial disorders are a group of clinically heterogenous conditions affecting
multiple systems with a prevalence that is estimated to affect 1 in 4,300 individuals.
Mitochondrial function is under the control of both the mitochondrial and nuclear
genomes which encode >1200 mitochondrial proteins. Manifold biochemical pathways
and possible gene targets contribute to the highly variable genotype-phenotype
correlations observed in mitochondrial patients, posing distinct challenges in reaching a
genetic diagnosis. Whole Exome Sequencing (WES) is a gene agnostic approach that has
been hugely powerful in diagnosing mitochondrial disease patients and broadening the
genotypic spectrum of disease. Mitochondrial genetic disease is largely understudied in
Kuwait where levels of consanguinity reach 50% in the community. Studying the genetics
and aetiology of mitochondrial disorders in Kuwait presents huge potential in identifying
novel Mendelian causes of disease.
I custom-designed mitochondrial disease criteria to evaluate and recruit patients
suspected of mitochondrial disease in Kuwait. WES led to the diagnosis of 14 out of 22
recruited families: 8 families harboured variants in known mitochondrial disease genes
(SLC19A3, PDHX, SURF1, MPC1, TTC19, NDUFA13, NDUFB9 and RRM2B), 2
harboured variants in a novel mitochondrial disease gene (LETM1), and 4 were diagnosed
with phenocopies of mitochondrial disease (RNASEH2C, TREX1, VPS13B and ATP8A2).
Functional validation of novel variant pathogenicity was performed in patient fibroblasts
from 4 families. Functional validation was also carried out on additional mitochondrial
patients from Newcastle and external collaborators (COX15, TTC19, NDUFAF3 and
NDUFC2). Complexome profiling helped characterise the effect of NDUFC2 variants (a
novel candidate gene) on Complex I assembly while a controlled lentiviral rescue
experiment partially recovered protein expression and validated variant pathogenicity.
My work highlights the potential of employing WES to identify novel causes of disease
in understudied consanguineous populations and emphasises the importance of
establishing functional pipelines alongside the genetic studies in the Kuwait Medical
Genetics CentreGovernment of the State of Kuwai
Breastfeeding and weaning practices of African mothers living in North East England
Get PDFPhD ThesisAbstract
Breastfeeding provides optimal nutrition for the healthy growth of infants and is the highest preventive measure in reducing under-five mortality. It is also beneficial to the health of the mother and reduces maternal deaths from breast cancer. Evidence shows that mothers migrating from regions of higher breastfeeding rates, to high-income countries of lower breastfeeding rates, tend to breastfeed less the more acculturated they get to the new environment. With the United Kingdom (UK) having one of the lowest rates of breastfeeding globally, the impact of acculturation on the breastfeeding practices of Africans living in the UK has been understudied.
This study aimed to investigate the breastfeeding and weaning practices of African mothers living in North East England. The first phase of this study was a systematic review of existing international literature on the breastfeeding knowledge and practices among African immigrant mothers living in high-income countries. Thirty-five studies were included in this review. The second and third phases of this study used qualitative interviews and thematic analysis to explore the breastfeeding experiences of 19 African mothers and the perception of 18 health professionals providing breastfeeding support to Africans in the UK. Pierre Bourdieu’s theory of practice was applied to interpret the findings.
Three key themes were identified: breastfeeding as a culture, gathering and navigating information sources, and the essentiality of support. Key differences between the views of mothers and health professionals were observed and highlighted. Social norms and practices within the UK influence the beliefs and practices of African mothers as they are faced with conflicting opinions and suggestions regarding choices of infant-feeding. An awareness of the cultural practices of African mothers, recognising their challenges to breastfeeding in the UK and offering more intentional support including education on exclusive breastfeeding and strategies to overcome barriers is required to improve breastfeeding outcomes
Temporary space in Amman: a co-creation of everyday activism and state flexibility
Get PDFPh. D. ThesisThe literature about temporary urbanism has been generally focused on the context of North
America and Europe. It is undeniable, though, that temporariness happens more in the global
South than in the North. While such literature offers the opportunity for comparative reflection
across the global North-South ‘divide’ there is still an imminent need towards exploring and
learning from different contexts and divergent urban experiences. Also, very little is said about
the production process of temporary urban spaces, the power relations between actors and how
these relations are affected by the actors’ intentions and resources. This research aims to address
this gap through exploring three cases of temporary urban space in Amman, Jordan: Ras AlAin market, Nour Al-Barakeh community garden and the ‘Vista’ at Jordan street. This
qualitative research draws on documents, audio, visual and digital material as well as semistructured interviews with a range of key actors and field observations.
By exploring the intentions of the temporary space activists and the state’s response to them,
the case studies demonstrate that temporary space is a co-creation of everyday actions and state
flexibility. This meant that on the one hand the temporary urban spaces were steered by different
types of marginalized social groups that were able to come together to achieve their needs
around various types of social capital. On the other hand, these temporary urban spaces also
came to exist due to opportunities of agency which arise as a result of a gap in the planning
framework and the state’s flexibility to urban policy sanctions. Hence, through conscious
everyday actions, the social groups pursue their needs with no aim of direct confrontation with
the state. These everyday actions enable these social groups to achieve those needs and have
taken various forms according to: the category, resources and aims of the social group as well
as the regulatory frameworks within which they exist. Whereas flexibility is a state response
to the various social groups and is a strategy towards urban governance in which urban
sanctions are extended, or suspended. This measure is taken by the state to preserve social order
in relation to certain social groups and to resolve urban issues
Does the presence of disseminated tumour cells in bone marrow or circulating tumour cells predict early tumour recurrence in patients with oesophagogastric cancer?
Get PDFMD ThesisOesophagogastric cancer is associated with poor long term survival; overall five-year survival is 10-15%. A significant proportion of patients who undergo curative surgery subsequently develop metastatic disease. Evidently, local control of the tumour does not eliminate the risk of haematogenic recurrence. The cancer cell biology of disseminated disease and the mechanisms of its development remain poorly understood. The presence of disseminated tumour cells in blood and bone marrow of patients with breast, colorectal and lung carcinoma are associated with poor prognosis.
The aims of the study were to develop a reliable assay to identify circulating tumour cells (CTCs) in the blood and disseminated tumour cells (DTCs) in the bone marrow and characterize the prevalence, biology and heterogeneity of the cells in patients with oesophagogastric cancer by high resolution imaging flow cytometry and fluorescence activated cell sorting. The objective of the thesis was to evaluate the prognostic significance CTCs and DTCs in oesophagogastric cancer.
Blood samples were taken from patients undergoing curative and palliative treatment for oesophagogastric cancer. Bone marrow from the rib section excised as part of an open oesophagetomy was collected. CTCs and DTCs were isolated from the blood and the bone marrow by red cell lysis and immunomagnetic removal of white blood cells. Enriched cells were incubated with antibodies against epithelial, mesenchymal and predictive biomarkers. CTCs and DTCs were identified based upon their morphology and biomarker expression by high resolution imaging flow cytometry.
CTCs and DTCs were present in all patients undergoing curative and palliative treatment for oesophagogastric cancer. Post curative surgery, patients with 100 or more CTCs in the blood had a significant reduction in relapse free survival (p=0.012).
The study highlights the prognostic potential of CTCs in the blood and DTCs in the bone marrow of patients undergoing curative and palliative treatment for oesophagogastric cancer
Non-aqueous protonation, protonolysis and related reactions of polyoxometalates
Get PDFPh. D. Thesis.A sound knowledge of the complex solution processes underlying molecular metal oxide aggregation and structural inter-conversion is crucial to developing efficient synthetic routes to accessing unprecedented structures and understanding their properties. The nonaqueous aggregation of (TBA)2[W6O19] from WO(OMe)4 and (TBA)2[WO4] was studied systematically using 17O NMR spectroscopy. The study resulted in the development of a novel and efficient synthetic route to a series of heterometallic Lindqvist POMs, {M′M5} from [M6O19]2– anion. Using a combination of NMR techniques (1H, 17O and 2D 1H EXSY) and DFT calculations, the hydrolysis and condensation of (TBA)3[(MeO)TiW5O18] and (TBA)3[(MeO)SnW5O18] were investigated to understand factors influencing subtle differences in the POMs. The study led to the isolation and characterization of (TBA)3[(HO)TiW5O18] - a new member of the [M′M5] family. The study further revealed that the tin hydroxido POM, (TBA)3[(HO)SnW5O18] was more readily accessible than the titanium anologue, (TBA)3[(HO)TiW5O18] under similar conditions whereas the tin oxo-bridge dimer, (TBA)6[(μ-O)(SnW5O18)2] was less stable and hence more difficult to isolate compared to the titanium oxo-bridged POM, (TBA)6[(μ-O)(TiW5O18)2]. These behaviours were ascribed to differences in relative free energies. Furthermore, the surface oxygen basicity of the POMs, (TBA)3[(MeO)TiW5O18] and (TBA)6[(μ-O)(TiW5O18)2] and the behaviour of (TBA)6[(μ-O)(TiW5O18)2] towards a range of electrophiles were explored using 17O NMR spectroscopy. Generally, it was demonstrated that the TiOW oxygens are the most basic sites in (TBA)6[(μ-O)(TiW5O18)2]. The reactions resulted in the isolation and characterization of the dmso adduct (TBA)4[(μ-O)(TiW5O18H)2(dmso)] and (TBA)4[(μ-O)(TiW5O18)2(SnMe2)] wherein Sn(IV) is bonded to the Ti-O-W oxygens suggesting the possibility of “POM-pincer” complexes. Additionally, [O=TiW5O18]4–, which is possibly the first member of the oxo-titanium Lindqvist family was isolated and characterized in an excellent yield by treating [(CH3O)TiW5O18]3– with an organic base. The new POM was subsequently reacted with alkyl and aromatic isocyanate providing insights into the reactivity of the titanyl bond. Generally, products were further characterised by FT-IR, Multinuclear NMR (1H, 13C, 31P, 119Sn, and 183W) and/or single crystal XRD. While DFT calculations provided support for experimental observations.COST Action PoCheMoN, Newcastle Universit