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Genetic risk factors and disease mechanisms in mitochondrial optic neuropathy
Get PDFPhD ThesisMitochondrial vitality is dependent upon continued cooperation between nuclear
(nDNA) and mitochondrial genomes (mtDNA) and defects in either genome can result
in a broad spectrum of human disease.
Leber’s Hereditary Optic Neuropathy (LHON) is an inherited blindness caused by
the selective loss of retinal ganglion cells (RGCs) which predominantly affects young
males. In 90% of LHON, cases harbour one of three primary mutations, m.11778G>A,
m.3460G>A and m.14484T>C, which affect Complex I subunits of the electron transport
chain. mtDNA mutations alone cannot explain the characteristic reduced penetrance,
gender bias and secondary phenotypes of LHON. Recent work on LHON has often,
unsuccessfully, focused on the identification of a nuclear-encoded modifying gene
which would explain these characteristics.
Thus, the aims of this PhD were, i) to improve our estimates of disease penetrance
and investigate other potential disease modifiers, ii) to use LHON patient cell lines to
investigate the effect ofmtDNAmutations on cellular processes whichmay contribute to
LHONand iii) to evaluate retinal degeneration in a mtDNAmutation animal model. By
investigating these key components of LHON we hope to improve our understanding
of the disease process.
Using a large familial cohort, we were able to update and improve current LHON
penetrance estimates, with our analysis indicating that the penetrance in females is
much higher than previous estimates. In addition, we were able to show that LHON
cases have differential genetic variants and methylation patterns, identifying ~45nDNA
modifier genes that may explain the variable penetrance and gender bias in LHON.
Primary LHON fibroblasts were further characterised across a range of biochemical
mechanisms.
Analysis of an mtDNA mutation mouse has improved our understanding of the
effect of mtDNA mutation on RGC loss. Work in this PhD has identified a technical
framework that could be applied to other models with optic neuropathy
Novel molecular mechanisms of transcription targeting antibiotics
Get PDFPhD ThesisTranscription, the first stage of gene expression, is performed by the multi-subunit RNA polymerase (RNAP). The indispensable nature of transcription and sequence divergence from eukaryotic counterparts make bacterial RNAP an excellent target for antibiotics. Yet very few clinical antibiotics target RNAP. The growing prevalence of antibiotic resistance amongst pathogenic bacteria demands the identification of novel antibacterial compounds acting through novel molecular mechanisms. This work consists of four distinct projects in which we investigated the molecular mechanisms of several previously uncharacterised transcription inhibitors.
(i) Most clinical antibiotics are derived from the natural products of actinomycete bacteria. Consequently, we screened a library of actinomycetes compiled by our industrial collaborators DemurisTM for producers of novel inhibitors of bacterial transcription. From this screen we identified Antibiotic A39079S-1, produced by Streptomyces strain DEM40380, as an inhibitor of bacterial RNAP. The compound is an ansamycin type antibiotic with a previously uncharacterised mechanism of action. Here, we show the compound inhibits bacterial RNAP through a steric occlusion mechanism typical of rifamycins.
(ii) Recently, the antibiotic ureidothiophene (Urd) was identified within a commercial screen of synthetic compounds in which inhibition of S. aureus RNAP was analysed. Here, we characterised the molecular mechanism of action by which Urd inhibits bacterial RNAP. We show the inhibitor targets regulatory sub-region 1.2 of the sigma subunit to prevent melting of the -10 promoter element. Consequently, Urd inhibits formation of the open promoter complex, a key step in transcription initiation.
(iii) A prior screening program conducted by DemurisTM had identified the rifamycin type natural product kanglemycin A (KglA) as an inhibitor of rifampicin resistant RNAPs. Here, we show the unique ansa-bridge substituents of the compound act to form new binding contacts with RNAP. We also show
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KglA inhibits transcription through a unique steric occlusion mechanism by preventing extension of the nascent transcript at an earlier stage than rifampicin.
(iv) Finally, we investigated ADP-ribosylation as a mechanism of KglA inactivation by Mycobacterium smegmatis and Mycobacterium abscessus Rifampin ADP-ribosyltransferase (Arr) enzymes. We show KglA is not a substrate of the rifampicin inactivating Arr purified from Mycobacterium smegmatis, but remains a substrate of Arr purified from the extensively drug resistant pathogen Mycobacterium abscessus
The physical health and quality of life of patients with X-linked agammaglobulinaemia in England and Wales
Get PDFPh. D. Thesis.Background
Patients with X-linked agammaglobulinaemia (XLA) have absent peripheral circulating Blymphocytes and agammaglobulinaemia caused by defects in BTK. Treatment consists of
life-long immunoglobulin replacement therapy. This only contains the IgG isotype containing
little or no IgA or IgM. Patients may still, therefore, experience recurrent infections and
complications. Novel therapies are potentially available, most notably gene therapy and
newborn screening.
I aimed to examine the clinical health outcomes and quality of life for patients with XLA in
England and Wales to evaluate current practices and the potential role for novel therapies.
Methods
This is a retrospective, longitudinal observational study of patients with a definite diagnosis of
XLA (BTK mutation or absent BTK expression), in England and Wales.
Retrospective clinical data were collected from patients’ records, including conversion of lung
function results to Z-scores. Patients and/or their families were invited to complete
questionnaires on their health-related quality of life (HRQoL) and psychological health.
Results were compared against UK norms and UK patients with cystic fibrosis.
Results
Fifty-four patients were enrolled in the study (21 children, 33 adults). Median age at
diagnosis was 2.59 years with no statistically significant improvement seen since 1990.
Twenty-two patients (44%) had evidence of bronchiectasis on high-resolution computerised
tomography. Patients with bronchiectasis were diagnosed with XLA significantly later than
patients without bronchiectasis. Neither infection incidence nor IgG trough levels were
associated with an increased risk of bronchiectasis.
In the absence of bronchiectasis, XLA patients had normal HRQoL results. HRQoL was
strongly correlated with respiratory symptoms and lung function.
Conclusions
Recurrent respiratory tract infections and bronchiectasis remain a major burden for this cohort
despite modern therapy. In the absence of bronchiectasis, patients have a normal HRQoL.
Curative therapy, such as gene therapy or bone marrow transplantation, may provide the only
option for improving outcomes in XLABubble Foundation, Jeffrey Modell Foundation,
UKPI
The Urinary 1-hydroxypyrene Biomarker in Street Janitors Exposed to Traffic Related Benzo[a]Pyrene During One Islamic Calendar Year in Madinah, Saudi Arabia
Get PDFPh. D. ThesisBackground
Madinah is the second most important Muslim holy city in Saudi Arabia. Therefore, during
the holy months, traffic congestion and consequent air emission of several pollutant compounds,
mainly carcinogenic Benzo[a]pyrene (BaP), are expected to reach high levels in certain places. The
emission of these pollutants into the atmosphere increases their concentration, which in turn
increases the risk of exposure through inhalation. Inhalation of air BaP is the main route of entry
into the human body, and so the aim of the present study was to assess exposure to air BaP through
urinary 1-hydroxypyrene (1-OHP) in street janitors. The study also aimed at finding the relation
between this biomarker and daily exposure to air BaP with traffic volume and meteorological
factors, such as wind speed, temperature, wind direction, relative humidity, and precipitation.
Methods
The study was performed during one Islamic calendar year from 1/4/1438 – 30/3/1439
(December 2016 to November 2017) in Madinah City. The research was designed to include three
traffic locations with high traffic density and two control locations with low traffic activity. In these
locations, BaP was measured daily in the breathing-zone air of the street janitors using PM2.5
personal samplers, and in the air of the five studied locations using high-volume air samplers. Daily
measures including traffic volume (cars, medium and heavy vehicles), temperature, humidity, wind
speed, and precipitation, were recorded at the five studied locations. 20 male participants (street
janitors) aged between 25 and 35 years were recruited in the study from each of the five studied
locations. In total, 100 street janitors were involved in the study. Two urine samples were obtained
from each participant after their work shift and in the early morning of the next day to measure 1-
OHP. Summary statistics, including medians, were used to describe the data. Graphs, including
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calendar and time series plots were used to visually present the data. Data were analysed using
methods that included Spearman’s rank correlation, Mann-Whitney, and Kruskal-Wallis tests.
Results
It was found that BaP and 1-OHP concentrations were significantly higher in the heavy
traffic locations compared to the control locations. The research also found that BaP and 1-OHP
concentrations were significantly higher during the holy months of the Islamic calendar than the
normal months. For example, the median concentrations of 1-OHP in traffic locations during the
holy months of Ramadan, Dhu al-Qadah, Dhu al-Ḥijjah and Muḥarram were 0.98, 0.99, 1.01 and
0.82 μmol/mol, respectively. The median concentrations of 1-OHP in normal months ranged
between 0.71–0.74 μmol/mol. The median ambient BaP concentration during the holy months
ranged between 0.59–0.63 ng/m3
, while in the normal months it ranged between 0.36–0.39 ng/m3
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It was also found that morning 1-OHP concentration was higher than after work 1-OHP
concentration. The research also found that ambient BaP concentration was generally higher than
the individual BaP concentration. Correlation results indicated that ambient BaP is positively
correlated with CO (r = 0.59, p<0.001) and traffic volume (r = 0.74, p<0.001. It was also found
that individual BaP is positively correlated with CO (r = 0.56, p<0.001) and traffic volume (r =
0.85, p<0.001). Meteorological conditions such as temperature, relative humidity and precipitation
were found to have no effect on the concentration of both ambient and individual BaP. Conclusion
The research found that the higher the traffic volume in Madinah, especially during the holy
months, the higher the ambient BaP pollutants, and the higher the chance of people being exposed
to BaP pollutants (individual BaP). Therefore, it is recommended that efforts to reduce the number
of vehicles in Madinah be adopted. Two approaches are suggested: enacting laws preventing
traffic, tax law in traffic areas to reduce the number of cars in the traffic areas, and encouraging the
use of hybrid buses, electric trains and cars in Madinah. It is, however, important to note that the
higher correlations found in this study were likely due to the controlled environment.Saudi Ministry of Education and the Saudi Cultural Bureau
in Londo
Double stator switched reluctance machine with mutual coupled windings
Get PDFPh. D. ThesisThis thesis describes the development of a four-phase double-stator switched-reluctance motor (DSSRM) with mutually coupled windings. This machine, which has a topology that combines features of both the mutually coupled and double-stator switched-reluctance motor, will be shown to have the potential to offer improved torque productivity under the same ohmic loss limitation; though experimental mechanical and thermal confirmation remains to be completed.
It is widely thought that the electrification of vehicle traction will be an essential element in the automotive industry in the next decade. An eventual sales ban on diesel and petrol vehicles has recently been reported to be planned by several industrial countries. Even disregarding these political declarations, which could only be considered as expectation rather than strict policy, the spread of hybrid powertrain technology and the continuous fall of Li-ion battery prices will effectively motivate an increasing number of automotive manufacturers to participate in this revolution. Since the cost of rare-earth materials is expected to continuously rise and the mining of these resources has left an enormous impact on the environment, the development of a high-performance SRM as an alternative to a permanent magnet synchronous machine (PMSM) is worthy and necessary.
In addition to the enhancement of electromagnetic performance, this thesis discusses the mechanical properties and rotor structure of the DSSRM. The rotor support, which holds the rotor segments together, should be strictly of non-conducting material to prevent significant eddy current loss. Since most materials struggle to offer a compatible stiffness to steel, methods have been investigated to meet the challenge of designing a rotor support able to endure the centrifugal force of rotor segments during high-speed spinning. Thermal issues pose another challenge for this prototype but simulations indicate that the DSSRM could have better cooling capability than the conventional SRM configuration.
A prototype was built and compared with a 12/16 segmental-rotor SRM, previously developed at Newcastle University. The results indicated that the prototype machine gave a promising torque performance with a relatively low copper loss
Characterisation of circulating tumour cells in metastatic prostate cancer
Get PDFProstate Cancer, The Royal College of Surgeons of England and Cancer ResearchIn the UK, prostate cancer is the most prevalent male cancer and approximately 40% of men will have metastatic disease at diagnosis. The treatment pathway in metastatic prostate cancer is offered often without any histological or genetic knowledge of the tumour and there is currently no reliable biomarker to monitor response to treatment.
Over the past decade, there has been increasing interest in circulating tumour cells (CTCs). These cells, which have broken away from the tumour of origin and can be captured via a simple blood test, can be quantified, sequenced or examined for antigen expression. Stem cell marker expression, specifically Oct4, SOX2 and Nanog, has been found to correlate with aggressive disease when looking at solid prostate tissue. Consequently, exploring the expression of these markers in circulating tumour cells could enable the development of a new biomarker in prostate cancer.
This study had three aims. The first was to optimise an assay using flow cytometry to enable detection of the stem cell markers Oct4, SOX2 and Nanog, alongside epithelial and mesenchymal markers in CTCs from patients with metastatic prostate cancer. The second aim was to explore the prognostic role of these markers and the final aim was to culture CTCs from patients to enable downstream utilisation.
Blood was obtained from seventy-eight patients with different stages of prostate cancer and processed using two flow-cytometry based methods; one on the Imagestream, a combined flow cytometer and high-resolution microscope, and the second using conventional multi-channel FACS. Enumeration of total number of CTCs in addition to the individual marker positive cells was correlated with existing clinical data (PSA and Alkaline Phosphatase level, in addition to survival). Cells from six patients were successfully maintained in culture for up to a year. Attempts to prove the genotype of these cells included real time qPCR, SNP Array and whole exome sequencing experiments. Cells from one patient were implanted into five NSG mice and experiments to look at both chemokine expression and the Young’s modulus of the cells were also performed.
No correlation was found between either CTC count or antigen expression and the clinical outcome of the patients. Unfortunately, due to a lack of good quality DNA the sequencing
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experiments didn’t yield any data and despite one of the mice developing a hind leg paralysis, there was no histological evidence of an engrafted tumour. The chemokine receptor and Young’s modulus experiments showed promising early results and form part of some ongoing collaborative projects.
The first aim of developing an assay to detect prostate cancer CTCs was achieved, and survival data at a later time point is going to be collected to ascertain whether the markers explored in this study have a prognostic role. Whilst cells were successfully cultured, the genetic signature of these is still unclear and future plans to continue this work are currently in progress
The Effect of Twitter Dissemination on Cost of Equity: Evidence from Financial and Carbon Information
Get PDFPh. D. ThesisThe recent development of communication technology through social media, more
particularly Twitter, has changed the way that firms communicate with investors. This
channel has created an opportunity for firms to disseminate their information to investors on a
real-time basis directly. This thesis aims to investigate whether firms can influence their cost
of equity (COE) by broadly disseminating their information over Twitter. Employing a
sample of non-financial firms with a Twitter account, listed on the US NASDAQ stock
exchange over the period 2009-2015, the thesis comprises several objectives. The first
objective investigates the effect of firms’ dissemination of financial information (iDisc) over
Twitter on the cost of equity. The results find evidence that firms’ dissemination of financial
information over Twitter (iDisc) significantly reduces the cost of equity. These results are
pronounced for less visible firms that are relatively small in size, have a low analyst
following and a small number of investors. Highly visible firms are less likely to benefit from
iDisc influencing their cost of equity as other communication channels may have widely
disseminated their financial information. Additionally, further tests are employed to
investigate the influence of news magnitude on the examined relationship. The findings of the
additional tests show a significant negative association between iDisc and cost of equity. The
second objective is to investigate whether firms’ dissemination of carbon-related information
over Twitter, referred to as iCarbon, influences their cost of equity. The study finds that
iCarbon is significantly and negatively associated with COE. Also, additional tests are
applied to investigate whether Bloomberg's environmental (ENV) and environmental, social
and governance (ESG) disclosure scores influence the relationship between iCarbon and cost
of equity. The findings show a consistent negative association between iCarbon and cost of
equity after determining the effect of ENV and ESG score. The findings of the thesis
encourage managers to consider the benefits of directly disseminating financial and carbon
information to stakeholders and potential investors over Twitter to reduce firm equity
financing.Saudi Cultural Bureau and King Faisal Universit
Long-term landscape development in south-east Northumberland using orientation analysis
Get PDFPh. D. ThesisThe following thesis will explore the extent to which antecedent rural landscape features endured into later periods in south-east Northumberland using bespoke models built in GIS, and statistical analysis to calculate and compare the orientation of linear human-made features including boundaries, settlements and agricultural traces, and assess their relationship with underlying terrain.
The extent to which antecedent systems of land use may have influenced those which came after has preoccupied archaeologists and historians for many years in other parts of England; but has never been discussed in the current study area, which lay beyond the northern frontier of the Roman Empire. Chronological gaps in settlement and land-use have been identified, between the second and sixth-century AD; and between the ninth and twelfth-century AD. It is the purpose of the current research to produce evidence which may help to shed light on how people lived and worked in the landscape at these times. Numerous large-scale, developer-funded excavations during the last twenty years have transformed our knowledge of the region. The rich grey literature resource resulting from these projects are here placed into a research context which reaches beyond the chronological constraints of the sites and features themselves. The results of these analyses, in light of recent research in the region and beyond, has led to a proposition that if boundary features present on 1st edition Ordnance Survey maps share orientation with nearby ancient boundaries and settlement enclosures, they could represent long-standing land-divisions from at least the late Iron Age to the nineteenth-century and in some cases into the present. In general, however, many boundaries present on 1st edition ordnance survey mapping conform to underlying slope direction, which makes the above claim more ambiguous
Process planning methodology and evaluation of tool life for micromilling with an application to the fabrication of thin wall structure
Get PDFPh. D. Thesis.The scaling down effect on feature geometries and tools used in micromilling results in low feature stiffness and excessive tool wear. To achieve the required costs and tolerances, optimisation of the machining processes and their associated parameters are necessary which requires a thorough understanding of machining characteristics. Furthermore, the compensation must be sought for downscaling issues that arise at the process planning stage. Hence, the effect of the characteristics of the cutting tool, workpiece material and machining parameters are investigated in this research through a critical review of the literature followed by a numerical and experimental study of the impact of process variables. The research findings are used in the development of a process planning methodology for micromilling of components with application to high aspect ratio structures, to assist machine operators and to fill the gap between industrial and academic machining knowledge.
From the investigation of machining sequences, the study of machining layer strategy considering the sequence of removal of excess material using numerical simulation, strategic planning of machining layers in relation to feature stiffness is required, in particular to the machining of high aspect ratio features. The results from numerical simulation recommend an improved layer strategy for micromilling of thin wall structures, which were then experimentally validated in relation to machining time and geometrical and surface accuracy. The importance of planning tool entry and exit position in relation to feature rigidity was highlighted. The increase in depth of cut shows to improve the tool engagement reducing the thin wall deflection by 168 μm and appearance of the burr along the wall edge indicated by up to 200% drop in burr width. The investigation of tool paths showed the suitability of strategies for machining of circular and linear geometries. Also, the experimental findings emphasise on considering the feature geometry type in the selection of tool paths to achieve a balance between high-performance machining and improved productivity.
This study also investigates tool life, associated with flank wear rate, surface roughness, volumetric tool loss and the degradation of the cutting edge radius for micro endmills where a direct correlation between cutting speed and tool wear rate has been found. The new procedure for tool life prediction in conjunction with clear tool rejection criteria for the micro end mill is recommended. Along with standard procedure for the evaluation of tool change intervals to avoid tool failure and consequential defects in parts produced. In addition to the findings in the literature on machine process planning and findings from the study of machining sequence on the thin wall structure and tool life investigation conducted, a new process planning methodology for micromilling has been proposed. The process planning methodology includes
four distinct modules i.e. feature recognition, tool selection, machining parameter selection and machining sequence planning. The feature recognition module proposes a new approach to identify key feature faces and their corresponding machining attributes required for tasks in process planning. In the tool selection module, a new methodology for the evaluation of the machinability index and the tool replacement strategy for micro endmills are proposed to guide the operator in the task of tool selection and estimating tool replacement intervals. The machining parameter module provides a systematic approach for the selection spindle speed, feedrate and depth of cut. The machine sequence planning module assists the operator in selecting a suitable tool path and tool layer strategy along with a compensate technique for tool path errors.
An artefact with thin wall features has been fabricated using the methodology proposed and the conventional process planning method. The results show the part processed using the proposed methodology achieved better geometrical tolerance, and improved repeatability. It also show a 17% improvement in mean surface roughness, which demonstrates the effectiveness of the proposed methodology
The effect of explicit instruction and auditory/audio-visual training on Chinese EFL learner's perception of intonation
Get PDFPh. D. Thesis (Integrated)Intonation accounts for a big part in speech intelligibility and is notoriously difficult to be
acquired by L2 learners. The bulk of research on L2 intonation has focussed on the
examination of learners’ intonational performance at the phonetic and phonological levels
using perceptual and/or production tasks; however, empirical studies on whether and how
intonation training can help improve learners’ performance are surprisingly scarce. This study
fills this gap by devising instruction and training materials which were meticulously tailored
for Chinese learners of English, the largest population of English learners in the world. The
participants were 60 English-related majoring students from Newcastle University, most of
whom wanted to become English teachers following their studies. They were pseudorandomly
mapped into three groups according to their overall English proficiency. Two of the
groups were taught explicitly on the forms and functions of English intonation but one selfpracticed
auditorily on Audacity whereas the other audio-visually on Praat. The third group,
which served as control, did not get any intonation training. Learners’ competence of
intonation was assessed by a comprehension task before, immediately after, and two months
after the three-week training course. Ten native speakers of Southern British English were
recruited for the pre- and post-test to set a baseline for the analysis of learners’ performance.
The results are: 1. Chinese EFL learners did significantly worse than native speakers in terms
of understanding intonation meanings contrasted by accentuation, phrasing, and tone. 2.
Learners’ comprehension ability was improved immediately after the training for all three
aspects. 3. The training effect remain in the delayed post-test. 4. The audio-visual group did
not perform significantly better than the auditory group. The results indicate that certain
aspects of intonation are teachable and learnable, and tailor-made instruction and materials
are effective and applicable in use. This study provides English teachers in China with novel
ways to equip Chinese EFL learners with greater intonational competence