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The role of miR-140 AND miR-455 in murine skeletal development and osteoarthritis pathogenesis
Get PDFPh. D. Thesis.Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder, which is
complex and characterised by degradation of articular cartilage of joint tissue. Epigenetic
changes alter transcriptional regulation and disrupt signalling pathways involved in cartilage
homeostasis leading to OA, and dysregulation of and by microRNA (miRNA) (e.g. miR-140
and miR-455) correlate with bone and OA development. Bioinformatic analysis of RNAsequencing (RNA-seq) could reveal the mechanism of the function of miR-140 and miR-455
in murine skeletal development and OA pathogenesis.
Aim: Generation of Mir140-null and Mir455-null mouse model to understand their role in
relation to murine skeletal and OA development.
Material and Methods: By CRISPR/Cas9 technology we generated two mouse lines, for
Mir140 (miR-140-/-
) and Mir455 (miR-455-/-
). These mice were interbred to derive mice with
deletions of both Mir140 and Mir455 (DKO). On the mutant mice, we performed
phenotyping of skeletal X-ray, weight growth curve, and tibial growth plate analysis,
including histological staining and immunocytochemistry (BrdU labelling to monitor
proliferation and TUNEL labelling to monitor apoptosis). Destabilisation of the medial
meniscus (DMM) surgery was performed on mutant mice to assess knee articular cartilage
integrity following joint trauma. Differential expression of genes within the growth plate of
the mutant mice were identified by RNA-seq and potential upregulated miRNA predicted
targets validated by 3’UTR luciferase analysis. The expression of two growth plate targets
(Creb3l1 and Nrf2) were assessed by western blot analysis in isolated chondrocytes.
Transcriptome analysis was also performed on micro-dissected knee articular cartilage (preand post-DMM surgery) and on laser micro-dissected growth plates.
Results: Genotyping analysis confirmed the deletion of miRNAs in our mutant mice.
Phenotyping of our Mir140-null mice matched that of previously published work, including
mild short stature, domed skull, and a short tail. Our Mir455-null mice were dwarf when
compared to control mice, however, Mir455-null mice housed at our collaborator were
undisguisable from age-matched control mice. Our DKO mice shared a similar phenotype
III
with Mir140-null mice. Histological staining revealed abnormal tibial growth plate
morphology and delayed secondary ossification in 1-week and 3-week-old mice. A reduction
in tibial growth plate chondrocyte proliferation and increased apoptosis was detected in
mutant mouse lines. Mutant mice exhibited different transcriptome profiles of costal
chondrocyte by RNA-seq, and three miR-140 growth plate predicted target genes (Creb3l1,
Nrf2 and Zeb1) were confirmed to be targets and to attenuate chondrocyte proliferation by
WST-1 assay analysis.
Following DMM-surgery all mutant mice showed higher OA score with either Glasson
scoring system or OARSI scoring system, and all exhibited significant proteoglycan staining
loss compared to control mice. Comparison of transcriptome profiles before and after DMM
surgery had led to understand the function of miR-140 during OA pathogenesis.
Discussion and conclusion: Our mutant mice showed clear growth phenotypes compared to
control mice, however the phenotyping of our Mir455-null mouse line was different to
recent literature and to similar animals at our collaborator facility. Histological and
immunohistochemistry analysis confirmed that mutant mice exhibited abnormal growth
plate morphology, with reduced chondrocyte proliferation and increased apoptosis.
Transcriptome profiling from costal chondrocyte and knee cartilage chondrocyte (pre and
post-DMM surgery) represents an important tool to identify miRNA target genes and,
herein, to gain insights about miR-140 and miR-455 in skeletal developments and OA
pathogenesis
Physiological and molecular responses to water-stress in local Saudi wheat cultivars
Get PDFPhD ThesisThe kingdom of Saudi Arabia (KSA) is among the top countries in terms of agriculture in desert areas, the country uses highly developed agricultural technologies to grow different crops under challenging environmental conditions. The global climate change and the consequent increase in temperature and drought especially in the arid and semi-arid regions made the situation even more challenging. This work aimed at determining the physiological and molecular mechanisms underpinning drought-tolerance in wheat using six local Saudi wheat cultivars. Understanding the phycological responses and gene regulations under water stress could contribute to improving wheat cultivation in Saudi Arabia. My thesis was divided into four main experimental chapters, each chapter describes one experiment. The first experiment was to assess drought tolerance in a collection of six known wheat Cultivar grown in different regions of the KSA by comparing their growth and yield under well-watered conditions and water-stress conditions. Shoot weight and length, Root weight and length, Root: shoot ratio, RWC, proline content, soluble sugar content and protein content in addition to yield were used as selection criteria for drought resistance. The results allowed to group the studied wheats into two groups, drought resistant (193 Najran (Cv2) and 357 Sama (Cv3) ) and drought sensitive (181 Jizan (Cv1), 377 Rafha (Cv4), 562 Ma’ayah (Cv5) and 981 Najd (Cv6)). In the second experiment, RNA sequencing was performed in leaf samples harvested from water control and PEG-treated plants of one drought resistant 193 Najran (Cv2) and one drought sensitive cultivar (377 Rafha (Cv4)) at the vegetative, flowering and grain filling stage. Transcriptomic analysis aimed at finding differentially expressed genes and associated metabolic pathways in the two Saudi wheat cultivars under water stress at the three growth stages. This yielded 24.2 GB of sequence data. At least 40 million reads of 85 bp each were obtained per sample, Although genes from different pathways changed expression under water-stress, the increase in expression of genes associated with Photosynthesis, Amino acid metabolism and Secondary metabolism such as phenylpropanoid biosynthesis seemed to be the most important. The third experiment investigated the drought induced changes in the proteome in drought resistant 193 Najran (Cv2) and drought sensitive cultivar (377 Rafha (Cv4)) at the vegetative, flowering and grain filling stage, Proteomics analysis showed tangible changes in protein levels indicated a general regulation trend of plant defence under water stress, such as Stress/defence/detoxification proteins, Photosynthesis proteins, Carbohydrate metabolism proteins and Amino acid metabolism proteins. In a fourth experiment, based on the results of the transcriptomics and proteomics results together with the literature, the expression of four drought
related genes The four genes included Dehydrin gene (DHn3), Bidirectional sugar transporter (Sweet), Phenylalanine ammonia-lyase (Pal5) and Serine hydroxy methyl transferase (Shmt) found to be various regulation in the six wheat cultivars based on their response to water stress. using qRT-PCR. The qRT-PCR analysis confirmed increased expression levels of these genes known to be up-regulated under water stress.
From this work, I could conclude the following:
1. There is a big difference in the speed of response to water-stress between wheat Cultivars, this difference is associated with variation in transcript and protein expression levels at three growth stages.
2. The vulnerability of wheat plants to water stress is higher at the flowering stage compared to the vegetative and grain filling stages. Attempts to improve drought tolerance in wheat should be targeted to this growth stage.
3. The phenylpropanoid metabolic pathway plays a key role in resistance to water-stress in wheat and might be a target for improving drought resistance in this crop
Deciphering liver fibrosis with next generation omics
Get PDFPh. D. Thesis.Fibrotic diseases underpin around 45% of all chronic diseases and deaths in the western world.
In particular, chronic liver diseases represent a major threat to the public health affecting
around 1.5 billion people in the world and accounting for 2 million deaths annually.
Hepatic fibrosis is a progressive pathology characterized by a dysregulation of the woundhealing response, resulting in extracellular matrix (ECM) accumulation. Hepatic Stellate cells
(HCSs) are the major contributor to ECM deposition in liver diseases. Thus, HSCs are crucial
for liver fibrosis progression. Understanding molecular mechanisms of HSC activation is
therefore of major interest in study of liver fibrosis. To this end, miRNA sequencing during
HCSs activation time-course was employed, which showed significant differences in miRNA
expression which may be important for fibrosis progression.
2D cultures are widely used in the study of liver disease. However, they do present limitations,
which can be overcome by the use of Bioreactor-cultured Precision cut liver slices (PCLSs)
technology developed in our lab. Using this technology, human PCLSs were created and RNA
sequencing, proteome and secretome analysis performed. In this thesis, I showed that fibrotic
PCLSs present differential gene/protein expression patterns that could be associated with the
fibrotic outcome.
Previous studies in our lab reported an epigenetic mechanism that generated heritable
adaptation in wound healing response in male rats. The adapted rats showed significant
fibrogenesis/fibrosis reduction. I was interested in studying the mechanism behind the
adaptation. Using omics techniques, I analysed gene expression pattern in adapted livers and
differences in DNA methylome and proteome in sperm, as potential mechanism for
adaptation transmission. Moreover, I studied the potential of adaptive inheritance
transmission via female lineage.
This study provides evidence of i) the importance of miRNA during HSCs activation, ii)
differential gene/protein expression patterns associated with the fibrotic outcome in human
liver, iii) differential gene expression in adapted livers and epigenetic modifications in sperm
as a putative mechanism of the adaptive response transmission. This adaptive response was
absent in female offspring, suggesting only males can transmit the adaptation.Dr Lorna Jones Legacy gif
The Adoption of Pervasive Technology in Private Spaces: Exploring Pre-Exposure Beliefs and Post-Exposure Outcomes Using Cognitive Dissonance Theory
Get PDFPh. D. Thesis.The development of pervasive technology for homes has always revolved around ever-growing consumer needs for comfort, a better home experience and the convenience of technology operation. Smart home technologies promise to deliver financial, environmental and health-related benefits through real-time control and management of resource consumption, remote monitoring and support, and other tailored services for users. However, despite the benefits of the technology for its users, the adoption rate is still low. Low adoption incurs the risk that the technology’s potential will never be realised, decreasing its positive implications for individuals and society in general. Against the backdrop of the low implementation of smart homes and their fast-paced development, it is important to examine technology utilisation from the user’s perspective, focusing on beliefs that underpin the acceptance and the perceived outcomes of performance. Given that new technology raises high expectations, which may undermine post-performance evaluation, it is important to consider the psychological factors that the perception and experiences of the promised performance entail. This will provide valuable evidence about the conditions which lead people to continue with or abandon the technology.
The academic community has intensified its efforts to examine the concept of the smart home, its technological capabilities, its implications and the impact on people’s lives, but the literature still lacks empirical evidence about the users’ perspective on the utilisation of technology. Users’ beliefs, such as the expected benefits and risks which may facilitate or inhibit trialling the technology in private spaces have been under-researched. Studies have examined interaction with technologies irrespective of the context, thus decreasing the validity of the analysis of situational behaviour. However, the utilisation of technology in private settings is contingent on psychological factors, the perception of outcomes, motives and beliefs. Those factors affect the perception of the values and risks that the use of the technology might entail. Secondly, there is still a lack of insight into the outcomes of the use of technology when the performance falls short of initial expectations. The behavioural and cognitive responses following poor technology performance and the coping mechanisms that users deploy to ameliorate negative consequences are under-researched.
Given the gaps in the literature, the first focus of the thesis was to examine the user’s perspective on smart home utilisation by examining the beliefs that underpin the adoption of the technology. The research adopted the Task Technology Fit (TTF) model, integrated
with the constructs that pertain to the users’ perception of technology performance, such as perceived usefulness and perceived ease of use. While TTF stresses the importance of the “fit” factor when it comes to task-related behaviour, perceived usefulness and perceived ease of use explain the attitudinal underpinnings of the behaviour. Additionally, the model aimed to explain whether utilitarian, hedonic values, privacy and financial risks influence the users’ perception of task-fit. The second focus of this thesis was to explore individuals’ behaviour when technology performance falls short of expectations. The hypotheses were drawn from the literature in the confirmation-satisfaction and cognitive dissonance domains. Such an approach made it possible to examine psychological, behavioural and cognitive factors following a negative experience with technology. Post-performance dissonance arousal reflecting the psychological discomfort induced by the discrepancy between performance and expectations was examined. Furthermore, the adoption of cognitive dissonance theory aimed to explore the role of different types of emotions associated with dissonance and their role in post-dissonance behaviour. The motivational roles of each emotion in predicting coping strategies for reducing dissonance, such as behaviour change, attitude change and information seeking, were investigated.
Two online surveys were conducted to address the objectives of the thesis. The first survey focused on examining the antecedents of pervasive technology adoption by smart home users. The data for the first survey was collected from 422 respondents located in the United States. The focus of the second questionnaire was to examine the behaviour following disconfirmed expectations. Therefore, only smart home users who had had a negative experience with using smart home technologies were eligible to participate in the survey. After filtering non-eligible cases, the final sample consisted of 387 responses. Both questionnaires consisted of two parts: 1) questions related to the socio-demographic characteristics of the respondents, and 2) questions designed to measure the variables for the model. For the analysis of the data, structural equation modelling was utilised.
Results indicated that hedonic and utilitarian beliefs are critical for the perception of task fit, whereas privacy and financial factors were found not to be significant. The fit between tasks and technology demonstrated its significant role in predicting perceived usefulness, perceived ease of use, use behaviour and satisfaction. Lastly, use behaviour showed a positive correlation with satisfaction. When it came to examining the outcomes of performance following disconfirmed expectations, results indicated that weak technology performance induces dissonance due to the discrepancy between expected and actual
technology performance. Dissonance triggered feelings of anger, guilt and regret. The arousal of those emotions activated distinctive dissonance reduction mechanisms aimed at reducing psychological discomfort through attitude change, behaviour change or information-seeking mechanisms. Behaviour change was selected when people felt anger and regret, while consonant information-seeking and attitude change were selected when people felt guilt. The coping mechanisms, in turn, had different effects on satisfaction and wellbeing. Satisfaction and wellbeing were achieved when people coped with dissonance by changing their attitude to the technology or searching for information to justify the use of the technology. The withdrawal of behaviour increased the likelihood of feeling dissatisfaction and reduced the likelihood of perceiving wellbeing.
The results of this thesis make several contributions. The findings contribute to the literature on the acceptance of pervasive technology in private spaces. Evidence on the role of beliefs pertaining to technology utilisation (i.e. task-technology fit, perceived usefulness and perceived ease) in private spaces moves forward the theoretical front in the domain of smart homes. In addition, the examination of psychological beliefs (i.e. hedonic value, utilitarian value, privacy and financial risks) with the task-technology fit factor explained the facilitating and inhibiting conditions in which the technology is most likely to be perceived to be compatible with users’ needs. Secondly, insight into consumer experience after technology widens the boundaries of the research on innovative technology acceptance, which has predominantly focused on the underpinnings of adoption as opposed to the outcomes of initial use. The results of the thesis provide evidence about behavioural outcomes following the utilisation of technology when performance falls short of expectations. Such an approach adds to the literature adopting the expectation disconfirmation paradigm, by providing a different perspective on the behavioural outcomes of disconfirmed expectations. In contrast to prior research, the results indicate that the disconfirmation of expectations can lead to positive outcomes, such as satisfaction and perceived wellbeing. Thirdly, the results widen the application of cognitive dissonance theory, by identifying the complex psychological, cognitive and behavioural processes following the evaluation of technology performance. As far as practical implications are concerned, the results inform practitioners about the factors to focus on when developing technology to satisfy a broader user segment. Also, they provide suggestions on marketing and communication strategies that may eliminate the likelihood or the consequences of disconfirmed expectations
A Monte Carlo simulation study of the factors influencing the performance of flood early warning systems
Get PDFPhD ThesisIn recent decades, flood early warning systems (FEWSs) have been widely used as
complementary non-structural mitigation measures in order to improve the
population resilience to floods. FEWS research focusses mainly on flood
forecasting techniques or social aspects of warning response, and end-to-end
modelling frameworks that represent the entire FEWS forecast-decisionresponse/impact chain are rarely developed.
A generic Monte Carlo simulation framework has been developed that represents
an end-to-end FEWS in a versatile way, allowing factors influencing FEWS
performance to be explored which cannot be analysed easily based on limited realworld data. The framework has been applied to a simulated generic fluvial case,
where factors influencing FEWS performance in terms of reliability and economic
effectiveness are explored. A new reliability performance measure based on
inundation maps has been proposed. The framework has also been used to explore
factors controlling the performance of a simulated FEWS representing an urban
polder in Nanjing, China, with performance metrics based on waterlogging and
pumping costs.
For the generic fluvial case, the main results show that: i) the correlation between
forecasts and observed values controls reliability; ii) probabilistic forecasts based
on optimising a probabilistic threshold are robust to forecast biases in the mean and
variance, iii) a FEWS based on uncertain forecasts is characterised by an optimal
lead time that represents a balance between an adequate time to act in response and
a reasonably good forecast; iv) the performance of the proactive action is the most
important factor influencing the economic effectiveness of a FEWS. For the
simulated flood-prone polder system case study, the results show that probabilistic
forecasts of storm rainfall and runoff volume can considerably enhance the
waterlogging and pumping metrics.
The results of this research can be used to improve the performance of fluvial
FEWSs, and to design FEWSs for polder systems.Ph.D. scholarship from
the Secretary for Higher Education, Science, Technology and Innovation
(SENESCYT) of the Government of Ecuador
Investigation of oral health assessment measures for the care planning of dependent adults
Get PDFPh. D. Thesis.Dependent adults (i.e. eighteen-year-old or older who need or receives assistance due to
a reduction in mental capacity or physical capability) can experience oral health
problems. Whilst the reasons behind these problems are complex, evidence suggests
that caregivers’ limited knowledge about oral health and care may play a role. Oral
health measurement instruments could provide one means by which to help caregivers
establishing appropriate oral care plans for dependent adults and, thus, preventing or
managing these problems. This project aimed to investigate oral health assessment
measures for oral care planning of dependent adults through three separate studies.
A qualitative evidence synthesis was undertaken to establish an empirical conceptual
model of oral health in dependent adults. It showed that oral health in dependent adults
is a dynamic and multidimensional construct that consists of four domains: the
intactness and cleanliness of oral structures; oral pain and discomfort; oral functions;
and noticeable oral health aspects.
A systematic review was undertaken to critically appraise published oral health
measurement instruments for dependent adults. It revealed many instruments have
been proposed, but none of these instruments has been adequately and
comprehensively tested to establish strong evidence in relation to their measurement
properties, feasibility and interpretability.
Finally, a qualitative interview study was undertaken to explore healthcare service
providers’ perspectives on barriers and implementation issues in relation to measuring
oral health in dependent adults. The consistent theme was that the implementation of
oral health measurement instruments may not be easy due to multiple potential
barriers. These barriers therefore need to be considered during implementation,
including those related to instruments, service providers, dependent adults and
implementation environment.
The findings in this thesis support a recommendation that multiple perspectives need to
be considered when designing and evaluating oral health measurement instruments used
to guide oral care planning for dependent adults. By doing this, these instruments are
more likely to meet different stakeholders’ needs and, thus, can be successfully
implemented.King
Saud bin Abdulaziz University for Health Science
Philanthropy and the socioeconomic development of Pakistan
Get PDFPhD ThesisThis thesis evaluates the present role and future potential of philanthropy in the socioeconomic
development of Pakistan. It is the first in-depth study on the topic and adds to the wider
literature on socioeconomic development in developing countries beleaguered by poverty,
inequality and seemingly relentless population growth. The principal objective is to better
understand the role played by philanthropically funded third sector organizations in addressing
developmental and inequality challenges in countries like Pakistan. In doing so, the study
identifies the motivations of philanthropic actors in establishing charitable organizations,
examines the governance and management practices of such organizations, and explores the
socially innovative practices of five case study organizations and the magnitude of their
contributions to social well-being.
The study is focused on answering a core research question - what is role of
philanthropy in the ongoing socioeconomic development of Pakistan? – and takes an
interpretivist approach, carefully considering both quantitative and qualitative evidence and
guided by appropriate theory, notably Bourdieusian social theory. Quantitative data were
gathered from international development agencies and survey results published by the Pakistan
Centre for Philanthropy. Qualitative data were gathered from (a) a group of development
experts and policy makers and (b) five case study organizations by means of semi-structured
interviews, collection of documents and field observations. The quantitative data were used to
create charts and tables and proved valuable in assessing the importance of different forms of
philanthropic funding relative to government spending and overseas development assistance.
The qualitative data were analyzed thematically through coding and application of the Gioia
method of qualitative data reduction. My analysis is framed by four subsidiary research
questions, one for each thematic chapter.
The study makes four main empirical contributions to the literature on philanthropy
and socioeconomic development in Pakistan. First, I show that philanthropy has played and
continues to play a supporting role to government and that in quantitative terms the supply of
philanthropic funds has struggled to keep pace with demand. Second, I demonstrate that
philanthropy is inextricably linked to social activism with funds raised from the many not the
few to support front-line charitable organizations in addressing fundamental social needs.
Third, I find that there is a variable approach to governance within the philanthropic sector and
make the case for more regulation as a precondition for increased philanthropic funding.
Fourth, I show that the main contribution of philanthropy to the socioeconomic development
of Pakistan stems from social innovations that help in satisfying unmet social needs in
healthcare, education and community development. At a more abstract, theoretical level, I
identify and specify the role of indigenous social activists as pivotal actors in delivering the
social innovations at the heart of the development process and move beyond viewing
philanthropy as a monolithic construct to identify the agentic nexus between philanthropy and
social activism as crucial in creating and embedding new organizational models and practices
that deliver beneficial economic and social change.Charles Wallace Trust UK for the grant of Charles Wallace Fellowshi
Development of Novel PET-MRI Methodologies for Dementia Research
Get PDFPh. D. Thesis.Changes to cerebral mitochondrial function have been implicated in neurodegenerative
pathology and in the process of healthy ageing. Phosphorus MR spectroscopy (31P-MRS)
with magnetisation transfer (MT) can be used to make direct in vivo measurements of
mitochondrial function. By combining 31P-MT with simultaneous FDG-PET imaging
it is possible to assess the relationship between glucose hypometabolism, which is a
characteristic feature of Alzheimer’s disease, and mitochondrial function.
The primary aim of this project was to develop and evaluate the methodology for
simultaneously measuring glucose metabolism and mitochondrial function with PETMR.
An accelerated technique for 31P-MT was implemented, utilising kinetic modelling to
reduce the acquisition time. This facilitates a multi-voxel implementation, allowing the
rate of the creatine kinase reaction, k1, to be measured in multiple locations across the
brain within a single scanning session. This approach was tested in phantoms and in vivo
through an assessment of repeatability and comparison with a more established technique.
The methodology for performing PET attenuation correction with non-standard MR RF
coils was also developed and evaluated.
The 31P-MT methodology was applied to investigate regional changes in mitochondrial
function during the healthy ageing process. A global reduction in k1 was observed between
young and middle-aged subjects, followed by a stabilisation between the middle-aged and
elderly groups. This pattern is driven by voxels in the anterior region of the brain, with
mitochondrial function remaining consistent across the age groups in the posterior region.
This suggests that normal cognitive function in old age is associated with the maintenance
of k1 in the posterior region of the brain.
The combined PET-MRS methodology was applied in a small number of healthy subjects
to demonstrate the approach to probing cerebral bioenergetics. This methodology has
the potential to provide new insights into the pathological mechanisms of mitochondrial
disease and Alzheimer’s disease.GE Healthcar
Evolution, environmental distribution, and engineering of the abyssomicin biosynthetic gene cluster
Get PDFPh. D. Thesis.Evolution, environmental distribution, and engineering of the abyssomicin biosynthetic gene
cluster.
Microbial secondary metabolites constitute a great source of pharmaceutically interesting
biomolecules. In particular, the tetronate family of natural products is a structurally and
functionally diverse group of secondary metabolites whose potent bioactivities make them
attractive targets for clinical and industrial exploitation. The abyssomicins are an actively
growing family of small spirotetronate natural products that has been widely studied due to the
unique structural features and bioactivities that some of its members exhibit, including
antimicrobial activity against Gram-positive bacteria such as methicillin- and vancomycinresistant Staphylococcus aureus and different Mycobacteria strains, HIV inhibitory and
reactivator properties and anti-influenza A virus activity.
Abyssomicin C and its atrop- isomer, produced by the slow growing marine Actinobacteria
Micromonospora maris AB-18-032 T
, are type I polyketide antibiotics that inhibit the formation
of p-aminobenzoic acid, a constituent of the folate pathway. Abyssomicin biosynthesis is highly
amenable to reengineering, as the enzymes involved in the synthesis of the tetronate (AbyA1)
and the spiro-tetronate-forming Diels-Alderase (AbyU) are both capable of accepting
structurally diverse substrates. The aim of this project was to set up the grounds for the
discovery and production of novel abyssomicins with applications in the biopharmaceutical
industry. First, in order to understand the environmental distribution and evolution of the
abyssomicin biosynthetic gene clusters (BGCs) present in nature, an analysis of publicly
available genomic and metagenomic data was carried out. The strategy of selecting a pathwayspecific enzyme to direct the mining proved to be an excellent strategy; 74 new Diels–Alderase
homologs were identified and a surprising prevalence of the abyssomicin BGC within terrestrial
habitats, mainly soil and plant-associated, was unveiled. Five complete and 12 partial new
abyssomicin BGCs and 23 new potential abyssomicin BGCs were also identified, suggesting
that a plethora of abyssomicins remain to be discovered. A preliminary study on the
abyssomicin production potential of five of the strains containing potential abyssomicin BGCs
was also carried out although no abyssomicins were found.
After that, with the final goal of producing abyssomicins of various lengths and different
saturation/oxidation patterns, it was necessary to express the aby BGC of M. maris AB-18-032
in a well-established heterologous host. This cluster was successfully moved into E. coli and
various Streptomyces species, the abyssomicin production potential of these strains was
evaluated in various conditions and some of the hosts were promoter engineered to force the
expression of the aby BGC. Active gene expression was demonstrated, but despite the efforts,
none of the heterologous hosts produced abyssomicins. Later analysis unveiled the presence of
several mutations within abyB1, the first polyketide synthase gene in the aby BGC, suggesting
this could be the reason for the lack of production.
Since the approach to heterologously produce abyssomicins was not fruitful, this work then
focused on increasing abyssomicin production in M. maris AB-18-032 and developing genetic
tools for this system. First, through ribosome engineering, a library of M. maris drug-resistant
mutants capable of producing up to 3.4-fold abyssomicin C in comparison to the wild-type
strain was generated. Then, using statistical Design of Experiments (DOE), an efficient
electroporation protocol that could accelerate targeted genetic manipulations in M. maris was
developed. Together, increased abyssomicin production and a quick and easy electroporation
protocol for M. maris, will facilitate future engineering of the aby BGC directly in M. maris to
produce diverse non-natural abyssomicin
Multiphase wireless dynamic charging systems for electric vehicles
Get PDFPhD ThesisElectric vehicles (EVs) have been intensively developed as an attempt to reduce carbon-dioxide emissions caused by fossil-fuel vehicles. EVs require expensive batteries and power electronics for charging and discharging the battery. Unfortunately, battery technology, such as lithium-ion batteries requires substantial improvements to effectively compete with fossil-fuel cars in price. Also, batteries are usually heavy, take up large space and still have range limitation. Wireless Dynamic Charging (WDC), while the vehicles are in motion, is seen as an alternative to overcome the drawbacks associated with batteries. Due to the continues charging when driving, batteries can become smaller as most of the traction energy comes from the grid directly. WDC is fundamentally developed based on inductive power transfer (IPT) technology, where a time-varying magnetic field is generated by transmitter coils, which are installed underneath the road surface, to wirelessly power receiver coils, that charge the EV’s battery continuously.
Presently, there are several technical challenges associated with WDC, which hinders commercialization. The output power fluctuation along the driving direction is one of the most serious problems. These fluctuations cause reduction in constant energy transfer thus requiring larger batteries. Also, batteries lifetime is significantly reduced as a result of increasing internal heating. Several studies attempted to realise constant output power for WDC. However, proposed methods so far, have disadvantages such as high cost, complexity or unable to sustain constant output power throughout the charging process.
The work in this thesis proposes a multiphase WDC system to simultaneously achieve constant and high output power for EV applications. The proposed WDC system utilizes multiple primary windings that guarantee a homogeneous mutual magnetic flux for the receiver along the driving direction. This results in a constant induced voltage across the receiver and hence constant output power to charge the EV battery. High output power capability is attained by using multiple transmitter windings arranged in a novel winding method. The effectiveness of the proposed system is analytically described, simulated and demonstrated experimentally using a 3-kW laboratory prototype with the three-phase transmitter. The proposed system requires only simple control, eliminates communications between the primary and secondary sides and delivers 125% higher power transfer capability compared to conventional single-phase WDC systems