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Stem cell bandages – an investigation into the therapeutic effect of adipose-derived mesenchymal stem cells in corneal wound healing
Get PDFPh. D. ThesisHuman corneal regenerative capacity is not enough to heal deep injuries that
causes scars, such abrasions result in the formation of the irregular extracellular
matrix that leads to scar and often results in corneal transparency and vision
reduction. Because of the limited number of corneal donors for transplantation, there
has been an increased interest in cell therapy. Adipose-derived stem cells (ASCs)
showed a promising choice for they are environmentally responsive in wound repair.
ASCs can secrete paracrine factors and extracellular molecules that have
immunomodulatory, angiogenic, trophic, anti-fibrotic, and antimicrobial activity. This
thesis explores the formation of bandages; such bandages are formed of sodium
alginate. Alginates are present in nature and extracted from brown seaweed.
Generally, this material is safe, cost-effective, and wildly used in biomedical
applications due to its biocompatibility and ease of handling. Hypothermic
temperatures are also explored in here for bandage storage. Hence, this project
hypothesized the formation of hypothermically stored ASCs bandages and their
beneficial effect on corneal injuries. ASCs bandages were found to improve in
vitro corneal scratch wounds, while storage at 15°C presented a favorable condition
to store the bandages to vastly enhance their therapeutic potential. This storage is
suggested to be a hypoxia-induced environment that augments cells ability to
produce paracrine factors. Thus, hypoxia-induced ASCs bandages showed a
promising therapeutic ability in healing corneal injures. Gene assessment and
protein array revealed the production of several paracrine factors including HGF,
TSG-6, IL-8, MCP1, TSP-1 that aid in wound healing. In vivo, hypoxia-induced ASCs
bandages were transferred to adult male mice corneas following a chemical burn,
where reduction of immune cell infiltration was observed. Thus, supporting this thesis
hypothesis
The role of starch in the day/night re-programming of stomata in plants with Crassulacean Acid Metabolism
Get PDFPh. D. ThesisCrassulacean acid metabolism (CAM) is a specialised type of photosynthesis characterised by the unique inverted stomatal rhythm, which increases water use efficiency (WUE) and enhances the potential for sustainable biomass production in warmer and drier conditions. Starch turnover in the mesophyll of CAM species supports nocturnal CO2 assimilation and CAM activity. In C3 plants, starch metabolism has been reported to play an important role in determining stomatal behaviour; in this case, guard cell starch degradation is triggered by blue light, producing osmolytes that promotes stomatal opening. Based on the importance of starch and the little knowledge regarding CAM stomatal behaviour, this study tested the hypothesis that starch metabolism has been re-programmed in CAM plants to enable nocturnal stomatal opening, by using biochemical and genetic characterisation of wild type and RNAi lines with curtailed starch metabolism in the constitutive CAM species Kalanchoë fedtschenkoi.
Measurements of guard cell starch content over 24 hours in wild type plants of K. fedtschenkoi indicated a day/night shift in starch turnover compared to C3, evidenced by an increment over the first hours of the day and a diminution at the beginning of the night. The characterisation of the RNAi lines confirmed that curtailed starch metabolism affected nocturnal CO2 fixation, besides the phosphorolytic starch degradation has a pivotal role for driving nocturnal CAM activity evidenced, in the same way, in both mesophyll and epidermis proteome datasets. Furthermore, higher levels of soluble sugars in the epidermis of the RNAi lines appeared to curtail completely stomatal closure during the day, indicating that guard cell starch biosynthesis is an important sink for carbohydrates, ensuring day-time stomatal closure.
Finally, this thesis constituted an approach for the understanding of starch metabolism in CAM stomata and together with further studies lead to the potential engineering of CAM into C3 to enhance WUE.Government of Colombia through the Administrative Department of Science, Technology and Innovation – Colciencias and the Newton-Caldas fund partnership
RC/GC developing tools for the remote control of genetic circuits
No full textPhD ThesisWith the advent of industrial biotechnology, we can use microbes to produce myriad valuable compounds with a range of useful applications. These may be drugs, flavourings preservatives or many others. With contemporary synthetic biology approaches we are able to bring together enzymes from a variety of different genomic contexts and build pathways that don’t yet exist in nature. This naturally proposes an increase in complexity in pathway design and function, that will require more complex methods to regulate and control.
One way to achieve this regulation is using magnetic fields. Physical stimuli like this hold many advantages over the chemical induction methods used contemporarily. Every cell could be reached simultaneously using physical stimuli, promoting metabolic synchronicity. There is also no risk of some cells sequestering chemical inducers, leaving some cells experiencing greater levels of induction, and others none at all.
A magnetic stimulus could penetrate an entire culture and stimulate each cell simultaneously and repeatedly. Magnetic nanoparticles when placed in oscillating magnetic fields have their magnetic dipoles forcibly switched to align with oscillations. At high frequency magnetic oscillations, the MNP loses some energy as heat. By conjugating magnetic nanoparticles with temperature sensitive proteins it may be possible to produce stimulus of specific genetic elements with hitherto unseen precision.
To achieve this, microbes must be equipped with two things – a magnetic “aerial” module which can receive the magnetic stimulus, and genetically encoded heat-sensitive apparatus that responds to changes in temperature induced by the magnetic field.
This thesis has surveyed the potential for Pd nanoparticles to perform the role of a magnetic aerial, and heterologous gene expression with genes from magnetotactic bacterial genomes to produce magnetic material de novo. This strand of work illuminated the potential for a model contrary to how Pd-NPs are made in E. coli. This being that instead of microbial hydrogenases directly nucleating production of a nanoparticle in the active site of the enzyme, hydrogen produced by microbial hydrogenases reacts with soluble Pd(II) precipitating Pd nanoparticles.
Work performed in parallel has provided a novel mechanism to produce magnetic material de novo using heterologous expression of the mms6 gene from Magnetospirulum gryphiswaldense.
This work has also used contemporary synthetic biology approaches to produce genetic programs that respond to temperature changes, using the genes tlpA, mogR and gmaR, with mogR and gmaR being new
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additions to the pantheon of genetic elements used in synthetic gene circuits. This thesis contains designs, characterisation and optmisation of functional novel genetic circuits which utilise mogR and gmaR in the pMOGMAR-20K plasmid.
Using genetic elements characterised in this work in concert, the apparatus to begin producing novel magnetoreceptive mechanisms in E. coli is now available
Narrating employability from English Studies : an ethnographic study
Get PDFGovernment policy assumptions about employability characterise students as rational economic actors, motivated by maximising the return on their investment in higher education. Furthermore, policy documents emphasise the greater economic significance and potential returns from studying Science, Technology, Engineering and Mathematics (STEM) and more vocational disciplines. Beginning with a review of the literature and the political and historical developments which have led to the marketization of higher education, I trace the rise of employability as a high stakes issue. My thesis challenges government policy assumptions about employability with a specific focus on the non-vocational discipline of English Studies at Northern University. I explore the complexity and range of motivations which underlie individual choices of discipline, university and career and provide an account of employability from English through a longitudinal ethnographic study. I draw on my ethnographic fieldwork and semi-structured interviews conducted over five years to create case studies of the lives and early careers of students and graduates. In doing so I produce richly nuanced data about how employability is perceived, developed, embodied and articulated. I argue that employability is more than just getting a job, as government narratives imply; it is a complex, socially-embedded process of situated, lifelong learning. Using an analytical frame which draws on Bourdieu’s concepts, I explore the impact of social structures on choices and perceptions of employability. I offer participant narratives which evidence that career motivations are linked to identity and subjective considerations rather than simply maximising economic outcomes. Those narratives contextualiseskills within practice. My research confirms that English can lead to a wide range of careers. I place my findings in the context of changing workplace demands as the pace of technological change accelerates and conclude that the skills and capabilities which are developed by studying English are increasingly valuable for 21st century workplaces
Stroke thrombectomy : new devices, imaging, scoring system and outcomes in the older population
Get PDFPh. D.In the past several years there have been major developments in the interventional treatment of acute ischaemic stroke. This thesis consists of four projects that I developed throughout my graduate studies.
I started by working on setting up a phase 2 multicentre, randomised, controlled trial to assess the technical safety and efficacy of two new devices to be used in endovascular treatment: the ERIC retriever and the SOFIA distal access catheter. The trial has not yet finished, but partial results are presented.
I went on to investigate the role of imaging in hyperacute stroke management. I developed a validated case archive of computed tomography angiography scans which was used for a radiological course. Assessing trainees reports pre- and post-training showed that there was a significant improvement in rates of major errors and this study concluded that an intensive hands-on radiological course was effective.
While the trial was running, I developed a new technical index of thrombectomy difficulty. This score uses computed tomography angiographic images to evaluate key factors for predicting the expected procedural difficulty. The results demonstrated an excellent inter-rater reliability making this potentially a powerful tool to help with decision making and procedural planning.
Due to limited evidence for mechanical thrombectomy in the older population, I worked on a study to assess the safety and efficacy of this treatment. Patients had more comorbidities, more tandem occlusions, but still good reperfusion rates with a similar safety profile to the younger population. Clinical results at 3 months were poorer than in the younger population, but milder presenting clinical deficits and good reperfusion rates were predictors of good outcomes.
This thesis will first discuss stroke with a review of the evidence for performing mechanical thrombectomy. The projects undertaken during my studies will be discussed and I will finish with a succinct conclusion
Modelling the cryogenic properties of germanium for emerging liquid hydrogen power applications
Get PDFPh. D. ThesisIn recent years, there has been an increase in research focused towards the reduction and/or
elimination of greenhouse emissions from applications used in everyday life. In addressing
this, liquid hydrogen has been highlighted as an attractive alternative fuel source for commercial
vehicles due to it’s lower weight, higher power density and zero greenhouse emissions in
comparison to petrol and diesel fuels. Incorporating such a fuel source however introduces
a cryogenic environment of 20 K affecting the power electronics used to deliver the power
from source to load.
Herein, the physical properties of semiconductors influencing the overall efficiency of
devices within an H-bridge circuit are considered. From this, germanium is hypothesised to
be the most suitable semiconductor for power devices at or near temperatures of 20 K.
Closed-loop models are developed for the carrier concentration, carrier mobility, carrier
velocity, for both electrons and holes as a function of doping concentration and temperature
with critical analysis of the range of suitability for each. Multiple models are also developed
for both carrier concentration and carrier mobility which offer a trade off depending on
whether one requires accuracy or simplicity in calculation.
A significant influence on the device characteristics of MOSFETs is that of the oxide/semiconductor
interface. For the first time, ZrO2 is fabricated directly on germanium substrates
through the thermal oxidation of zirconium on germanium. The interface state density of
these capacitors are comparable to literature values offering a much cheaper and simpler
fabrication method for high-κ dielectric formation on germanium substrates. The leakage current density of the ZrO2 MOS capacitors are low in comparison to reported values and
are shown to decrease with decreasing temperature.
With the physical models of both bulk and interfacial germanium, multiple PiN germanium
diodes are simulated using technology computer aided design (TCAD) that show the
potential for germanium power devices with breakdown voltages in excess of 800 V at room
temperature and 400 V at 20 K. Simulations of vertical power MOSFETs incorporating
a ZrO2 interlayer show great promise for low temperature power electronics at or near
20 K where other commercial devices experience significant resistive losses. With the work
conducted here, vertical power MOSFETs fabricated using germanium and ZrO2 open the
gateway for low voltage applications incorporating liquid hydrogen fuel cells.Engineering and Physical Sciences Research Counci
Characterising lymphocyte mediated mechanisms of genetic risk in rheumatoid arthritis
Get PDFPh. D. ThesisRheumatoid arthritis (RA) is an immune-mediated inflammatory disease of the synovial joints
with a global prevalence of 1%, causing pain, work instability and disability. The condition’s
genetic aetiology is complex, and genome-wide association studies have highlighted over 100
susceptibility loci. The vast majority of implicated variants are noncoding, posing a major
challenge in defining genetic mechanisms of cell-mediated immune dysregulation. The precise
contribution to this process of epigenetic factors at a cellular level, such as the addition of
methyl groups to DNA, also remains to be deciphered. By conducting comprehensive molecular
profiling of circulating immune cells from early arthritis patients, my project aimed to elucidate
mechanisms of genetic risk and prioritise causal disease genes in RA. To address this, genomewide
DNA methylation, transcriptome and genotype data were available from peripheral blood
CD4+ T cells and B cells of treatment-naïve early arthritis patients. Firstly, a comparison of
DNA methylation between patients with early RA and those with other arthropathies was
undertaken. Subsequently, the capacity of RA-associated variants to influence DNA
methylation by mapping methylation quantitative trait loci (meQTLs) was confirmed. Here, it
was observed that disease variants preferentially modified DNA methylation at sites mapping
to lymphocyte enhancers and regions flanking transcription start sites, as well as positions
bound by the NFκB transcription factor. By integrating transcriptomic data and employing a
statistical approach to infer causality, loci were identified at which genetically conferred
modifications in DNA methylation regulate transcription of genes including FCRL3,
ANKRD55, IL6ST, and JAZF1 in CD4+ T cells. Finally, in vitro assays were used to validate
meQTLs at loci of interest, and to confirm regulatory mechanisms. This work highlights genes
and pathways of potential relevance to lymphocyte-mediated pathology in early RA and,
potentially, other immune mediated diseases. It has implications for the functional
interpretation of genome/epigenome-wide association studies.JGW Patterson Foundatio
An investigation of economic evaluation methods for public health interventions : meeting the needs of public health decision-makers
Get PDFPhD ThesisMarket failure typically arises from the provision of public goods in private markets due to their being non-excludable and non-rivalry in consumption. Consequently, public goods will generally be under-provided in private markets and government intervention is commonplace to help ensure efficient provision. Programmes for prevention in health can be considered public goods. Prevention largely falls within remit of public health which, since 2013, has been the responsibility of local authorities (LAs) in England.
Due to limited resources to fund public health activities in England, the prioritisation of resources is paramount. Economic evaluation can assist in guiding resource allocation decisions. Recommendations for economic evaluation methods to appraise public health interventions are less clear than for health technologies. Identifying relevant methodologies for public health appraisal is important to address the complexity of public health programmes and the LA setting of public health decisions in England. This study aimed to engage public health decision-makers (PHDMs) to identify the most beneficial economic evaluation tool(s) to meet their needs.
This research focused on a sub-sect of public health: interventions related to alcohol consumption. A systematic review was conducted to identify economic evaluations of interventions to reduce alcohol misuse since 2006. The majority of evaluations identified were cost-utility analyses (CUAs). Limited consideration of methodological challenges specific to public health was found.
A qualitative interview study with PHDMs in North-East England was then conducted which identified limited use and knowledge of health economic tools amongst the PHDMs. A desire for the incorporation of broader outcomes in evaluations to incorporate the local decision-making context was established.
Building on the outcomes from the review and qualitative study, a cost-benefit analysis (CBA) and social return on investment (SROI) were conducted to evaluate a brief alcohol intervention in schools. In order to conduct the CBA, a contingent valuation (CV) study was carried out to obtain a monetised measure of benefit. Despite debate in the literature on the suitability of the CV method to elicit true economic values for public goods, particularly for goods offering limited private consumption value, CV has been used in the field of environmental economics for decades to ascertain non-use values for goods. The method
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has also been used to value healthcare goods. The outcomes of the CV survey additionally provided information on the mechanics of the decisions made by the public via examination of predictors of willingness-to-pay for the intervention. It can also help elucidate reasons given in support of the intervention (or lack of), potentially relevant to PHDMs. Such examination help bridge the gap between the fields of health economics and psychology.
Both the CBA and SROI identified positive societal benefit from the intervention. These results were shown to PHDMs at a workshop, alongside results from a previously conducted CUA and a cost-consequence analysis (CCA) of the same intervention. The workshop elicited PHDM attendees’ preferences for the practical use of methodologies. SROI was most preferred, yet concern remained over its use and interpretation by PHDMs. A “one-tool-fits-all” approach was doubted as being appropriate by some attendees. Combining CCA alongside another evaluation was proposed favourably by PHDMs. Providing locally relevant analysis via additional sensitivity analyses was also regarded as highly beneficial by attendees.Fus
Investigating the effect of endurance exercise on mitochondrial function in the ageing heart
Get PDFPhD ThesisMitochondria are organelles present in most cells of the body responsible for the production of cellular energy in the form of adenosine triphosphate (ATP), through oxidative phosphorylation (OXPHOS). Mitochondrial function is particularly important in the heart due to its high requirement for ATP, and a decline in mitochondrial function with age is implicated in the pathogenesis of age-related cardiovascular disease. Exercise interventions can induce mitochondrial biogenesis and increase OXPHOS capacity; however, it is unknown whether such interventions are successful if started in later life. I wanted to investigate the effect of an endurance exercise intervention in 2 mouse models; 1) a model of accelerated ageing due to enhanced mitochondrial DNA mutagenesis (PolgAmut/mutmice); 2) C57Bl6/J mouse model of physiological age at 14 months of age.
Study 1: Following baseline characterisation of PolgAmut/mutmice compared with age-matched controls, male PolgAmut/mutmice underwent a 6-month endurance exercise programme from 16 weeks of age to investigate the effects of exercise on physiological phenotype, behavioural activity, and cardiac mitochondrial function. I found no improvement in clinical score, locomotor activity, cardiac hypertrophy and fibrosis and mitochondrial function in the exercised mice. This suggests that six months of endurance exercise is unable to improve the clinical phenotype and cardiac mitochondrial function in PolgAmut/mutmice due to the severity of the genetic defect.
Study 2: To investigate whether starting an exercise programme in later life is beneficial, I first characterised the effect of age on cardiac function in sedentary mice. Between 14 and 22 months of age, I found a significant decline in neuromuscular coordination, decreased cardiac function (measured by cardiac MRI), and diminished aerobic capacity. Six months of endurance exercise improved physiological function and neuromuscular coordination, decreased cardiac fibrosis and apoptosis and improved mitochondrial OXPHOS activity. This suggests that starting an exercise programme later in life can still have beneficial effects.The MRG, CAV and Barbour foundatio
Synthetic Biology Toolkit for Cupriavidus necator H16: An Industrially Relevant Microbe
Get PDFPhD ThesisCupriavidus necator is a Gram-negative soil bacterium of great biotechnological interest. It
is known as a producer of the bioplastic 3-polyhydroxybutyrate, has been used in
bioremediation efforts, and its lithoautotrophic capabilities raise the possibility that it could
function as a microbial factory upgrading renewable resources to fuels and chemicals.
However, appropriate experimental resources to permit controlled bioengineering and
system optimisations with the bacterium are not well established. In this study, statistical
Design of Experiments (DoE) was used to identify how key media components and their
interactions affect cell growth. The model resulting from this approach is predictive and
was experimentally validated against novel media compositions at different cultivations
scales. Specifically, interaction between histidine and CuSO4 are important for reliable
robust growth prediction. Further, plasmid parts (replication origins, antibiotic cassettes
and reporter proteins) were characterised for improved transformation efficiency,
segregational stability and reporter protein expression in C. necator. Modular minimal
plasmid sets (pCAT) were constructed for C. necator using these well-characterised
biological parts, which were assembled by Golden gate method. The resulting plasmids
were delivered to C. necator via electroporation, and the transformation efficiency
obtained was more than 3000-fold higher in comparison to that obtained with the existing
plasmid, pBHR1. More importantly, the resulting Golden gate restriction-ligation products
can be delivered directly to C. necator via electroporation with high transformation
efficiency and can co-express more than one functional protein carried on a single
plasmid restriction-ligation product. pCAT plasmids can stably propagate for more than six
generation (144 h) without the addition of antibiotics. Furthermore, the application of the
toolkit was demonstrated by engineering C. necator for improved tolerance to ethanol
using directed evolutionary approach. The toolkit established in this study will be crucial in
making future bioengineering applications in C. necator more efficient, controllable and
predictable