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A Col9a3 exon 3 skipping mouse as a novel model for multiple epiphyseal dysplasia
Get PDFPh. D. ThesisA Col9a3 EXON 3 SKIPPING AS NOVEL MODEL FOR MULTIPLE EPIPHYSEAL DYSPLASIA.
Introduction: Multiple epiphyseal dysplasia (MED) is generally an autosomal dominant chondrodysplasia characterised by early-onset degenerative joint disease. Its genetic background is complex and heterogeneous and among the mutated genes are those encoding for the pro-α chains of the collagen type IX, COL9A1, COL9A2 and COL9A3, where the majority of mutations lead to the skipping of a syngenic exon.
Aim: Reproduce in a mouse model the skip of exon 3 in the COL9A3 gene to understand its pathogenic role in relation to MED.
Material and Methods: By CRISPR/Cas9 technology we generated two mouse lines, one carrying a deletion of Col9a3 exon 3 (Col9a3Δex3/Δex3), reproducing the splicing events reported in a MED patients group and a Col9a3-null mouse (Col9a3-/-). On the mutant mice, we performed skeletal X-ray phenotyping and growth plate analysis, including immunohistochemistry and BrdU labelling to monitor proliferation. Ultrastructure of growth plate was visualised using Transmission electron microscopy (TEM). Sequential protein extraction and Atomic force microscopy (AFM) were used to evaluate growth plate cartilage stability, whereas articular cartilage integrity was assessed by destabilisation of the medial meniscus (DMM) surgery and during ageing. Bone density and ultrastructure was assessed by Microcomputed tomography (μCT). Mutant transcriptomes were obtained by RNAseq.
Results: The phenotyping of CRISPR/Cas9 generated offspring through DNA and cartilage RNA analysis had led to the establishment of two transgenic mouse lines, one splicing as predicted (Col9a3Δex3/Δex3) and a second lacking the Col9a3 transcript and collagen type IX protein (Col9a3-/-). Both lines are viable, however only Col9a3-/- mice displayed detectable phenotypic abnormalities: mild short stature and hip dysplasia, abnormal tibial epiphysis morphology and delayed ossification of femoral head in 18-week old mice. A reduced level of growth plate chondrocyte proliferation was detected in both mutant mice compared to WT, along with softening of the proliferative zone shown by AFM indentation measurements on new-born and 6 weeks old animals. Interestingly, both mutant mice exhibited similar transcriptome profiles. Only in Col9a3-/- mice immunoblotting of sequentially extracted matrilin-3 and COMP proteins revealed different cartilage extractability from controls. Articular cartilage integrity seemed not to be affected by either mutation.
Discussion and conclusion: Analysis confirmed the production of a shorter transcript from cartilage of Col9a3Δex3/Δex3mice. However, these mice still produced collagen type IX protein and had no overt phenotype apart from reduced chondrocytes proliferation and softer proliferative cartilage extracellular matrix. The Col9a3-/- mice had a mild skeletal phenotype and express no Col9a3 transcript or protein, resulting in overall more unstable cartilage.
Both mutant mice will represent an important tool to gain insights on collagen type IX role into the matrix. In particular, the Col9a3Δex3/ Δex3 line, by recapitulating human Col9-MED, can add to our understanding of MED disease mechanism.SYBIL consortiu
Molecular diversity from N-phenylquinoneimine
Get PDFPhD ThesisDiversity-oriented synthesis (DOS) continues to grow as an area of importance in the disciplines of organic synthesis and chemical biology.1-3 It involves the preparation of diverse scaffolds of varying complexity and enables the exploration of a greater range of ‘chemical space’ than hitherto possible through multicomponent-coupling and tandem reactions.
Tandem reactions, also known as cascade or domino reactions, link several transformations together in a single process step. Simple starting materials can be used to rapidly build complex structures with high selectivity. The use and production of toxic and/or hazardous intermediates can be avoided/minimised and novel avenues of research and new reactions can be developed.4
In this work, azaquinone 1 has been studied as a new key intermediate in multicomponent coupling and tandem reactions (Scheme 1). As all six carbon atoms in the quinoid ring are different, it has the potential to serve as a novel building block to access a range of useful structural motifs as part of scaffold diversity synthesis and drug discovery.
Conjugate addition to azaquinone 1 with nitrogen nucleophiles (aromatic amines) has been investigated, giving di-substituted arylamino compounds 2 in good yields (56–68%). Although the basic process has been reported,5 the regiochemistry of these compounds has now been confirmed by 2D NMR (COSY and NOESY) and also by determination of the crystal structure of the products. The reaction of aromatic amines with azaquinone occurred by both 1,4-conjugate addition to the α,β-unsaturated imine and the α,β-unsaturated ketone, resulting only in di-addition products. These diarylamino products 2 are coloured compounds which may be of importance in the dye industry, and those interested in development of diagnostic tools. Further reactions of compounds 2 could result in the production of azine, oxazine, and thiazine dyes.Newcastle University and TetFun
Investigation of (super)hydrophilic coatings for marine antifouling applications
Get PDFPh. D. ThesisThe undesirable colonization of artificial structures at sea, termed marine biofouling, has significant
economic and environmental impacts.
The marine antifouling (AF) market is currently dominated by biocidal coatings that release toxic
heavy metals or organic biocides, limiting the attachment and growth of fouling organisms. The
central aim of this thesis was to explore the potential of environmentally inert, (super)hydrophilic
surfaces as next-generation AF coatings. In contrast to previous research in this area, the coatings
presented here were prepared as industrially relevant copolymers and tested in the laboratory against
two major fouling species; cypris larvae of the barnacle Balanus improvisus and cells of the diatom
Navicula incerta, as well as a multispecies biofilm and field testing in temperate and tropical
locations.
The laboratory assays revealed that, in general, barnacles settled in lower numbers on bulk polymers
containing ionic functionalities. The surfaces containing zwitterionic sulfobetaine methacrylate
(SBMA) and anionic 3-sulfopropyl methacrylate (SPMA) were found to not only inhibit cyprid
settlement but also promote high removal of juvenile barnacles, diatom cells and biofilm. The
exceptional AF and fouling-release performance of these two chemistries was explained by the large
degree of swelling when hydrated and presence of nonfreezable water at the interface. The surfaces
presented showed limited AF efficacy when subjected to the real-world marine environment. Using
behavioural analysis and imaging surface plasmon resonance (iSPR) experiments, it was shown that
barnacle cyprids explored all surfaces. However, in the case of SBMA, the exploratory events were
very short and the amount of retained temporary adhesive was low.
In conclusion, this work demonstrates the potential of bulk hydrophilic copolymers as a starting point
for the development of next-generation, green AF coatings. It is hoped that the findings of this work
will encourage greater industrial participation in the development of fouling-control technologies that
function via non-biocidal means.AkzoNobel,
Marine and Protective Coatings (International Paint)
New perspectives on war memorialisation: North-East England, 1854-1910
Get PDFPhD ThesisThis study focuses on three clusters of conflicts — the Crimean War and Indian Rebellion in the 1850s, the ‘small wars’ of the 1880s, and the Boer War (1899-1902) — to determine how far reactions to different conflicts shaped their memorialisation. The research utilises the methodology of the historiographical debate on war memorialisation, concerned primarily with the First World War, and extends it to the relatively-neglected arena of nineteenth-century conflicts. Examining aspects of the memorialisation process such as organisation, form, function and narrative, it questions the motivations that underpinned these communal endeavours. By considering wars over a protracted timeframe, it can identify threads of continuity in the memorialisation process but also reveal a transformation in intent and purpose: from ill-defined, triumphal trophies of the Crimean War to apparently sombre monuments to ordinary soldiers after the Boer War, transmitting didactic narratives of the virtues of good citizenship in a more democratic society; including, if necessary, the ultimate sacrifice.
The memorialisation process is placed within the historiographical framework of municipal political culture, assessing the influence of local socio-political tensions and the correlation between patriotism and civic pride. The thesis investigates the relations of power that determined how wars were represented and asks how far memorials can be considered a hegemonic device that transmitted the civic elite’s values and beliefs to an acquiescent community. This thesis makes important contributions to the historiographical debate on the memorialisation of war, gauging why civic war memorials were produced and what they reveal about changes in contemporaneous society
Finite element modelling of cell mechanics and cell-material interactions
Get PDFPh.D thesisUnderstanding cell mechanics subjected to external stimuli is important to design microniche to direct cell migration, differentiation and proliferation. However, previous models have not elucidated the mechanisms during the mechanotransduction process. Therefore, the main objective of this thesis is to develop different types of cell models including structure-based and continuum-based models to study the cell response during interactions with external stimuli. The structure-based cell model consisting of discrete cellular components was adopted to study the cellular responses during atomic force microscope (AFM) indentation tests, which revealed the significant contribution of stress fibres (SFs) to apparent modulus.
A continuum-based model has been developed to examine the effect of substrate thickness, lateral boundary and neighbouring cell on cell responses. In this model, the active behaviour of the cell was described by a SF formation model. Focal adhesion (FA) model driven by the SF contractility was implemented to account for the interactions with substrate. It has revealed that the thin layer of substrate enhanced the SF and FA formation. The SF concentration and integrin density decrease exponentially with increasing substrate thickness. Higher substrate stiffness attenuates the cell responses to thickness variation. Larger cell sizes promote the formation of SFs and enable deeper thickness sensing. Fixed lateral boundary of the substrate influences the SF and FA formation as well as the SF orientation. Soft substrate enables cells to sense the lateral displacement field created by another cell while stiff substrate hinders the cell-cell communication. Cell orients its SFs towards the neighbouring cell and could be influenced to polarize in this direction.
These predictions are consistent with experimental findings. Furthermore, the physics underpinned by the modelling has improved our understanding of the substrate boundary sensing and mechanics regulated cell-cell communications. This modelling framework could be potentially adopted for rational design of biomaterials in tissue engineering
Analysing and visualising data sets of cybercrime investigations using structured occurrence nets
Get PDFPh. D. Thesis.Structured Occurrence Nets (SONs) are a Petri net based formalism for
portraying the behaviour of complex evolving systems. As a concept,
SONs are derived from Occurrence Nets (ONs). SONs provide a powerful
framework for evolving system analysis and are supported by the existing
SONCraft toolset. On the other hand, modelling of cybercrime investigations has become of interest in recent years, and large-scale criminal
investigations have been considered as complex evolving systems. Right
now, they present a significant challenge for police investigators and analysts. The current thesis contributes to addressing this challenge in two
different ways: (i) by presenting an algorithm and an implemented tool
that visualise data sets using maximal concurrency; and (ii) by detecting
DNS tunnelling through a novel SON-based technique and tool. Moreover,
the theoretical contribution of this thesis focuses on model extensions and
abstraction; in particular, it introduces a new class of SONs based on
multi-coloured tokens
An Energy Efficient non-volatile FPGA Digital Processor for Brain Neuromodulation
Get PDFPhD ThesisBrain stimulation technologies have the potential to provide considerable clinical benefits for people with a range of neurological disorders. Recent neuroscience studies have shown that considerable information of brain states is contained in the low frequency local field potential (If-LFP; below 5Hz) recordings with application in real-time closed-loop neurostimulation for treating neurological disorders. Given these signals can be sampled at low sampling rate and hence provide sparse data streams, there is an opportunity to design implantable neuroprosthesis with long battery lifecycles which enables enough processing power to implement long-term, real-time closed loop control algorithms. In this thesis, a closed-loop embedded digital processor has been created for use in rodent neuroscience experiments. The first contribution of this work is to develop a mathematical analytical design approach of feedback controller for suppressing high-amplitude epileptic activity in the neuron mass model to form a better understanding of how to perform a better closed-loop stimulation to control seizures. The second contribution and the third contribution are combined to present an exploratory energy-efficient digital processor architecture built with commercial off-the-shelf non-volatile FPGAs and microcontroller for sparse data processing of brain neuromodulation. A digital hardware design of an exemplar PID control algorithm has been implemented on this proposed digital architecture. A new power computing diagram of this time-driven approach significantly reduced the power consumption which suggests that a digital combined control system of non-volatile FPGAs and microcontroller outweighs a digital control system of microcontroller with microcontroller regarding computing time cost and energy consumption supposing one microcontroller is always required. Taken together, this digital energy-efficient processor architecture gives important insights and viewpoints for the further advancements of neuroprosthesis for brain neurostimulation to achieve lower power consumption for sparse sampling data rate
Development of a wrist-worn accelerometer feedback intervention to enhance arm recovery after stroke
Get PDFPh. D. Thesis.Background
Arm recovery after stroke is enhanced by frequent practice of functional activities. Remaining motivated to practice and remembering to integrate the impaired limb into daily activities can be difficult.
Methods
A mixed methods approach developed a novel intervention to promote use of the impaired arm after stroke:
1. Systematic review examined reports of self-directed interventions for arm rehabilitation after stroke.
2. Development of a novel intervention using a wrist-worn accelerometer with vibrating alert to prompt arm activity.
3. An un-blinded observational proof of concept study within 4 weeks of stroke refined the intervention.
4. A multi-centre, observer-blind pilot randomised controlled trial (RCT) evaluated feasibility of the intervention for 8 weeks within 3 months of stroke and provided descriptive clinical and biomarker data.
Results
The systematic review showed that high doses of independent practice are possible, with benefits from functional task practice.
A novel intervention was developed consisting of feedback from a wristband accelerometer to prompt increases in functional therapy practice within daily routines. The proof of concept study showed that feedback was acceptable with refinements to the technology and therapy programme.
Thirty-three participants were recruited to the Pilot RCT. Research assessments were completed for 28/29 and 25/28 patients at four and eight weeks. Wristbands were worn for 79% of the recommended time with a median of 8[IQR: 6-10] prompts delivered per participant/day.
ii
Clinical outcomes were better for intervention participants and continued to improve post-intervention although the small cohort size precluded statistical analysis.
Statistical analysis of the accelerometer data showed impaired arm activity increased for the intervention group and continued to increase further over the follow-up period. In contrast, arm activity in the control group changed marginally.
Conclusion
Feedback from a wristband accelerometer to prompt greater use and independent practice of the impaired arm after stroke is feasible and should be considered for further research evaluation.Stroke Associatio
Mitochondria and microRNAs in obesity and after weight loss
Get PDFPh. D. ThesisColorectal cancer (CRC) is the 3rd most common cancer worldwide. CRC is initiated in colonocytes and, with advancing age, colonocytes accumulate mitochondrial mutations which contribute to age-related dysfunction and to increased cancer risk. Obesity, and its lifestyle determinants, physical inactivity and poor diet, also increase CRC risk. However, the effects of weight loss by bariatric surgery on mitochondrial defects in human colonoctyes and on CRC risk are unclear. Epigenetic mechanisms involving microRNAs that lead to dysregulated gene expression may mediate the effects of obesity and weight loss on CRC risk.
I hypothesised that mitochondrial defects and microRNAs are i) elevated and aberrantly expressed in obese individuals compared with healthy non-obese individuals and ii) reduced and modulated by significant weight loss following bariatric surgery, respectively.
Colorectal mucosal biopsies of obese patients listed for bariatric surgery were collected at baseline and six months post-surgery and at baseline only from non-obese Controls. Mitochondrial oxidative phosphorylation proteins complex I and IV and mitochondrial mass were quantified by immunofluorescence. Using Next Generation Sequencing and bioinformatics i) mitochondrial DNA was sequenced and ii) a panel of 8 microRNAs was selected and validated by quantitative PCR in colorectal mucosal biopsies.
Greater adiposity and advancing age resulted in significantly more complex I and IV deficient crypts in the human colorectal mucosa but, at least after 6 months, weight loss following bariatric surgery had no significant effect on these mitochondrial defects. Neither excess adiposity nor significant weight loss resulted in differences in mtDNA mutations between the study groups. Expression of miR-31, miR-215, miR-3196 and miR-4516 was significantly higher in obese than in non-obese individuals. Weight loss reduced expression of miR-31, miR-215 and miR-3196 significantly to expression levels that were comparable with those in Controls. These differentially expressed microRNAs are implicated in pathways linked with inflammation, obesity and cancer. This research enables the broadening of our knowledge on the mechanistic pathways of obesity related CRC risk and provides novel evidence on the effects of intentional weight loss by bariatric surgery on these biomarkers in the colon.Medical Research Counci
Building a 3D model of the human keratoconic cornea
Get PDFPhD ThesisKeratoconus is a corneal disease characterized by the affected tissue adopting a conical shape, leading to loss of vision and compromised structural integrity. The disease is manageable in its early stage with lenses or glasses and can be treated in its later stages with surgery or crosslinking. The underlying cause of the disease is still poorly understood, a model would aid the exploration of this disease. In this study, primary stromal cells harvested from healthy and keratoconic corneas using a variety of extraction methods were cultured in compressed collagen gels and serum-free medium containing retinoic acid to simulate the cornea’s natural environment. The survival of stromal cells and their gene expression was assayed, paying special attention to genes related to differentiation and ECM maintenance. Among healthy cells, differences were identified between limbal and central population while central keratoconic cells more closely resembled healthy limbal cells. Use of retinoic acid as a medium supplement allowed for the culture of cells in serum-free conditions and caused gene expression that resembled in vivo behaviour. The final addition of ECM in the form of curved, compressed collagen gels to this culture system caused the stromal cells to radically change their behaviour and reaction to retinoic acid. Overall, this study identified a stromal tissue model as well as the multiple considerations in the construction of such a model. The use of this model in keratoconus research may lead to breakthroughs in attempts to better understand the disease