Search

Cutaneous melanoma remains an increasing world health problem, emphasised by the lack of credible biomarkers able to identify the risk of disease progression of seemingly low risk tumours, or stratify such patients for early novel adjuvant precision-based therapies to prevent metastasis. Data leading to the present study identified the loss of epidermal differentiation proteins, AMBRA1 and Loricrin as putative prognostic biomarkers for AJCC stage I melanomas and their associated loss of expression with increased tumoural secretion of isoform specific TGF-β2. The aim of the present study was to validate the combined loss of AMBRA1 and Loricrin (AMLo) overlying AJCC stage I melanomas as a prognostic and companion biomarker marker to stratify high risk tumour subsets for novel adjuvant treatment strategies targeting TGF-β2 signalling. Semi-quantitative immunohistochemical analysis of AMLo expression in 236 AJCC stage I melanomas identified a high risk subset (defined as tumours with complete loss of AMBRA1 and any break in epidermal Loricrin expression), for which univariate analysis demonstrated a significant decrease in disease free survival in tumours stratified as high risk. Addtionally, semi-quantitative immunohistochemical analysis of TGF-β2 in high risk AJCC stage I melanomas revealed the significant correlation between increased tumoural TGF-β2 expression and loss of epidermal AMLo. Furthermore, high risk AJCC stage I melanomas were associated with a concurrent loss of peri-tumoural endothelial AMBRA1 where treatment of endothelial cells with exogenous TGF-β2 induced the significant downregulation of AMBRA1 and junctional proteins VE-Cadherin or Claudin-5, the effect of which was partially rescued by the stable knockdown of TGF-β2 in melanoma cells. Combined bioinformatics of the AMBRA1 promoter, the analysis of TGF-β2 receptor expression and Smad signalling in endothelial cells revealed the contribution of non-canonical TGF-β2 signalling to enhanced proliferation and loss of endothelial integrity. Finally, combined studies of ALK1 and ALK5 inhibition suggest, targeting ALK1 in high risk AJCC stage I melanomas may provide a novel adjuvant therapeutic strategy to prevent metastasis, for which the loss of AMLo is a companion biomarker.

We envisage a fine-grained access control solution that allows a user's access privilege to be linked to the confidence level (hereafter referred to as the assurance level) in identifying the user. Such a solution would be particularly attractive to a large-scale distributed resource sharing environment, where resources are likely to be more diversified and may have varying levels of sensitivity and resource providers may wish to adjust security protection levels to adapt to resource sensitivity levels or risk levels in the underlying environment. However, existing electronic authentication systems largely identify users through the verification of their electronic identity (ID) credentials. They take into account neither assurance levels of the credentials, nor any other factors that may affect the assurance level of an authentication process, and this binary approach to access control may not provide cost-effective protection to resources with varying sensitivity levels. To realise the vision of assurance level linked access control, there is a need for an authentication framework that is able to capture the confidence level in identifying a user, expressed as an authentication Level of Assurance (LoA), and link this LoA value to authorisation decision-making. This research investigates the feasibility of estimating a user's LoA at run-time by designing, prototyping and evaluating an authentication model that derives an LoA value based upon not only users' ID credentials, but also other factors such as access location, system environment and authentication protocol used. To this aim, the thesis has identified and analysed authentication attributes, processes and procedures that may influence the assurance level of an authentication environment. It has examined various use-case scenarios of authentication in Grid environments (a well-known distributed system) and investigated the relationships among the attributes in these scenarios. It has then proposed an authentication model, namely a generic e-authentication LoA derivation model (GEA-LoADM). The GEA-LoADM takes into account multiple authentication attributes along with their relationships, abstracts the composite effect by the multiple attributes into a generic value called the authentication LoA, and provides algorithms for the run-time derivation of LoA values. The algorithms are tailored to reflect the relationships among the attributes involved in an authentication instance. The model has a number of valuable properties, including flexibility and extensibility; it can be applied to different application contexts and supports easy addition of new attributes and removal of obsolete ones. The prototypes of the algorithms and the model have been developed. The performance and security properties of the LoA derivation algorithms and the model are analysed here and evaluated based on the prototypes. The performance costs of the GEA-LoADM are also investigated and compared against conventional authentication mechanisms, and the security of the model is tested against various attack scenarios. A case study has also been conducted using a live system, the Multi-Agency Information Sharing (MAIS) system.

This thesis concerns the modified and improved, time-stepping, dynamic reluctance mesh (DRM) modelling technique for machines and its application to multiple machine systems with their control algorithms. Improvements are suggested which enable the stable solution of the resulting complex non-linear equations. The concept of finite element (FE) derived, overlap-curves has been introduced to facilitate the evaluation of the air-gap reluctances linking the teeth on the rotor to those on the stator providing good model accuracy and efficient computation. Motivated industrially, the aim of the work is to develop a fast and effective simulation tool principally for evaluating salient pole generator system designs including the generator, exciter and the automatic voltage regulator (AVR). The objective is to provide a modelling system capable of examining the detail of machine operation including saturation of main and leakage flux paths, slotting and space harmonics of the windings. Solutions are obtained in a sufficiently short computational time to facilitate efficient iterative design procedures in an industrial design office. The DRM modelling technique for electrical machines has been shown in this thesis to be a fast and efficient tool for electrical machine simulation. Predicted results for specific machine and system designs have been compared with FE solutions and with experimental results showing, that for engineering purposes, the technique yields excellent accuracy. The DRM method has a great advantage in multiple machine simulations. This is because magnetic field calculations are limited to evaluating only the most important information so saving computation time. A brushless generating system including the excitation system and control scheme has been modelled. Additionally a cascaded, doubly fed induction generator for wind generator applications has also been modelled. These different applications for the dynamic reluctance mesh method have proved that this approach yields an excellent machine and machine-system evaluation and design tool.

Initiation and effectiveness of neuron migration to the right places are critical for the development and repair of central nervous system. It is now confirmed that mammalian, including human central nervous system (CNS) contains stem cells and nerve progenitor cells. Upon brain injury either from ischemia or trauma, those cells must be able to proliferate and migrate to the damaged parts in order to repair the damage. To understand the underlying mechanisms and find new techniques of directing neuron growth are of both scientific and medical significance. Endogenous electric fields (EFs) are widespread in developing and regenerating tissues. During embryonic development, endogenous EFs exist rostrocaudally and mediolaterally at the neural plate and neural fold stages. The size, location and developmental timing of EFs correlate with active neuron migration. Therefore, electric fields may provide a signal to direct cellular behavior of nerve cells during nervous system development and reparation. Application of EFs directs cell migration of many types of cells. However, whether neurons migrate directionally in applied electric fields has not been demonstrated. I have investigated the effects of applied physiological EFs on cultured hippocampal neurons and the underlying mechanisms. Hippocampal neurons from rats and mice were cultured on poly-L-lysine and laminin coated dishes and were identified with MAP-2 (a specific marker for neuron) staining. The neurons were exposed to small applied electric fields and the migration and division were recorded with an imaging system. 1. In a DC electric field, dissociated rat hippocampal neurons migrate to the cathode. The migration direction was reversed when the EF polarity was reversed. Neuronal migration in EFs involes the same steps as that in non-EF cultures, namely leading process extension, nuclear translocation and retraction of trailing process. 2. The cathodal migration of hippocampal neurons is voltage and time dependent. A DC EF does not have significant influence on neuronal migration speed. This result shows that the effect of EFs is different on neuronal migration speed and direction of migration. 3. The guidance effect of EFs is also seen in neuronal migration from hippocampal micro-explants and neuronal migration along glial process. Both types of migration are towards the cathode. 4. Electric fields direct growth cone path finding and neurite orientation. EF induced neurite orientation showed time and voltage dependence. Leading process branching, turning and swapping over of leading and trailing processes are the main types of neurite re-orientation. Guided neuronal migration and neuron leading process orientation are not accompanied with neuronal size change. 5. In an applied EF, MAP-2 (microtubule associated protein 2), p-Akt, Golgi apparatus and centrosome redistribute asymmetrically to the cathode facing side of hippocampal neurons. 6. Inhibition of ROCK (Rho-associated protein kinase) and Pi3k (phosphoinositide-3 kinase) with inhibitors decreases leading neurite orientation and Golgi polarization in the neurons in response to the EFs. They also decrease the directedness and speed of guided neuronal migration in EFs. 7. Null mutation of p110gamma, which encodes the catalytic domain of Pi3k gamma significantly decreased the electric field directed neuron migration. These results suggest that ROCK and Pi3k regulate EFs directed neuronal migration. 8. Application of electric fields also affects cell division of postnatal hippocampal neurons. The neuronal phenotype of divided cells was confirmed with MAP-2 and GFAP staining. Most cells divided with a cleavage plane perpendicular to the EF vector. 9. Using vibrating probe techniques, we show that there is a significant change in electric currents upon wounding hippocampus in vitro. In conclusion, this study demonstrates that hippocampal neurons from both rat and mice respond to applied EFs by directional migration and directional division. The electric field directed neuronal migration involves ROCK and Pi3k signaling. This raises the possibilities that 1) there may be a neglected role for endogenous electric fields in directing neuron migration and division; 2) electric fields may be used as a potential cue to direct neuronal migration in repair of the central nervous system.

The catalytic allylation of aldehydes, ketones, and imines is a very useful reaction for the formation of a new carbon-carbon bond in synthetic organic chemistry. There have been several successful reports of catalytic asymmetric reactions that use aldehydes as the substrate. However, there have been very few successful examples with ketones. Herein, a nickel-catalyzed allylation of dienones with the pinacol ester of allylboronic acid is presented. Based on 3,3’-reductive elimination, the relationship between the dienone structure and 1,2- and 1,6-regioselectvity has been studied. The development of a catalyzed asymmetric 1,2 allylation of dienones is also presented.

Heat shock response in Drosophila melanogaster is a complex process causing changes of the cellular environment in reaction to the heat shock induction. RNA transcription is dramatically changed during heat shock response and many regulatory factors are required. Chromatin insulators are found to be involved in the transcriptional regulation process, but much remains to be understood on how insulator function. Described herein are multiplexed selections and characterization of inhibitory RNA aptamers against several Drosophila insulator proteins using an efficient selection method, RAPID (RNA APtamer Isolation via Dual-cycles), and high-throughput sequencing. Background binding sequences are identified through a series of comprehensive analyses on the sequencing dataset. I have also examined the binding affinity of the top aptamer candidates and designed assays to map the binding elements. Aptamers generated here can be utilized as a very powerful approach to dissect the insulator function in vivo in the well-characterized heat shock system.

The Sonchinae is the most widely distributed subtribe in the tribe Cichorieae, with a discontinuous, almost cosmopolitan, and very peculiar phytogeographic distribution. In particular, genus Sonchus subgenus Sonchus, appears to be responsible for the origin of several island endemics both in the Pacific Ocean (Dendroseris, Thamnoseris, Actites, Kirkianella and Embergeria ) and the Atlantic Ocean (woody Sonchus alliance in the Macaronesian Islands). So polyploidy Sonchus and their relative Pacific polyploidy endemics are an ideal system to study the polyplodization for the island plants. Several hypotheses have been proposed based on the morphology and karyology. We use the cloning technique to retrieve the different ITS repeat types in order to test the origin of several polyploidy Sonchus as well as polyploidy in the related Pacific islands. The Pacific endemics (Dendroseris, Embergeria, Kirkianella, and Actites) are all formed the monophyletic clade with all the their repeat types which support the single origin of these endemics. S. arvensis has been identified as a potential allopolyploid. The Stebbins hypothesis of allpolyploid origin for S. oleraceus is partially supported with confirmation of S. asper as one donor. The data also indicate a hybrid origin for S. hydrophilus which might have subsequently homogenized with ITS copies from the hybrid ancestor via concerned evolution.The woody Sonchus alliance is one of the largest Macaronesia diverse endemic groups with 6 genera and approximately 31 species. Determining accurate phylogenetic relationships among the members of the woody Sonchus alliance presents challenges because of insufficient level of molecular variation and the convergent evolution of similar morphological traits in island settings. All taxa of woody Sonchus alliance were sampled to investigate the phylogenetic relationships as well as to test the potential role of hybridization and introgression using three independent low-copy nuclear genes: glyceraldehyde 3-phosphate dehydrogenase (G3pdh) , B12 and calmodulin (Cam) . B12 and CAM phylogeny is not well resolved due to the limited informative sites. The G3PDH data set was not significantly different from that of B12 and CAM,and subsequent combined analysis provided a better resolved and supported phylogeny within the alliance. In the MP combined tree, the basal lineages of monotypic genera were not identified. The all Taeckholmia species except T. arborea formed a well-support clade. It partly supports Boulos' classification to treat Taeckholmia as a genus. But the Dendrosonchus is highly polyphyletic which does not support either Boulos' or Aldridge's classification of tree Dendrosonchus. Assessment of the role of hybridization and introgression was limited due to low sequence variability of B12 and CAM genes, however, potential hybridization has been recognized comparing independent gene regions. The members of woody Sonchus alliance display extensive morphological, ecological, and anatomical diversity, but all taxa have a uniform chromosome number. Furthermore, all the members have no crossing barriers, there are extensive opportunities for hybridization between these species. We include two independent low-copy nuclear genes: glyceraldehyde 3-phosphate dehydrogenase (G3PDH) and Calmodulin (CAM) with the cloning with several key species to test the possible homoploid hybrid speciation. There is no evidence in our data to support a hybrid origin for the monotypic species (Babcockia platylepis, Sventenia bupleuroides and Lactucosonchus webbii). The hybridization between Dendrosonchus and Taeckholmia is not supported in our data. S. tuberifer might have hybrid origin between herbaceous and small woody species.