Investigating the role of N-WASP in the development of colorectal cancer

Colorectal cancer (CRC) is the second most common cancer in Europe, with the second highest mortality rate. Although prognosis is improving, survival rates remain poor for those presenting with the most advanced stages of the disease. There is therefore a need for improved early diagnosis and thus a greater understanding of the early stages of the development of colorectal tumours is desirable. Additionally, as most deaths in colorectal cancer are due to advanced metastatic disease, it is of great interest to explore any potential mechanisms by which metastatic disease can be inhibited. N-WASP is a ubiquitously expressed protein with multiple intracellular roles including actin regulation and maintaining stability of epithelial cell-cell junctions. Through its role as an actin regulator, it has been implicated in the processes of invasion and metastasis of multiple cancer types. Its role in the development and progression of colorectal cancer however has not been fully explored. This thesis will present a series of in vitro and in vivo studies that were carried out with the aim of answering the following questions: • Does N-Wasp have a role in normal intestinal homeostasis? • Does N-Wasp knockout affect the development of tumours in a mouse model of intestinal tumourigenesis? • Does N-Wasp knockout affect the invasive properties of intestinal cancer in vitro? • Does N-WASP correlate with prognosis or other indicators in human colorectal cancer TMAs? Findings from the in vivo experiments, using an inducible, gut-specific knockout model, have uncovered potential roles for N-Wasp in regulating differentiation and migration of intestinal epithelial cells. Although it had no effect in short term models of intestinal hyperproliferation, N-Wasp knockout increased tumour burden and decreased survival in an established in vivo model of intestinal tumourigenesis, in which there is heterozygous loss of Apc (Apcfl/+). No effect was seen on tumour development or survival when additional N-WASP knockout was introduced into a more rapid model, with heterozygous loss of Apc and mutation of Kras (Apcfl/+ KrasG12D/+). N-WASP expression in human colorectal cancer was assessed using immunohistochemical staining of two tissue microarrays. Low levels of N-WASP expression were found to be associated with presence of MMR deficiency. There was no statistically significant difference in overall or cancer specific survival based on N-WASP expression. Collectively, the data presented here suggest a previously unreported role for N-WASP in regulation of intestinal epithelial differentiation and indicate that it may act as a tumour suppressor against development of benign precursor lesions of colorectal cancer. Further research is warranted to delineate the mechanisms underlying these processes.

NETCODE: an XOR-based warning dissemination scheme for vehicular wireless networks

The next generation of vehicles will be equipped with automated Accident Warning Systems (AWSs) capable of warning neighbouring vehicles about hazards that might lead to accidents. The key enabling technology for these systems is the Vehicular Ad-hoc Networks (VANET) but the dynamics of such networks make the crucial timely delivery of warning messages challenging. While most previously attempted implementations have used broadcast-based data dissemination schemes, these do not cope well as data traffic load or network density increases. This problem of sending warning messages in a timely manner is addressed by employing a network coding technique in this thesis. The proposed NETwork COded DissEmination (NETCODE) is a VANET-based AWS responsible for generating and sending warnings to the vehicles on the road. NETCODE offers an XOR-based data dissemination scheme that sends multiple warning in a single transmission and therefore, reduces the total number of transmissions required to send the same number of warnings that broadcast schemes send. Hence, it reduces contention and collisions in the network improving the delivery time of the warnings. The first part of this research (Chapters 3 and 4) asserts that in order to build a warning system, it is needful to ascertain the system requirements, information to be exchanged, and protocols best suited for communication between vehicles. Therefore, a study of these factors along with a review of existing proposals identifying their strength and weakness is carried out. Then an analysis of existing broadcast-based warning is conducted which concludes that although this is the most straightforward scheme, loading can result an effective collapse, resulting in unacceptably long transmission delays. The second part of this research (Chapter 5) proposes the NETCODE design, including the main contribution of this thesis, a pair of encoding and decoding algorithms that makes the use of an XOR-based technique to reduce transmission overheads and thus allows warnings to get delivered in time. The final part of this research (Chapters 6--8) evaluates the performance of the proposed scheme as to how it reduces the number of transmissions in the network in response to growing data traffic load and network density and investigates its capacity to detect potential accidents. The evaluations use a custom-built simulator to model real-world scenarios such as city areas, junctions, roundabouts, motorways and so on. The study shows that the reduction in the number of transmissions helps reduce competition in the network significantly and this allows vehicles to deliver warning messages more rapidly to their neighbours. It also examines the relative performance of NETCODE when handling both sudden event-driven and longer-term periodic messages in diverse scenarios under stress caused by increasing numbers of vehicles and transmissions per vehicle. This work confirms the thesis' primary contention that XOR-based network coding provides a potential solution on which a more efficient AWS data dissemination scheme can be built.

Simulation tools for the analysis of single electronic systems

Developments in theory and experiment have raised the prospect of an electronic technology based on the discrete nature of electron tunnelling through a potential barrier. This thesis deals with novel design and analysis tools developed to study such systems. Possible devices include those constructed from ultrasmall normal tunnelling junctions. These exhibit charging effects including the Coulomb blockade and correlated electron tunnelling. They allow transistor-like control of the transfer of single carriers, and present the prospect of digital systems operating at the information theoretic limit. As such, they are often referred to as single electronic devices. Single electronic devices exhibit self quantising logic and good structural tolerance. Their speed, immunity to thermal noise, and operating voltage all scale beneficially with junction capacitance. For ultrasmall junctions the possibility of room temperature operation at sub picosecond timescales seems feasible. However, they are sensitive to external charge; whether from trapping-detrapping events, externally gated potentials, or system cross-talk. Quantum effects such as charge macroscopic quantum tunnelling may degrade performance. Finally, any practical system will be complex and spatially extended (amplifying the above problems), and prone to fabrication imperfection. This summarises why new design and analysis tools are required. Simulation tools are developed, concentrating on the basic building blocks of single electronic systems; the tunnelling junction array and gated turnstile device. Three main points are considered: the best method of estimating capacitance values from physical system geometry; the mathematical model which should represent electron tunnelling based on this data; application of this model to the investigation of single electronic systems. (DXN004909)

Shaping surface acoustic waves for cardiac tissue engineering

The heart is a non-regenerating organ that gradually suffers a loss of cardiac cells and functionality. Given the scarcity of organ donors and complications in existing medical implantation solutions, it is desired to engineer a three-dimensional architecture to successfully control the cardiac cells in vitro and yield true myocardial structures similar to native heart. This thesis investigates the synthesis of a biocompatible gelatin methacrylate hydrogel to promote growth of cardiac cells using biotechnology methodology: surface acoustic waves, to create cell sheets. Firstly, the synthesis of a photo-crosslinkable gelatin methacrylate (GelMA) hydrogel was investigated with different degree of methacrylation concentration. The porous matrix of the hydrogel should be biocompatible, allow cell-cell interaction and promote cell adhesion for growth through the porous network of matrix. The rheological properties, such as polymer concentration, ultraviolet exposure time, viscosity, elasticity and swelling characteristics of the hydrogel were investigated. In tissue engineering hydrogels have been used for embedding cells to mimic native microenvironments while controlling the mechanical properties. Gelatin methacrylate hydrogels have the advantage of allowing such control of mechanical properties in addition to easy compatibility with Lab-on-a-chip methodologies. Secondly in this thesis, standing surface acoustic waves were used to control the degree of movement of cells in the hydrogel and produce three-dimensional engineered scaffolds to investigate in-vitro studies of cardiac muscle electrophysiology and cardiac tissue engineering therapies for myocardial infarction. The acoustic waves were characterized on a piezoelectric substrate, lithium niobate that was micro-fabricated with slanted-finger interdigitated transducers for to generate waves at multiple wavelengths. This characterization successfully created three-dimensional micro-patterning of cells in the constructs through means of one- and two-dimensional non-invasive forces. The micro-patterning was controlled by tuning different input frequencies that allowed manipulation of the cells spatially without any pre- treatment of cells, hydrogel or substrate. This resulted in a synchronous heartbeat being produced in the hydrogel construct. To complement these mechanical forces, work in dielectrophoresis was conducted centred on a method to pattern micro-particles. Although manipulation of particles were shown, difficulties were encountered concerning the close proximity of particles and hydrogel to the microfabricated electrode arrays, dependence on conductivity of hydrogel and difficult manoeuvrability of scaffold from the surface of electrodes precluded measurements on cardiac cells. In addition, COMSOL Multiphysics software was used to investigate the mechanical and electrical forces theoretically acting on the cells. Thirdly, in this thesis the cardiac electrophysiology was investigated using immunostaining techniques to visualize the growth of sarcomeres and gap junctions that promote cell-cell interaction and excitation-contraction of heart muscles. The physiological response of beating of co-cultured cardiomyocytes and cardiac fibroblasts was observed in a synchronous and simultaneous manner closely mimicking the native cardiac impulses. Further investigations were carried out by mechanically stimulating the cells in the three-dimensional hydrogel using standing surface acoustic waves and comparing with traditional two-dimensional flat surface coated with fibronectin. The electrophysiological responses of the cells under the effect of the mechanical stimulations yielded a higher magnitude of contractility, action potential and calcium transient.