An investigation of the role of TRIB2 in steady state and stressed haematopoiesis

TRIB2 is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Here, we studied murine haematopoiesis after Trib2 ablation under steady state and proliferative stress conditions, including genotoxic and oncogenic stress. At the steady state, we found that TRIB2 loss did not adversely affect peripheral blood cell counts and populations. No detectable significant differences were found in the populations of haematopoietic stem and progenitor cells. However, Trib2-/- mice had significantly higher thymic cellularity due to the increased proliferation of Trib2-/- developing thymocytes which give rise to increased number of mature thymic subsets. During stressed haematopoiesis, Trib2-/- developing thymocytes demonstrate hypersensitivity to 5-fluorouracil-induced cell death. Nevertheless, Trib2-/- mice exhibit accelerated thymopoietic recovery post 5-fluorouracil treatment due to increased cell division kinetics of developing thymocytes. In an experimental murine T-cell acute lymphoblastic leukaemia (T-ALL) model, Trib2-/- mice had reduced latency in vivo which associated with aggressive T-ALL phenotypes and impaired activation of mitogen-activated protein kinase. Gene set enrichment analysis showed that TRIB2 expression is elevated in immature subtype of human T-ALL enriched with mitogen-activated protein kinase signalling. However, TRIB2 expression is suppressed in mature subtype of human T-ALL. Thus, TRIB2 emerges as a novel regulator of thymocyte cellular proliferation, important for the thymopoietic response to genotoxic and oncogenic stress, and possessing tumour suppressor function. In Drosophila, Tribbles promotes degradation of String which is an orthologue of mammalian CDC25 phosphatases in order to arrest cell cycle during embryonic development. Here, we showed that the role of Tribbles-induced degradation of String is evolutionarily conserved in TRIB2. We found that TRIB2 interacts with CDC25B/C but not CDC25A isoform. Overexpression of TRIB2 promotes polyubiquitination and degradation of CDC25C. Hence, future works are warranted to examine TRIB2-CDC25C interaction in the context of developing thymocytes and in T-cell acute lymphoblastic leukaemia, the malignant counterpart.

Applications of finite element computer modelling and thermal infrared remote sensing to the study of geothermal anomalies

Buried heat sources can be investigated by examining thermal infrared images and comparing these with the results of theoretical models which predict the thermal anomaly a given heat source may generate. Key factors influencing surface temperature include the geometry and temperature of the heat source, the surface meteorological environment, and the thermal conductivity and anisotropy of the rock. In general, a geothermal heat flux of greater than 2% of solar insolation is required to produce a detectable thermal anomaly in a thermal infrared image. A heat source of, for example, 2-300K greater than the average surface temperature must be a t depth shallower than 50m for the detection of the anomaly in a thermal infrared image, for typical terrestrial conditions. Atmospheric factors are of critical importance. While the mean atmospheric temperature has little significance, the convection is a dominant factor, and can act to swamp the thermal signature entirely. Given a steady state heat source that produces a detectable thermal anomaly, it is possible to loosely constrain the physical properties of the heat source and surrounding rock, using the surface thermal anomaly as a basis. The success of this technique is highly dependent on the degree to which the physical properties of the host rock are known. Important parameters include the surface thermal properties and thermal conductivity of the rock. Modelling of transient thermal situations was carried out, to assess the effect of time dependant thermal fluxes. One-dimensional finite element models can be readily and accurately applied to the investigation of diurnal heat flow, as with thermal inertia models. Diurnal thermal models of environments on Earth, the Moon and Mars were carried out using finite elements and found to be consistent with published measurements. The heat flow from an injection of hot lava into a near surface lava tube was considered. While this approach was useful for study, and long term monitoring in inhospitable areas, it was found to have little hazard warning utility, as the time taken for the thermal energy to propagate to the surface in dry rock (several months) in very long. The resolution of the thermal infrared imaging system is an important factor. Presently available satellite based systems such as Landsat (resolution of 120m) are inadequate for detailed study of geothermal anomalies. Airborne systems, such as TIMS (variable resolution of 3-6m) are much more useful for discriminating small buried heat sources. Planned improvements in the resolution of satellite based systems will broaden the potential for application of the techniques developed in this thesis. It is important to note, however, that adequate spatial resolution is a necessary but not sufficient condition for successful application of these techniques.

Fabrication, characterisation and electroanalysis at 1-dimensional nanostructures

Integrated nanowire electrodes that permit direct, sensitive and rapid electrochemical based detection of chemical and biological species are a powerful emerging class of sensor devices. As critical dimensions of the electrodes enter the nanoscale, radial analyte diffusion profiles to the electrode dominate with a corresponding enhancement in mass transport, steady-state sigmoidal voltammograms, low depletion of target molecules and faster analysis. To optimise these sensors it is necessary to fully understand the factors that influence performance limits including: electrode geometry, electrode dimensions, electrode separation distances (within nanowire arrays) and diffusional mass transport. Therefore, in this thesis, theoretical simulations of analyte diffusion occurring at a variety of electrode designs were undertaken using Comsol Multiphysics®. Sensor devices were fabricated and corresponding experiments were performed to challenge simulation results. Two approaches for the fabrication and integration of metal nanowire electrodes are presented: Template Electrodeposition and Electron-Beam Lithography. These approaches allow for the fabrication of nanowires which may be subsequently integrated at silicon chip substrates to form fully functional electrochemical devices. Simulated and experimental results were found to be in excellent agreement validating the simulation model. The electrochemical characteristics exhibited by nanowire electrodes fabricated by electronbeam lithography were directly compared against electrochemical performance of a commercial ultra-microdisc electrode. Steady-state cyclic voltammograms in ferrocenemonocarboxylic acid at single ultra-microdisc electrodes were observed at low to medium scan rates (≤ 500 mV.s-1). At nanowires, steady-state responses were observed at ultra-high scan rates (up to 50,000 mV.s-1), thus allowing for much faster analysis (20 ms). Approaches for elucidating faradaic signal without the requirement for background subtraction were also developed. Furthermore, diffusional process occurring at arrays with increasing inter-electrode distance and increasing number of nanowires were explored. Diffusion profiles existing at nanowire arrays were simulated with Comsol Multiphysics®. A range of scan rates were modelled, and experiments were undertaken at 5,000 mV.s-1 since this allows rapid data capture required for, e.g., biomedical, environmental and pharmaceutical diagnostic applications.

Interaction of additive and quantization noises in digital PLLs

Phase-locked loops (PLLs) are a crucial component in modern communications systems. Comprising of a phase-detector, linear filter, and controllable oscillator, they are widely used in radio receivers to retrieve the information content from remote signals. As such, they are capable of signal demodulation, phase and carrier recovery, frequency synthesis, and clock synchronization. Continuous-time PLLs are a mature area of study, and have been covered in the literature since the early classical work by Viterbi [1] in the 1950s. With the rise of computing in recent decades, discrete-time digital PLLs (DPLLs) are a more recent discipline; most of the literature published dates from the 1990s onwards. Gardner [2] is a pioneer in this area. It is our aim in this work to address the difficulties encountered by Gardner [3] in his investigation of the DPLL output phase-jitter where additive noise to the input signal is combined with frequency quantization in the local oscillator. The model we use in our novel analysis of the system is also applicable to another of the cases looked at by Gardner, that is the DPLL with a delay element integrated in the loop. This gives us the opportunity to look at this system in more detail, our analysis providing some unique insights into the variance `dip' seen by Gardner in [3]. We initially provide background on the probability theory and stochastic processes. These branches of mathematics are the basis for the study of noisy analogue and digital PLLs. We give an overview of the classical analogue PLL theory as well as the background on both the digital PLL and circle map, referencing the model proposed by Teplinsky et al. [4, 5]. For our novel work, the case of the combined frequency quantization and noisy input from [3] is investigated first numerically, and then analytically as a Markov chain via its Chapman-Kolmogorov equation. The resulting delay equation for the steady-state jitter distribution is treated using two separate asymptotic analyses to obtain approximate solutions. It is shown how the variance obtained in each case matches well to the numerical results. Other properties of the output jitter, such as the mean, are also investigated. In this way, we arrive at a more complete understanding of the interaction between quantization and input noise in the first order DPLL than is possible using simulation alone. We also do an asymptotic analysis of a particular case of the noisy first-order DPLL with delay, previously investigated by Gardner [3]. We show a unique feature of the simulation results, namely the variance `dip' seen for certain levels of input noise, is explained by this analysis. Finally, we look at the second-order DPLL with additive noise, using numerical simulations to see the effects of low levels of noise on the limit cycles. We show how these effects are similar to those seen in the noise-free loop with non-zero initial conditions.

Design of digital differentiators

A digital differentiator simply involves the derivation of an input signal. This work includes the presentation of first-degree and second-degree differentiators, which are designed as both infinite-impulse-response (IIR) filters and finite-impulse-response (FIR) filters. The proposed differentiators have low-pass magnitude response characteristics, thereby rejecting noise frequencies higher than the cut-off frequency. Both steady-state frequency-domain characteristics and Time-domain analyses are given for the proposed differentiators. It is shown that the proposed differentiators perform well when compared to previously proposed filters. When considering the time-domain characteristics of the differentiators, the processing of quantized signals proved especially enlightening, in terms of the filtering effects of the proposed differentiators. The coefficients of the proposed differentiators are obtained using an optimization algorithm, while the optimization objectives include magnitude and phase response. The low-pass characteristic of the proposed differentiators is achieved by minimizing the filter variance. The low-pass differentiators designed show the steep roll-off, as well as having highly accurate magnitude response in the pass-band. While having a history of over three hundred years, the design of fractional differentiator has become a ‘hot topic’ in recent decades. One challenging problem in this area is that there are many different definitions to describe the fractional model, such as the Riemann-Liouville and Caputo definitions. Through use of a feedback structure, based on the Riemann-Liouville definition. It is shown that the performance of the fractional differentiator can be improved in both the frequency-domain and time-domain. Two applications based on the proposed differentiators are described in the thesis. Specifically, the first of these involves the application of second degree differentiators in the estimation of the frequency components of a power system. The second example concerns for an image processing, edge detection application.