A technique to randomise consecutive codons in a sequence of DNA using MAX oligonucleotides

Randomisation of DNA using conventional methodology requires an excess of genes to be cloned, since with randomised codons NNN or NNG/T 64 genes or 32 genes must be cloned to encode 20 amino acids respectively. Thus, as the number of randomised codons increases, the number of genes required to encode a full set of proteins increases exponentially. Various methods have been developed that address the problems associated with excess of genes that occurs due to the degeneracy of the genetic code. These range from chemical methodologies to biological methods. These all involve the replacement, insertion or deletion of codon(s) rather than individual nucleotides. The biological methods are however limited to random insertion/deletion or replacement. Recent work by Hughes et al., (2003) has randomised three binding residues of a zinc finger gene. The drawback with this is the fact that consecutive codons cannot undergo saturation mutagenesis. This thesis describes the development of a method of saturation mutagenesis that can be used to randomise any number of consecutive codons in a DNA strand. The method makes use of “MAX” oligonucleotides coding for each of the 20 amino acids that are ligated to a conserved sequence of DNA using T4 DNA ligase. The “MAX” oligonucleotides were synthesised in such a way, with an MlyI restriction site, that restriction of the oligonucleotides occurred after the three nucleotides coding for the amino acids. This use of the MlyI site and the restrict, purify, ligate and amplify method allows the insertion of “MAX” codons at any position in the DNA. This methodology reduces the number of clones that are required to produce a representative library and has been demonstrated to be effective to 7 amino acid positions.

Resonant two-wave mixing in photorefractive materials with the aid of dc and ac fields

A recently predicted resonant effect for the enhancement of two-wave mixing in photorefractive materials is investigated. The resonance occurs when the frequency of the applied ac field agrees with the eigenfrequency of the excited space-charge wave. Experimentally a clear resonance is found, as predicted by the theory, for high dc electric fields, but the resonance is smeared out for lower fields. A modified theory, taking into account the second temporal harmonic of the space-charge wave, shows good agreement with the experimental results.

Excitation of higher spatial harmonics by a moving light pattern in sillenites

We investigate numerically the dependence of higher harmonics of the space-charge field on the detuning frequency between the pump waves, which form a running interference pattern. Bistability and hysteresis of harmonics are predicted for a contrast of the interference pattern m =(0.25-0.3). For contrasts m˜1 and small detuning frequencies we show the existence of a narrow resonance, connected with the nonlinear excitation of a slowly decreasing sequence of spatial harmonics. For experiments we use a BSO crystal in the optical configuration which avoids nonlinear optical distortions. The experimental data show good qualitative agreement with theory.

A legal perspective of integrated surface mining, solid waste landfill reclamation and land conservation

In the area of international environmental law this thesis proposes the formulation of one-step planning and permitting regulation for the integrated utilisation of new surface mines as depositories for municipal solid waste. Additionally, the utilisation of abandoned and currently operated surface mines is proposed as solid waste landfills as an integral step in their reclamation. Existing laws, litigation and issues in the United Kingdom, the U.S. and Canada are discussed because of their common legal system, language and heritage. The critical shortage of approved space for disposal of solid waste has caused an urgent and growing problem for both the waste disposal industry and society. Surface mining can serve three important environmental and societal functions inuring to the health and welfare of the public: (1) providing basic minerals for goods and construction; (20 sequentially, to provide critically needed, safe burial sites for society's wastes, and (3) to conserve land by dual purpose use and to restore derelict land to beneficial surface use. Currently, the first two functions are treated environmentally, and in regulation, as two different siting problems, yet they both are earth-disturbing and excavating industries requiring surface restoration. The processes are largely duplicative and should be combined for better efficiency, less earth disturbance, conservation of land, and for fuller and better reclamation of completed surface mines returning the surfaces to greater utility than present mined land reclamation procedures. While both industries are viewed by a developed society and its communities as "bad neighbours", they remain essential and critical for mankind's existence and welfare. The study offers successful examples of the integrated process in each country. The study argues that most non-fuel surface mine openings, if not already safe, can economically, through present containment technology, be made environmentally safe for use as solid waste landfills. Simultaneously, the procedure safeguards and monitors protection of ground and surface waters from landfill contamination.

Automated assessment of visual fields and their inter-relation to evoked potentials in visual disorders

The Octopus Automated Perimeter was validated in a comparative study and found to offer many advantages in the assessment of the visual field. The visual evoked potential was investigated in an extensive study using a variety of stimulus parameters to simulate hemianopia and central visual field defects. The scalp topography was recorded topographically and a technique to compute the source derivation of the scalp potential was developed. This enabled clarification of the expected scalp distribution to half field stimulation using different electrode montages. The visual evoked potential following full field stimulation was found to be asymmetrical around the midline with a bias over the left occiput particularly when the foveal polar projections of the occipital cortex were preferentially stimulated. The half field response reflected the distribution asymmetry. Masking of the central 3° resulted in a response which was approximately symmetrical around the midline but there was no evidence of the PNP-complex. A method for visual field quantification was developed based on the neural representation of visual space (Drasdo and Peaston 1982) in an attempt to relate visual field depravation with the resultant visual evoked potentials. There was no form of simple, diffuse summation between the scalp potential and the cortical generators. It was, however, possible to quantify the degree of scalp potential attenuation for M-scaled full field stimuli. The results obtained from patients exhibiting pre-chiasmal lesions suggested that the PNP-complex is not scotomatous in nature but confirmed that it is most likely to be related to specific diseases (Harding and Crews 1982). There was a strong correlation between the percentage information loss of the visual field and the diagnostic value of the visual evoked potential in patients exhibiting chiasmal lesions.

A method for automatically eliciting node weights in a hierarchical knowledge-based structure for reasoning with uncertainty

Hierarchical knowledge structures are frequently used within clinical decision support systems as part of the model for generating intelligent advice. The nodes in the hierarchy inevitably have varying influence on the decisionmaking processes, which needs to be reflected by parameters. If the model has been elicited from human experts, it is not feasible to ask them to estimate the parameters because there will be so many in even moderately-sized structures. This paper describes how the parameters could be obtained from data instead, using only a small number of cases. The original method [1] is applied to a particular web-based clinical decision support system called GRiST, which uses its hierarchical knowledge to quantify the risks associated with mental-health problems. The knowledge was elicited from multidisciplinary mental-health practitioners but the tree has several thousand nodes, all requiring an estimation of their relative influence on the assessment process. The method described in the paper shows how they can be obtained from about 200 cases instead. It greatly reduces the experts’ elicitation tasks and has the potential for being generalised to similar knowledge-engineering domains where relative weightings of node siblings are part of the parameter space.

Orientation masking and cross-orientation suppression (XOS):implications for estimates of channel bandwidth

Most contemporary models of spatial vision include a cross-oriented route to suppression (masking from a broadly tuned inhibitory pool), which is most potent at low spatial and high temporal frequencies (T. S. Meese & D. J. Holmes, 2007). The influence of this pathway can elevate orientation-masking functions without exciting the target mechanism, and because early psychophysical estimates of filter bandwidth did not accommodate this, it is likely that they have been overestimated for this corner of stimulus space. Here we show that a transient 40% contrast mask causes substantial binocular threshold elevation for a transient vertical target, and this declines from a mask orientation of 0° to about 40° (indicating tuning), and then more gently to 90°, where it remains at a factor of ∼4. We also confirm that cross-orientation masking is diminished or abolished at high spatial frequencies and for sustained temporal modulation. We fitted a simple model of pedestal masking and cross-orientation suppression (XOS) to our data and those of G. C. Phillips and H. R. Wilson (1984) and found the dependency of orientation bandwidth on spatial frequency to be much less than previously supposed. An extension of our linear spatial pooling model of contrast gain control and dilution masking (T. S. Meese & R. J. Summers, 2007) is also shown to be consistent with our results using filter bandwidths of ±20°. Both models include tightly and broadly tuned components of divisive suppression. More generally, because XOS and/or dilution masking can affect the shape of orientation-masking curves, we caution that variations in bandwidth estimates might reflect variations in processes that have nothing to do with filter bandwidth.

An investigation into inductive parameter learning in complex hierarchical knowledge structures representing clinical expertise

This dissertation investigates the very important and current problem of modelling human expertise. This is an apparent issue in any computer system emulating human decision making. It is prominent in Clinical Decision Support Systems (CDSS) due to the complexity of the induction process and the vast number of parameters in most cases. Other issues such as human error and missing or incomplete data present further challenges. In this thesis, the Galatean Risk Screening Tool (GRiST) is used as an example of modelling clinical expertise and parameter elicitation. The tool is a mental health clinical record management system with a top layer of decision support capabilities. It is currently being deployed by several NHS mental health trusts across the UK. The aim of the research is to investigate the problem of parameter elicitation by inducing them from real clinical data rather than from the human experts who provided the decision model. The induced parameters provide an insight into both the data relationships and how experts make decisions themselves. The outcomes help further understand human decision making and, in particular, help GRiST provide more accurate emulations of risk judgements. Although the algorithms and methods presented in this dissertation are applied to GRiST, they can be adopted for other human knowledge engineering domains.

Transparent heterogeneous terrestrial optical communication networks with phase modulated signals

This thesis presents a large scale numerical investigation of heterogeneous terrestrial optical communications systems and the upgrade of fourth generation terrestrial core to metro legacy interconnects to fifth generation transmission system technologies. Retrofitting (without changing infrastructure) is considered for commercial applications. ROADM are crucial enabling components for future core network developments however their re-routing ability means signals can be switched mid-link onto sub-optimally configured paths which raises new challenges in network management. System performance is determined by a trade-off between nonlinear impairments and noise, where the nonlinear signal distortions depend critically on deployed dispersion maps. This thesis presents a comprehensive numerical investigation into the implementation of phase modulated signals in transparent reconfigurable wavelength division multiplexed fibre optic communication terrestrial heterogeneous networks. A key issue during system upgrades is whether differential phase encoded modulation formats are compatible with the cost optimised dispersion schemes employed in current 10 Gb/s systems. We explore how robust transmission is to inevitable variations in the dispersion mapping and how large the margins are when suboptimal dispersion management is applied. We show that a DPSK transmission system is not drastically affected by reconfiguration from periodic dispersion management to lumped dispersion mapping. A novel DPSK dispersion map optimisation methodology which reduces drastically the optimisation parameter space and the many ways to deploy dispersion maps is also presented. This alleviates strenuous computing requirements in optimisation calculations. This thesis provides a very efficient and robust way to identify high performing lumped dispersion compensating schemes for use in heterogeneous RZ-DPSK terrestrial meshed networks with ROADMs. A modified search algorithm which further reduces this number of configuration combinations is also presented. The results of an investigation of the feasibility of detouring signals locally in multi-path heterogeneous ring networks is also presented.

Interacting nonequilibrium systems with two temperatures

We investigate a simplified model of two fully connected magnetic systems maintained at different temperatures by virtue of being connected to two independent thermal baths while simultaneously being interconnected with each other. Using generating functional analysis, commonly used in statistical mechanics, we find exactly soluble expressions for their individual magnetization that define a two-dimensional nonlinear map, the equations of which have the same form as those obtained for densely connected equilibrium systems. Steady states correspond to the fixed points of this map, separating the parameter space into a rich set of nonequilibrium phases that we analyze in asymptotically high and low (nonequilibrium) temperature limits. The theoretical formalism is shown to revert to the classical nonequilibrium steady state problem for two interacting systems with a nonzero heat transfer between them that catalyzes a phase transition between ambient nonequilibrium states. © 2013 American Physical Society.

On the structural repertoire of pools of short, random RNA sequences

A detailed knowledge of the mapping between sequence and structure spaces in populations of RNA molecules is essential to better understand their present-day functional properties, to envisage a plausible early evolution of RNA in a prebiotic chemical environment and to improve the design of in vitro evolution experiments, among others. Analysis of natural RNAs, as well as in vitro and computational studies, show that certain RNA structural motifs are much more abundant than others, pointing out a complex relation between sequence and structure. Within this framework, we have investigated computationally the structural properties of a large pool (10 molecules) of single-stranded, 35 nt-long, random RNA sequences. The secondary structures obtained are ranked and classified into structure families. The number of structures in main families is analytically calculated and compared with the numerical results. This permits a quantification of the fraction of structure space covered by a large pool of sequences. We further show that the number of structural motifs and their frequency is highly unbalanced with respect to the nucleotide composition: simple structures such as stem-loops and hairpins arise from sequences depleted in G, while more complex structures require an enrichment of G. In general, we observe a strong correlation between subfamilies-characterized by a fixed number of paired nucleotides-and nucleotide composition. Our results are compared to the structural repertoire obtained in a second pool where isolated base pairs are prohibited. © 2008 Elsevier Ltd. All rights reserved.

Excitation of higher spatial harmonics by a moving light pattern in sillenites

We investigate numerically the dependence of higher harmonics of the space-charge field on the detuning frequency between the pump waves, which form a running interference pattern. Bistability and hysteresis of harmonics are predicted for a contrast of the interference pattern m =(0.25-0.3). For contrasts m˜1 and small detuning frequencies we show the existence of a narrow resonance, connected with the nonlinear excitation of a slowly decreasing sequence of spatial harmonics. For experiments we use a BSO crystal in the optical configuration which avoids nonlinear optical distortions. The experimental data show good qualitative agreement with theory.

Resonant two-wave mixing in photorefractive materials with the aid of dc and ac fields

A recently predicted resonant effect for the enhancement of two-wave mixing in photorefractive materials is investigated. The resonance occurs when the frequency of the applied ac field agrees with the eigenfrequency of the excited space-charge wave. Experimentally a clear resonance is found, as predicted by the theory, for high dc electric fields, but the resonance is smeared out for lower fields. A modified theory, taking into account the second temporal harmonic of the space-charge wave, shows good agreement with the experimental results.

Perceptions of parental pressure to eat and eating behaviours in preadolescents:the mediating role of anxiety

Previous research suggests that parental controlling feeding practices are associated with children's overeating and undereating behaviours. However, there is limited research addressing the link between children's mental health symptoms (specifically anxiety and depression) and their reports of eating behaviours, despite knowledge that these psychopathologies often co-exist. The current study aimed to identify the relationships between preadolescents' perceptions of their parents' feeding practices with reports of their own anxiety, depression and eating behaviours. Three hundred and fifty-six children (mean age 8.75 years) completed questionnaires measuring their dietary restraint, emotional eating and external eating, as well as their perceptions of their parents' use of pressure to eat and restriction of food. Children also completed measures of general anxiety, social anxiety and depression symptomology. Results indicated that preadolescents' eating behaviours were associated with their perceptions of the controlling feeding practices their parents used with them. Preadolescents' dietary restraint, emotional eating and external eating behaviours were positively associated with their reports of general and social anxiety, and depression symptomology. In addition, perceptions of parental pressure to eat were positively related to preadolescents' anxiety and depression levels. Child anxiety (general and social) was found to mediate the relationship between perceptions of parental pressure to eat and preadolescents' eating behaviours (dietary restraint, emotional eating and external eating). The results suggest that greater anxiety in preadolescents may explain why children who perceive greater pressure to eat by their parents are more likely to exhibit maladaptive eating behaviours. © 2014 Elsevier Ltd.

Dynamic complexity of chaotic transitions in high-dimensional classical dynamics:Leu-Enkephalin folding

Leu-Enkephalin in explicit water is simulated using classical molecular dynamics. A ß-turn transition is investigated by calculating the topological complexity (in the "computational mechanics" framework [J. P. Crutchfield and K. Young, Phys. Rev. Lett., 63, 105 (1989)]) of the dynamics of both the peptide and the neighbouring water molecules. The complexity of the atomic trajectories of the (relatively short) simulations used in this study reflect the degree of phase space mixing in the system. It is demonstrated that the dynamic complexity of the hydrogen atoms of the peptide and almost all of the hydrogens of the neighbouring waters exhibit a minimum precisely at the moment of the ß-turn transition. This indicates the appearance of simplified periodic patterns in the atomic motion, which could correspond to high-dimensional tori in the phase space. It is hypothesized that this behaviour is the manifestation of the effect described in the approach to molecular transitions by Komatsuzaki and Berry [T. Komatsuzaki and R.S. Berry, Adv. Chem. Phys., 123, 79 (2002)], where a "quasi-regular" dynamics at the transition is suggested. Therefore, for the first time, the less chaotic character of the folding transition in a realistic molecular system is demonstrated. © Springer-Verlag Berlin Heidelberg 2006.