Active whisking is an important model sensorimotor behavior, but the function of the cerebellum in the rodent whisker system is unknown. I have made patch clamp recordings from Purkinje cells, granule cells and interneurons in vivo to identify whether and how cerebellar activity encodes kinematic features of whisking including the phase and set point. I show that Purkinje cell spiking activity changes strongly during whisking bouts. On average, the changes in simple spike rate coincide with or slightly precede movement, indicating that the synaptic drive responsible for these changes is predominantly of efferent (motor) rather than re-afferent (sensory) origin. Remarkably, on-going changes in simple spike rate provide an accurate linear read-out of whisker set point. Thus, despite receiving several hundred thousand discrete synaptic inputs across a non-linear dendritic tree, Purkinje cells integrate parallel fiber and interneuron input to generate precise information about whisking kinematics via linear changes in firing rate. In addition, I demonstrate that evidence is consistent in both granule cells and interneurons activity within the circuit, providing a linear encoding regime of whisker set point.

Improvised explosive devices (IEDs) came to prominence in Iraq and became the primary weapon used by insurgent forces in Afghanistan against coalition troops and vehicles in the late 20th and early 21st century. IEDs caused over 50% of all UK combat fatalities in these conflicts, many of whom suffered extremity traumatic amputations (TAs). To date, understanding of the mechanism of blast-mediated TA has been based on limited anatomical and minimal radiological injury data. Blast-mediated TAs were first thought to be solely attributable to limb flail caused by a blast wind of sufficient velocity to bring about extremity avulsion. This injury mechanism theory was significantly modified in 1996 following UK military medical research: the shockwave – ‘primary blast injury’ – was thought to be pivotal in the creation of blast-mediated TAs, coupling directly into the limb and causing a long bone fracture prior to gross limb movement, with the blast wind – ‘tertiary blast injury’ - subsequently displacing the limb causing amputation through the aforementioned fracture. A strong link was believed to exist between TA and exposure to lethal levels of primary blast loading. Guillotine-type TAs were also seen due to large fragments energised by the blast (secondary blast injury). Modern battlefield blast casualty (survivors and fatalities) analysis, combined with incident data analysis of each blast event, has not shown the previously asserted link between TA and primary blast lung injury. Furthermore, the high proportion of through joint TAs (22.4% in fatalities vs. 1.3% reported previously), determined by postmortem CT imaging, has indicated pure flail as a valid injury mechanism. These injuries thus appear to have multiple blast injury mechanisms - primary and tertiary, secondary, and (previously unappreciated) pure tertiary – and a greater understanding of these injury modalities has significant implications for mitigation and prevention strategies.

Pattern formation is found widely throughout nature serving important roles in many different biological contexts. Multicellular organisms form patterns to enable new functions and pattern formation is also a crucial step for biological adaptation. The work presented here lays the foundations for the creation and control of multicellular growth patterns with a normally single-celled organism. The natural capacity for strains of Saccharomyces cerevisiae yeast to perform multicellular growth is here genetically and externally-controlled using synthetic biology tools. This allows S. cerevisiae grow in multicellular filamenting patterns, following a unique phenotype called pseudohyphal growth. Fine-control of the dynamic behaviour and gene expression of a colony of cells growing with this phenotype should enable fractal-like growth formations to be genetically-encoded. In this work, synthetic gene regulatory networks were first constructed in order to control genetic targets than induce or repress the pseudohyphal growth phenotype. This generated haploid and diploid yeast strains that can quickly switch to growing as filaments when simple chemical stimuli are externally provided or removed. In order to enable control over pattern formation with these strains, further genetic targets such as the BUD genes were investigated and the pseudohyphal growth phenotype was linked to previously described timer gene regulatory networks that dynamically change the expression of target genes over hours and days. These timer networks allowed cells to be programmed to transition from pseudohyphal growth back to normal yeast growth as the cells are grown in a colony over days. Finally, in an attempt to create controlled growth from pseudohyphal yeast so that fractal-like patterns could be made, new hybrid promoters with multiple modes of regulation were generated. These externally-inducible promoters were constructed to only express in mother cells after they have budded a daughter cell, and were designed in order to control how often filaments form new branches when S. cerevisiae grows in pseudohyphal form. While a full integration of the whole system to generate controllable fractal-like patterns was not achieved in the end, this study delivers several valuable new tools for yeast research and yeast synthetic biology.

Exploration is an active, closed loop process, where actions are coordinated to maximise sensory information gain through perception. Exploratory actions provide complementary and redundant sensory information, which our brain efficiently combines to reduce the uncertainty about the natural environment. As humans increasingly interact with machines, there is a growing need for human-machine interfaces to support natural interactions and efficient information display. The integration of sensory cues allows humans to resolve ambiguities in everyday natural interactions. Here, this mechanism is exploited to enhance information transfer of abstract tactile cues. This thesis develops a feedback method based on vibrotactile apparent motion, where an array of stimulators is excited in a particular spatio-temporal pattern to induce an illusion of motion across the skin. In the proposed approach, the speed of motion is coupled with additional cues to ease the discrimination between similar speeds. The increased throughput of information promises an efficient and convenient way for substitution of auditory or visual navigation cues. Sensory loss and dysfunctions, and cognitive disorders, such as blindness, tactile hypersensitivity and autism, often severely constrain one's ability to function. Assistive technology can greatly improve their life, such as in the case of tactile sensory substitution devices for visually and hearing impaired. However, as sensory impairments sometimes lead to cognitive dysfunctions, it is crucial to consider these relationships when designing assistive devices. Here, a case study investigated the use of vibrotactile cues to communicate with a deafblind autistic individual during equestrian therapy. The approach was validated by evaluating the individual's sensory perception and motor behaviour. Human ability to acquire and act upon sensory information trough touch is possible thanks to simultaneous control of arm motion, force and impedance. This capability remains absent in human machine interactions, such as in the case of VR and telerobotics, due to the complexity of arm impedance estimation. A novel approach is demonstrated here where impedance control is achieved by simplifying the model of human arm use. The benefits are demonstrated in virtual object manipulation. The improved control of contact dynamics promise more efficient exploration of virtual and remote environments. This thesis presents methods for efficient information transfer through tactile perception by both sensory feedback and motor actions. The capabilities and limitations of the human sensorimotor system are carefully considered and employed to design wearable interfaces applied to sensory substitution and telerobotics.

Biological cells organise their interior into compartments called organelles in order to function. The familiar ones are the mitochondria, the Golgi apparatus and the lysosomes, which are surrounded by a lipid membrane. There are also membrane-less organelles that are currently receiving intense attention from the biology and physics communities. Membrane-less organelles are ubiquitously present, from yeast cells to mammalian cells, and play key roles in biological functions. One of these are the stress granules (SG) that form in the cytoplasm when the cell is under stress, and are indispensable to the cell’s survival. Membrane-less organelles are proteinaceous liquid drops that assemble by phase separation in the cytoplasm. Phase separation under non-equilibrium conditions in the cell cytoplasm is poorly understood as a physical phenomenon, limiting our understanding of membrane-less organelles. In this thesis, we investigate the physics of non-equilibrium phase separation. Specifically, we study a ternary fluid model in which phase-separating proteins can be converted into soluble proteins, and vice versa, via ATP-driven chemical reactions. We elucidate using analytical and simulation methods how drop size, formation and coarsening are controlled by the reaction rates, and categorize comprehensively the qualitative behaviour of the system into distinct regimes. We then apply our formalism to SG formation. Guided by experimental observations, we consider minimal models of SG formation based on phase separation regulated by ATP-driven chemical reactions. We also provide specific predictions that can be tested experimentally. The model studied in this thesis is a minimal model of membrane-less organelle regulation in the cytoplasm, and can also be applied to chemically-controlled drops in emulsions in the engineering setting.

Anterior cruciate ligament (ACL) deficiency results in knee instability that includes an increase in internal tibial rotation and anterior tibial translation (ATT) as ACL is the primary restraint to anterior shear and internal rotation. Clinically, ACL deficient (ACLD) patients undergo surgery or/and rehabilitation programmes depending on their ability to cope or otherwise. However, the ACL reconstructed (ACLR) knees may still have residual instability in ATT and tibial internal rotation. Functional electrical stimulation (FES) has been used in conventional physiotherapy for ACL deficiency, including strengthening the muscles around the knee. The rehabilitation treatment focuses on strengthening the quadriceps muscle because it gets weakened after ACL injury or ACL reconstruction. However, stimulating the hamstrings, especially the biceps femoris long head (BFLH) with its insertion on the fibular head is a candidate to reduce the knee instability of ACLD and ACLR by applying a posterior pull and external rotation to the tibia. This thesis proposes that knee instability in ACLD subjects can be reduced by stimulating the BFLH muscle with FES. Here, a musculoskeletal modelling approach was used to simulate the function of FES. A new optimisation method was developed which allowed the inclusion of FES. There are three main studies present in this thesis. First, a pilot study was conducted in which healthy control subjects walked with and without FES of BFLH. It was found that selective activation of the BFLH can reduce the anterior tibial shear and tibial internal rotation torque at the knee in healthy subjects. Second, a validation study for the algorithm used in the musculoskeletal model was conducted in which the effect of FES stimulation of the BFLH on gluteus maximus activations was tested using electromyography (EMG). This study concluded that there were statistical correlations between peak and impulse of gluteus maximus activation between FES activation level and muscle activity of gluteus maximus as quantified by both EMG and the musculoskeletal model. In the final study, the validated model was used to compare the internal rotation torque, anterior shear force, speed and gluteus medius and gluteus maximus muscle activation between control, ACLD and ACLR groups during stance phase with and without FES stimulated on BFLH. This study found that the activation of BFLH with FES during stance phase was able to reduce the knee instability of the patient groups and triggered the compensatory mechanism for each patient group to react differently. Therefore, besides quadriceps, the rehabilitation treatment should focus on appropriate timed activation of the BFLH to improve the quality of life of patients.

Synthetic genomics is a new and fast emerging multi-disciplinary field of research, representing the largest scale of work underway in synthetic biology. The Saccharomyces cerevisiae version 2 (Sc2.0) project is currently the leading example of synthetic genomics research, and is an attempt to perform the first redesign, synthesis and assembly of a complete synthetic genome for an eukaryotic organism. Major changes are being made to the redesigned DNA sequence of the new yeast genome and these include a variety of different deletions, sequence recodings at every gene and also insertions of DNA motifs. The most significant insertion is the placement of a recombinase recognition site called loxPsym throughout the genome, placed downstream of all non-essential genes. These recombinase sites act as recombination hotspots for Cre recombinase, to bring about recombination-mediated genomic rearrangements of the synthetic chromosomes. Together the Cre recombinase and loxPsym inserts make up the inducible Synthetic Chromosome Rearrangement and Modification by LoxPsym Evolution (SCRaMbLE) system. This thesis describes the design, synthesis, hierarchical assembly and in vivo integration of synthetic DNA for the construction of synthetic chromosome XI for the Sc2.0 project. With the first 90 kb of the synthetic chromosome complete, the SCRaMbLE toolkit was then examined. It was hypothesised that along with causing gene deletions, inversions and duplications, this Cre-lox system could also be implemented to insert heterologous DNA into the synthetic chromosomes. This thesis shows that with the correct formatting of heterologous DNA, SCRaMbLE can be further developed to generate a new synthetic biology method called ‘SCRaMbLE-in’ suitable for the insertion of heterologous genes into synthetic chromosomes as they are rearranging to produce diverse synthetic yeast strains with novel functions. Having successfully developed and investigated SCRaMbLE-in, this method was then used for the simultaneous introduction of multiple genes that can confer a selective benefit to yeast. By providing three heterologous genes encoding enzymes that together reconstitute the oxidoreductase xylose-utilisation pathway, a synthetic yeast strain capable of growth on the lignocellulosic sugar xylose was produced by SCRaMbLE-in. This work thus demonstrates a new approach to constructing strains for metabolic engineering projects, where incorporation of heterologous genes and rapid evolution of the yeast genome can be done simultaneously in one pot.

Development of optical technologies aiming to reverse engineer neural circuits has been flourishing over the past two decades. Multi-Photon Laser Scanning Microscopy (MPLMS) together with the development of fast kinetic fluorescent calcium dyes has revolutionised the world of modern neuroscience. This technology enables mesoscale functional imaging in deep scattering brain tissues of large two (2D) and three dimensional (3D) neural networks. With single cell sensitivity in vitro as well as in vivo, it is one of the main contenders for deciphering higher brain functions. My approach in this thesis is to develop and test new scanning techniques for fast functional calcium imaging aiming to enhance the temporal precision of the acquisition. To avoid the slow and sequential "point" raster scanning nature of these Galvanometric Scanners (GSs) based microscopes, I developed new 2D and 3D scanning algorithms. These algorithms were developed in MATLAB with a simulation platform that models the main mechanical elements of the MPLSM. Both my 2D Adaptive Spiral Scanning (SSA) algorithm and my 3D Orbital Scanning Trajectory (OST) algorithm were developed to minimize the inertial slowdowns of the GSs and Electrical Tunable Lens (ETL) and therefore increase the temporal resolution of the acquisition. In 2D, I tested the SSA algorithm on in vitro hippocampal brain slices loaded with the synthetic calcium dye Cal520. To assess the performance of the scanning technique, I used the Cramer-Rao Bound (CRB) as a metric for signal quality. The CRB estimates the time of occurrence of an Action Potential (AP) from the calcium imaging data, taking into account the sampling frequency and the SNR of the acquisition. In this thesis, I show that the use of scanning strategies enables sampling rates one order of magnitude higher than traditional frame scanning in functional calcium imaging. I also show that frame scanning needs considerably higher SNR values than scanning strategies to reach the same temporal precision. In 3D, I implemented the scanning algorithms into the software and hardware of the MPLSM and recorded the trajectory of the focal point with a high-speed camera as a proof of principle. More analyses regarding the precision of the paths needs to be carried out in 3D for functional calcium imaging in vitro or in vivo. These software-based scanning strategies are attractive as they are inexpensive, easily transferable from one setup to another and enable fast functional calcium imaging with standard commercial MPSLMs. Finally, through this implementation of scanning strategies, I recorded multiple data sets of spontaneous and evoked activity in populations of Dentate Granular Cells (DGCs). This lead to the new beginning of a larger in vitro investigation at the microcircuit level on the functionality of the DG.

Plants are known to respond to various types of environmental stresses arising from physicochemical changes and other organisms. As plants often simultaneously experience multiple stress factors due to their immobility, capacity to appropriately regulate gene expression by integrating multiple stress signals is crucial for successful adaptation to hostile environments. Although significant progress has been made in elucidating the molecular mechanisms for regulation of stress response genes under single stress, little is known about the effects of combined stress signals on gene regulation and their associated mechanisms. This thesis aimed to contribute to the understanding of plant stress response by studying the signal integration mechanisms under various perspectives: first, the thesis explored how multiple stress signals affect the choices over discretised regulatory outcomes, such as up-regulation or down-regulation. We propose that processing of multiple signals can be described as logical operations, and subsequently investigate the mechanisms for each signal integration outcome by constructing logical model of intracellular signalling network. The resulting insight was applied to analyse a transcriptomic dataset from the model plant Arabidopsis thaliana, leading to novel hypotheses about potential crosstalk interactions that are missing between multiple stress signalling pathways. In parallel, the thesis also explored the cases where integration of multiple stress signals modulates dynamics of gene expression. An experimental study of the expression profile of Response-to-Dehydration 29A (RD29A), a model stress response gene, was conducted to show that combination of multiple stress inputs introduces a unique qualitative effect on dynamics of gene expression. The origin of this behaviour was investigated via a dynamical model of the RD29A regulatory network, which subsequently revealed potential interactions in the regulatory network that are currently unknown. Taken together, this thesis argues that systematic comparison between gene regulatory outcomes under single and combined stress inputs provides a crucial source of information for discovering functionally significant regulatory interactions in the stress signalling network.

The aim of this thesis was to develop novel carbon-based biosensors and sensors for real-time metabolite and drug detection, to provide the next generation of medical devices which can give clinicians relevant chemical information in real-time at the patient bedside. An autocalibration system was developed using LabSmith programmable components to give precise fluid delivery and excellent temporal control of multiple liquid streams. This enables a 5-point calibration to be carried out using two solutions in 12 minutes. Systems using chitosan, poly(ethylene glycol)diglycidyl ether hydrogel, electrodeposition and selfassembly to immobilise enzymes on a combined needle electrode surface were studied and their performances were investigated using a microfluidic platform. The autocalibration system was combined with the graphene oxide-based biosensors in a microchip coupled with a microdialysis probe and was examined as a proof-of-concept clinical on-line analysis system. A reduced graphene oxide-based sensor was fabricated using a combined needle electrode for on-line neurotransmitter detection of dopamine. Its performance was compared with that of a platinum electrode. A microfluidic sensor based on a carbon nanotube-epoxy composite was fabricated to detect the presence of carboplatin (anti-cancer drug) in healthy tissue in real time during chemotherapy. Detection of carboplatin was carried out using differential pulse voltammetry firstly in a beaker, in which carbon nanotube-epoxy composite electrodes performed better than glassy carbon electrodes for oxidation of free purine bases and than DNA-modified carbon nanotube-epoxy composite sensors for detection of carboplatin. Carboplatin detection was then performed in a microfluidic platform. The methodology for on-line carboplatin detection was optimised in terms of the analysis time and for the repeated determination of carboplatin using the same electrode. Microdialysis and microfluidic techniques have been combined to give a proof-of-concept system real-time carboplatin detection using the carbon nanotube-epoxy composite sensors.