The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is a major pest of agricultural and horticultural importance in most parts of the world. It damages crops by feeding and transmission of viruses. There is a need for sustainable IPM due to development of resistance to insecticides. Understanding the reproductive biology of this pest may suggest novel approaches to pest control. The aim of this research was to study the copulation behaviour of F. occidentalis and the possible role of pheromones. Female F. occidentalis copulated multiple times with virgin males either in rapid succession or over days, contrary to previous reports that females will not copulate again until after fifteen days. Subsequent copulations were, however, shorter than the first, probably due to detection of a barrier by males that may prevent sperm transfer. Culture reared males copulated with virgin females, but rarely with copulated females, and virgin males copulated with dead virgin females more often than with dead mated females of similar age, which showed that males could detect whether females had copulated. Observation suggested that during copulation, males were applying antiaphrodisiac pheromone by stroking or antennation, which may have been used for assessing females’ copulation status. This antiaphrodisiac pheromone may be the contact pheromone, 7-methyltricosane. Culture reared males were probably limited in reproductive resources due to old age or multiple copulation, thus they could have changed strategy to show preference for virgin females, copulating more with them than already copulated females, whereas, most young virgin males (with more resources) were not mate selective. It is also possible that the increased preference for virgin females resulted from male mating experience. Choice based on male copulation status was not observed in virgin females.

Guard cells controls the stomata through which exchange of gas takes place by balancing between CO2 uptake for photosynthesis and water loss through transpiration leading to ultimate plant water use efficiency (WUE). Climate change is predicted to lead to greater temperatures and reduced water availability resulting in adverse effect on plant productivity. Sustainable agriculture will therefore require a major reduction in plant water use hence stomata have become potential target for manipulation. Understanding the signal mechanisms of stomata in response to these changing environmental conditions is therefore critically important. In order to facilitate an understanding of stomatal regulation and how it is influenced by the surrounding mesophyll cells, we have used two approaches to find a possible coordination that links mesophyll and guard cell metabolism through the use of stomatal physiology and genetic engineering. The first approach used a novel epidermal mesophyll transfer experiment to monitor stomatal responses to dynamic environmental changes with and without the mesophyll present. The second approach used new molecular tools and techniques to manipulate chloroplast metabolism specifically in the guard cells to elucidate mesophyll-derived signals that coordinate mesophyll CO2 demands with stomatal behaviour towards crop improvement. The results presented have shown guard cells plays a role in stomatal function even though the degree of responsiveness is slower than when the mesophyll is present. Furthermore, the molecular approach demonstrated using Arabidopsis plants overexpressing Rieske and SBPase resulted in substantial and significant impacts on plant development coupled with increases in photosynthetic efficiency of photosystem II in the early stages of seedling development. The result obtained proves more opportunities await the exploitation of guard cells metabolism towards the improvement of plants.

Microtubules are long linear polymers that switch randomly between periods of growth and shrinkage, in a process known as dynamic instability. In vivo, dynamic instability is regulated by microtubule associated proteins (MAPs). One class of MAPS, the kinesins, move actively along microtubules, and some regulate microtubule dynamics. Kinesin-8, a kinesin, regulates microtubule dynamics in a wide range of eukaryotic cells. Schizosaccharomyces pombe (S. pombe) provides a well-characterised system in which to study microtubule regulation by MAPs. During interphase, microtubules grow from the centre of the rod-shaped cell until their plus ends reach and pause at the cell end, before undergoing catastrophe and shrinking. Shrinkage occurs predominantly at cell ends, even as the cell grows longer. I have studied the cell biology of kinesin-8-dependent interphase microtubule dynamics in S. pombe. I have identified an interphase-specific binding partner of S. pombe kinesin-8 (Klp5/Klp6); Mcp1. Mcp1 was required for Klp5/Klp6 accumulation at interphase microtubule plus ends and for Klp5/Klp6 induced interphase microtubule shrinkage. Tea2 (a kinesin) and Tip1 (CLIP170 orthologue) were found to stabilise interphase microtubules. Cells lacking Tea2 or Tip1 displayed interphase microtubules which, after reaching cell ends, underwent shrinkage sooner than wild type cells. Cells lacking Klp5/Klp6 or Mcp1 showed the opposite phenotype, microtubules which dwelt at cell ends longer than control cells before shrinking. Klp5/Klp6 accumulation on interphase microtubule plus ends steadily increased, peaking just before microtubule shrinkage. In contrast, Tea2 accumulated rapidly to newly nucleated interphase microtubule plus ends and was lost before microtubule shrinkage. I propose a model in which Tea2 prevents Klp5/Klp6 induced microtubule shrinkage until the interphase microtubule has grown to the cell end, where Tea2 is lost. At the cell end Klp5/Klp6 now induce shrinkage.

Autophagy is an evolutionarily conserved process of cellular material degradation, involved in development, starvation-induced nutrient-level control, degradation of aggregated proteins and pathogen removal. The specificity of this process is governed by selective autophagy receptors, messenger proteins which identify cargo and deliver it to Atg8a – a component of the core autophagic machinery. Receptors bind to Atg8-family proteins via the LIR motif, a short amino-acid sequence that is conserved across the animal kingdom. We performed a bioinformatics screen in order to identify new putative Atg8a interacting proteins. We searched the Drosophila proteome for proteins containing LIRmotifs and ubiquitin-binding domains. We identified the protein Kenny (homologue of human IKKg), which contains a LIR motif and a conserved UBAN domain. Kenny is a modulator of the Drosophila Immune deficiency (IMD) pathway, an innate immunity response targeted at gram-negative bacteria. Using biochemical approaches and in vivo studies in Drosophila we observed that Kenny interacted directly with Atg8a via its LIR motif and was selectively degraded by autophagy. We found that Kenny accumulated in autophagy depleted flies, which was accompanied by a constitutive activation of the IMD pathway and expression of antimicrobial peptides. This caused a hyperproliferation of stem cells in the midgut, reduced defecation rates and shortened the overall fly lifespan. Given sequence similarities between Kenny and another described receptor, Optineurin, we also investigated Kenny’s potential role in mitophagy and/or xenophagy. Kenny accumulated and localised with mitochondria in thorax muscles of flies, treated with FCCP. Kenny was found to localise in the vicinity of phagocytosed Staphylococcus aureus in larval haemocytes, cultured ex vivo. We propose that Optineurin could be a new functional mammalian orthologue of Kenny, in addition to the established mammalian homologue, NEMO.

In this thesis, a flexible low-cost quantitative phase imaging microscopy (LQPIM) system for imaging both thin and thick biological phase objects in a non-contact, non-invasive, and label-free manner is reported. LQPIM optics was developed based on classical Zernike’s phase contrast approach and an additional phase shifting module to introduce user-defined phase modulations by utilising standard optical components. The phase shifting was performed using twin concentric mirrors or laser cut apertures in the arms of a Michelson interferometer where the reference mirror can be moved in / n steps (n - number of steps) with a piezoelectric transducer. Hence, the optical phase shifting modules are 10 - 15% (approximately) of the cost compared to the more widely reported modules based on spatial light modulator. In the microscope implementation reported in this thesis, a total magnification of 25x was achieved utilising relay lenses in LQPIM optics together with a standard 10x objective lens. The imaging system was simulated in MATLAB, where two-beam interference equation with varying bandwidth (1 – 250 nm), centre wavelength (450 – 650 nm) of the illumination sources and a range of previously reported phase shift algorithms (PSA) were used. The simulation results confirm that the optimum phase resolution is achievable if a broadband source of bandwidth 30 - 50 nm is used for illuminating thin (i.e. ≤ 250 nm) and thick (i.e. ≥ 1250 nm) biological samples. The four frames at 90 PSA and six plus one frames at 60 PSA offer different compromises between image acquisition time, phase resolution and out-perform other PSAs. A phase resolution of 0.382 nm and 0.317 nm was achieved using four frames at 90 and six plus one frames at 60 PSAs, respectively for the broadband illumination from a green LED. A coherent, single longitudinal mode laser source with a rotating diffuser for speckle averaging, gave 0.667 nm and 0.512 nm phase resolution using the same algorithms mentioned above. The parasitic fringes resulted in reduced resolution; hence, incoherent LED illumination was preferred. Measurements are presented over a longer optical path difference (≥ 1250 nm) than hitherto reported for a similar microscope. The given exemplar data demonstrates an ability of LQPIM system to quantify cellular and sub-cellular structures at the nanoscale in epidermis cells of Allium cepa. Key words: Quantitative phase imaging, low-cost, optical microscopy, phase imaging and phase shift imaging.

The aim of synthetic biology is to create a new discipline of engineering based on biological parts, devices and systems. The availability of predictable, programmable tools to sense and to control gene expression is central to our ability to engineer such systems. Ribonucleic acid (RNA) is an attractive building material to create such programmable tools, as RNA-RNA interactions are predictable and RNA secondary structure prediction software has been developed. Design rules for creating such parts using RNA can be established, based on a standardised approach or on structural design rules into which function is implicitly encoded. In this latter case, RNA folding software can be used to create RNA sequence which satisfy generalisable structural characteristics, but are tailored to a specific application. In this work, new design rules for the creation of RNA-based sensors and actuators are developed. The actuator parts are based on riboregulators, but with a circular topology generated through splicing of a ribozyme. The ability of these circular riboregulators to activate transcription of gene expression in E. coli cells is demonstrated. A method for improving these actuators by directed evolution is then tested. Finally, design rules for creating sensors of RNAs based Clustered Regularly Interspaced Short Palindromic Repeat guide RNAs (CRISPR gRNAs) are developed. These gRNA-based sensors can switch states and repress gene expression through a CRISPR-Cas9 based platform, but only in the presence of an arbitrary "trigger" RNA. The rules developed for creating sensors and actuators are characterised in E. coli, but are based on general principles that could be used in other organisms including eukaryotic cells.

Saltmarshes are one of the most significant blue carbon sinks but there is a paucity of information regarding saltmarsh carbon stocks globally, consequently these habitats are not included in the global carbon budget. The aim of this study is therefore to better understand the spatial and temporal variation of saltmarsh sediment total organic carbon (TOC) content. Therefore, three saltmarshes along the salinity gradient of the Colne Estuary were studied. The effect of the study sites’ locations along the estuary, higher plant species distribution, above-ground biomass and aerobic respiration on TOC content was investigated. The spatial and temporal variation of sediment TOC content was investigated by monthly sampling from two habitats and three zones at each study site. There was a significant spatial variation in plant species distribution which could be due to zonation and the location of the sites along the estuary. Saltmarsh plants were the important driver of spatial and temporal variations in sediment TOC content. The sediment TOC content at the study sites in the lower (Colne Point) and the mid (Brightlingsea) estuary was significantly higher than the upper estuary (Wivenhoe) (P < 0.001, range: 9-25 Kg C m-2). The range of sediment TOC content of the studied saltmarshes was between 88% - 290% higher than other UK studied saltmarshes and between 4% -169% higher than the majority of the studied marshes in the Northern Hemisphere. Therefore, if the sediment carbon content of the similar saltmarshes to the Colne estuary were taken into account it would suggest that the UK and global saltmarsh sediment TOC estimate would increase. It will take possibly about 100 years for the realignment saltmarshes at Essex to reach the carbon storage capacity of Colne Point. Therefore, the Colne Point natural saltmarsh is a very significant carbon reservoir that has been overlooked.

Synthetic biology is the design and implementation of novel biological devices via the application of engineering principles to biological systems research. Mathematical modelling is an invaluable tool in developing our understanding of biological system dynamics and characterising small parts and circuits for the assembly of higher-level systems. In this thesis, mathematical modelling approaches are applied to three biological circuits of interest. A novel mechanistic model of the DNA recombination reactions comprising a genetic switch reveals the input criteria and operational specifications required of a digital data storage module. Specific layering of the components comprising recombinase-based genetic switches can provide cellular Boolean logic operations. A novel mechanistic model of a two-input temporal logic gate is able to simulate and predict in vivo dynamical responses captured by a large experimental dataset. Experimental implementation of recombinase-based circuitry is unpredictable and can lead to lengthy development times, providing clear evidence of the advantages of utilising mathematical models in synthetic biology. Antibiotic resistance has become one of the most prominent challenges facing medicine today, placing immense importance on the characterisation of new natural products. The rst detailed mathematical model of the methylenomycin A producing gene cluster in the bacterium Streptomyces coelicolor is developed through the application of model selection to a large set of candidate system architectures. Mathematical models presented in this thesis can be adapted and expanded to suit many different experimental conditions and system responses, facilitating the design of novel synthetic biological circuitry.

Cellular blebs are fast, pressure-driven protrusions of the cell membrane that are initially devoid of F-actin. Although blebs have often been overlooked as a functional part of cell motility, blebbing has been shown to play an important role in migration in 3D mechanically resistive environments, such as movement through densely packed tissues. The location of bleb nucleation sites is often assumed to be entirely stochastic, however, cells migrating using blebbing motility have been repeatedly observed to perform persistent, directional movement. Given the compelling evidence on the role of blebbing in directional cell migration, relatively little is known on the mechanisms of bleb site selection; how bleb sites are determined and directed to the cell front remains an open question. Previously, Tyson et al. found that chemotaxing Dictyostelium cells preferentially bleb from concave regions, where membrane tension could facilitate membrane-cortex detachment. Based on this, a biophysical model for curvature dependent bleb nucleation was proposed, hinting at the possibility that polarised blebbing was due to physical forces alone. We develop a novel image based modeling approach, using real cell contours from image data to initialise the model. This enables us to link quantitative experimental data and predictive modeling on the spatial distribution of blebs for the first time. We proceed to show that the extent to which cell geometry is a good predictor of bleb site selection is highly dependent on the degree of mechanical resistance the cells experience. For cells in highly resistive environments, where we observe the front and rear of the cell to be geometrically distinct, our novel modeling approach demonstrates that physical forces are sufficient to polarise blebbing activity in chemotaxing cells. For cells in low resistive environments however, where the front and rear of the cell are not geometrically distinct, we show that an additional mechanism is required to restrict blebbing to the cell front. We propose this additional mechanism to be a front-to-rear gradient in the linker protein TalinA. Creation of a TalinA-mNeon construct allows us to experimentally confirm the existence of an asymmetric linker distribution. We incorporate the observed exponential linker gradient within the model. Inclusion of this mechanism increases the predictive power of the model, with regard to the spatial distribution of experimentally observed bleb sites, through efficiently directing blebbing activity to the cell front. We have created a method which links quantitative experimental data and predictive modeling, this tool allows us to disentangle the role of physical forces and biological mechanisms in the prediction of bleb nucleation sites.

Scanning ion conductance microscopy (SICM) is a nanopipette-based technique that has historically been used for the topographical imaging of soft samples. This thesis demonstrates the development of SICM into a multifunctional tool, capable of providing a host of additional information about both biological and inert samples, whilst maintaining the structural mapping capability for which it is usually employed. Two approaches are taken to extend the functionality of SICM: (i) designing sophisticated potential, and positional, control functions that are then used with traditional single-channel nanopipettes; and (ii) incorporating an ion conductance channel into a multi-barrelled probe. In the single-channel setup, a pulsed-potential profile allows the extraction of surface charge density on extended substrates, and a ramped-potential profile permits spatially resolved mapping of redox reactions on an electrode substrate. When integrated into a more complex probe, SICM is used to study molecular uptake at cellular surfaces, and to print Cu microstructures on a Au substrate. While this thesis is primarily concerned with technique development, the studies herein have broad applications in cell biology, pharmaceuticals, materials science and beyond. In addition to developing imaging modes that allow the extraction of functional information at a surface, this thesis also contributes to the fundamental understanding of the SICM system. Finite element method simulations are performed alongside experimental studies, in order to fully understand the contributions of the pipette geometry, ion current rectification, and pipette-surface interactions on the measured ionic current. The theoretical treatment herein provides a foundation upon which future multifunctional SICM regimes could be designed, extending the scope of this increasingly powerful technique.