The sensor kinase PhoQ and its cognate response regulator PhoP constitute a two-component system, which is primarily responsible for sensing and responding to Mg2+ starvation in Escherichia coli. Additionally, there is growing evidence of regulatory links between PhoPQ and constituents of the outer membrane. Furthermore, it has been shown that PhoPQ is regulated negatively by MicA, an sRNA controlled by sigmaE. Encoded by rpoE, sigmaE is an alternative sigma factor that is activated in response to extracytoplasmic stress, specifically misfolded outer membrane proteins. Surprisingly, it was not possible to generate ΔphoP mutants, using P1vir transduction under standard conditions and kanamycin as the selective agent. Furthermore, a statistical analysis of these results indicates they cannot be explained by chance alone. The results show that PhoP is required for sigmaE activity in an RseA-independent manner, thereby suggesting that PhoP is a chief regulator of sigmaE activity. It is likely that diminished sigmaE activity in a phoP mutant, extracytoplasmic stress and OM deformation, caused by the reagents used in P1vir transduction, are responsible for the inability to transduce the phoP allele. Finally, evidence has been found relating to a second mechanism through which PhoP directly represses rpoE expression, thereby introducing further complexity into the regulator relationship that exists between sigmaE and PhoP.

Perceived exertional dyspnoea is reported to be the most common symptom among physically active individuals of all abilities and ages and/or performance in high level athletes, potentially impacting on performance and limiting enjoyment of sporting activities. Identifying the causes of the perceived symptoms requires careful assessment with a wide range of factors potentially contributing to the reported respiratory issues. The purpose of this thesis was to investigate different assessment approaches in the identification of breathing dysfunction in exercising adults. Elite swimming and boxing require athletes to achieve relatively high minute ventilation. In Chapter 4 (Study 1 of this thesis), thirty-eight elite boxers and 44 elite swimmers completed a thorough respiratory assessment that revealed a nine-fold greater prevalence of exercise-induced bronchoconstriction in swimmers when compared with boxers. These results suggested that the combination of a sustained high ventilation and provocative training environment may impact the susceptibility of athletes to this condition. Dysfunctional breathing may mimic and/or co-exist with exercise-induced bronchoconstriction. The use of specific questionnaires may improve the identification of this condition in athletes. In Chapter 5 (Study 2 of this thesis), 9% of the 428 healthy, physically active young adults who completed the Nijmegen Questionnaire had a score ≥ 23, suggestive of a dysfunctional breathing status. A separate cohort of 104 athletes underwent an indirect bronchoprovocation challenge and completed the Nijmegen questionnaire. The sensitivity, specificity, positive and negative predicted values suggested that the Nijmegen score was a poor predictor of a positive bronchoprovocation challenge in athletes and therefore is not suitable to detect dysfunctional breathing in athletes. The posture an athlete holds during exercise may alter breathing pattern and increase reported exercise induced respiratory symptoms. In order to investigate whether respiratory parameters are affected by different postural positions, in Chapter 6 (Study 3 of this thesis), 15 healthy male athletes performed a 10-minute, high intensity cycling test with normal shoulder position and with hunched shoulders. Results of this study showed that cycling with hunched shoulders at high intensities over a prolonged period leads to an increase in perceived dyspnoea and suggested that posture may contribute to reports of respiratory symptoms during exercise in the absence of cardio-pulmonary disease. With the aim of investigating the effect of different postural positions on the ventilatory excursion, in Chapter 7 (Study 4 of this thesis), 15 healthy male athletes performed baseline spirometric measurements and 10-minutes cycling challenges with normal shoulder position and with hunched shoulders, while undergoing simultaneous data collection with optoelectronic plethysmography. The findings of this study suggested that respiratory excursion and lung volume compartmentalisation at both rest and during high intensity exercise are affected by the position of the shoulders. In conclusion, athletes who train and compete in provocative environments at a sustained high ventilation have an increased susceptibility to airway dysfunction. No existing questionnaire is sensitive enough to identify dysfunctional breathing and differentiate it from other respiratory conditions, such as exercise-induced bronchoconstriction. Exercising for a prolonged period at high intensities with hunched shoulders triggers increased abdominal contribution to vital capacity and a subsequent increase in perception of breathing sensation without a significant effect on physiological markers of respiratory function. Further investigations should be undertaken in order to develop a new questionnaire that is more suitable for an athletic population and has higher accuracy in identifying symptoms associated with exercise induced breathing impairment. Precise detection of distortions between compartmental contributions in exercising individuals may play an important role in the differential diagnosis of dysfunctional breathing.

Myosin binding to actin, and thus muscle contraction, is regulated by Tropomyosin (Tpm), Troponin (Tn) and calcium (Ca2+). Tpm, is an α-helical coiled-coil dimer, which exists as a homo- or heterodimer. Two major isoforms of Tpm are found in striated muscle, α and β. Though it is known that different dimers exist, the mechanism by which they form and exchange is not fully understood. The thermal stability and exchange between dimers was explored with the use of circular dichroism and SDS PAGE densitometry analysis. Homodimers showed little exchange to form heterodimers at temperatures up to 20 °C . Dimer stability at these temperatures reduces the need for chemical cross-linking samples. While extensive exchange was seen at 37 °C . Reverse exchange of WT and mutant (E54K - dilated cardiomyopathy mutant) containing heterodimers to form homodimers did not show the same extent of exchange, suggesting a dimer preference. Results showed the ability to determine dimer content of a solution with the use of polyacrylamide gels and chemical cross-linking. The thermal melting curves of Tpm highlighted a significant destabilisation of β Tpm against the α isoform. Tpm heterodimer containing E54K mutant showed a decreased thermal stability. Notable differences were seen not only for isoforms and homo- and heterodimers, but also for buffer conditions and protein tags. Increased salt concentrations led to an increase in thermal stability. Crosslinking dimers increased thermal stability, whereas addition of His-tags led to a decrease in thermal stability. Changes in thermal stability highlighted the need for caution when tagging or cross-linking the protein. Dimer exchange on actin provided conflicting results between SDS PAGE cosedimentation assays and pyrene fluorescence cosedimendation assays, which highlighted limitations of cross-linking Tpm and using fluorescent labels. The stiffness of Tpm dimers was explored using atomic force microscopy (AFM) to image Tpm particles. Significant differences were seen between the relatively stiff α Tpm and less stiff β isoform. Changes in stiffness of Tpm affect its ability to cooperatively activate the thin filament, and provides insight into the assembly of dimers in vivo.

The ubiquity of software places emphasis on the need for techniques that allow us to ensure that software behaves as we expect it to behave. The most widely-used approach to ensuring software quality is unit testing, but this is arguably not a very efficient solution, since each test only checks that the software behaves as expected in one single scenario. There exist more advanced techniques, like property-based testing, model-checking, and formal verification, but they usually rely on properties, models, and specifications. One source of friction faced by testers that want to use these advanced techniques is that they require the use of abstraction and, as humans, we tend to find it more difficult to think of abstract specifications than to think of concrete examples. In this thesis, we study how to make it easier to create models that can be used for testing software. In particular, we research the creation of reusable models, ways of automating the generalisation of code and models, and ways of automating the generation of models from legacy unit tests and execution traces. As a result, we provide techniques for generating tests from state machine models, techniques for inferring parametrised state machines from code, and refactorings that automate the introduction of abstraction for property-based testing models and code in general. All these techniques are illustrated with concrete examples and with open-source implementations that are publicly available.

Background: Kv2.1 and TASK-1 channels are two main contributors of K+ currents in pulmonary artery smooth muscle cells (PASMC). Dysregulation of these channels has been implicated in the pathogenesis of pulmonary hypertension (PH). This thesis aims to delve deeper into the implications of the regulation of Kv2.1 by Kv9.3 in PH. Another subject of interest would be whether NADPH oxidase type 4 (Nox4), one of the major reactive oxygen species (ROS) producers in the PASMC, modulates Kv2.1, Kv9.3, and TASK-1 channels. The effects of several redox agents are also investigated as potential modulators of Kv2.1, Kv9.3, and TASK-1. In addition, this thesis also examined the effect of a Kv2-channel blocker, stromatoxin, on Kv2.1 and Kv9.3. Finally, since amphoterin-induced gene and open reading frame (AMIGO) proteins have recently been shown as novel Kv2.1-interacting partners, their effects on Kv2.1 and/or Kv9.3 are also explored in this study. Experimental approach: Whole-cell patch clamp electrophysiology was used to measure currents of the ion channels expressed in modified tsA-201 cells, in the absence and presence of Nox4 AMIGO and other regulatory molecules. Immunohistochemistry was deployed to visualize the distribution of Kv2.1 and Kv9.3 proteins in the rat lungs and hearts. Key results and Conclusions: This study supports the findings that Kv9.3 regulates Kv2.1 by increasing the current amplitude, shifting the activation threshold to a more negative voltage range, and prolonging the slow component of time constant of deactivation. These effects could be beneficial in PH as this would mean cells could be brought back to its resting membrane potential faster and the transduction of the next action potential can be delayed. Kv2.1 and Kv9.3 have also been detected at the endothelium and PASMC in rat lungs and hearts, further substantiating the claim that these channels are potential players in regulating PH. AMIGO1 and AMIGO2 proteins are confirmed as regulators of Kv2.1 and Kv9.3 proteins. Nox4 does not regulate Kv2.1, Kv9.3, and TASK-1 channels expressed in tsA-201 cells. While hydrogen peroxide (H2O2) does not have any effect on Kv2.1 and Kv9.3, it abolished the current reduction effect of AMIGO2 on Kv2.1/Kv9.3. Other redox agents used in this study such as dithiothreitol (DTT), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and chloramine T (Ch-T) are not modulators of these channels expressed in tsA-201 cells. The lack of effect from Nox4 and these redox agents could suggest that the redox regulation of different Nox subunit/Kv channels combination varies for different cell types due to the different regulatory proteins present in different heterologous expression systems. As with the case of H2O2 and AMIGO2, it is likely that the regulatory proteins, which could facilitate the hypoxia-sensing properties of Nox4 and the effects of the redox agents on the ion channels, are missing in our heterologous expression system, compared with other host cells.

The development of recent imaging modalities and of multimodal imaging may offer new perspectives for biomedical imaging, such as in-vivo cancer detection at early stages. By combining optical coherence tomography (OCT) and photoacoustic microscopy (PAM), complementary information is extracted from tissue: scattering and absorption. Non-invasive cross-sectional images with micrometre resolution are obtained. In this thesis, for the first time, encouraging results using a single SC source for OCT and PAM are obtained. Micrometre axial resolution is achieved using SC sources for OCT. The use of SC sources for PAM allows for multispectral PAM (MPAM) by using several excitation spectral bands. With MPAM, different absorbers are distinguishable and recognisable through their absorption spectra. In addition, for the first time, spectroscopic photoacoustic (sPA) measurements are demonstrated in the visible using a bandwidth narrower than 40 nm. These results were obtained with the first multimodal imaging system that combines sPA, PAM, MPAM and OCT. A single commercially available SC source is used for excitation. Diverse in-vitro and in-vivo samples are imaged to show the capabilities of such a configuration. In addition, the development of a novel fibre-based SC source with both increased energy density and pulse repetition frequency (PRF) is presented. The increased pulse energy allows reduction of excitations bands that leads to more accurate MPAM and sPA measurements, while the access to larger PRFs allows for both noise reduction and faster imaging rates in PAM and OCT. A tapered photonic crystal fibre (PCF) is used to generate the SC by nonlinear spectral broadening. The larger input core of the tapered PCF enables enhanced energy density, where more than 50-100 nJ is achieved with less than 30 nm wide bandwidth, over a broad spectrum extending from 500 nm to 1700 nm. Such a source can be used for in-vivo blood oxygen saturation determination, skin and other superficial organs imaging, which is critical to image tumours and diagnose early stage cancers. Such imaging modalities can also be beneficial during surgery and treatment.

No description available.

Physics and engineering principles have long been applied to the development of instrumentation and assays that have significantly advanced biological research. In particular, photonics based instruments and assays have proven to be powerful tools that enable researchers to investigate biological processes in vivo. The research described in this thesis covers a range of photonics based instruments and assays that expand the capabilities researchers have to investigate challenging biological problems. These advances give researchers new tools for directly visualising dynamic biological events in three ways: 1) How to look: novel microscope instrumentation; 2) What to look at: Novel imaging based assays to dissect the tumour microenvironment; and 3) Where to look: Novel surgical protocols to enable ultra-high-resolution optical imaging in living animals (intravital imaging). Application of these assays and instruments to the challenging problem of cancer metastasis has led to a new understanding of the process of intravasation and haematogenous dissemination, paving the way to new clinical diagnostics and therapies.

Human surfactant protein D (hSP-D) is a C-type lectin and member of the collectin family that is involved in pathogen recognition as part of the innate immune response. Recognition occurs through the carbohydrate arrays on the surfaces of the pathogens. This work focuses on characterising, at the atomic level, the recognition of oligosaccharide analogues of the carbohydrate arrays using a recombinant fragment of human surfactant protein D (rfhSP-D). The crystal structures of three rfhSP-D complexes with the α(1→6)-linked isomaltotriose, β(1→4)-linked cellotriose and β(1→3)-linked laminaritriose have been successfully solved in P21 using rigid body refinement. Isomaltotriose was refined to 1.96Å with refinement statistics: Rwork 0.1664 and Rfree 0.2084. Cellotriose was refined to 1.59Å with refinement statistics: Rwork 0.0.1879 and Rfree 0.2106. Laminaritriose was refined to 1.75Å with refinement statistics: Rwork 0.1808 and Rfree 0.2193. In combination with two previously solved malto-N-ose complexes, the structures reveal a new binding mechanism for β-D-glucoses in SP-D and identify a new extended binding surface. The crystal structure of rfhSP-D in complex with the disaccharide unit of peptidoglycan, muramyl disaccharide, extracted from the gram-positive bacteria, Micrococcus luteus has been solved in P21 and refined to 1.95Å with final refinement statistics of Rwork 0.1708 and Rfree 0.2169. This crystal structure provides the first insight into recognition of peptidoglycan, an important part of the bacterial cell wall. The rfhSP-D complex with the R7 rough mutant oligosaccharide from S. enterica minnesota has been redetermined from previous work in the group. The structure has been solved in P21 and refined to 1.75Å with final refinement statistics of Rwork 0.1730 and Rfree 0.1960. The crystal structure, along with the known R5 oligosaccharide complex, builds on the understanding of the flexibility and versatility of lipopolysaccharide recognition by hSP-D and the important role of the two flanking residues, Asp325 and Arg343.

Human transthyretin (TTR) is heavily implicated in a range of fatal amyloid diseases. The propensity to form amyloid varies between wild-type TTR and various mutants. In all cases this occurs by tetramer dissociation to a monomeric form, which in turn polymerises into intermediates that ultimately assemble into amyloid fibrils. Numerous X-ray crystal structures of TTR mutants have been determined over the last 40 years. These structures have all turned out to be very similar, shedding little light on the widely varying amyloid-forming properties. The goal of this PhD project was to investigate the neutron crystal structures of these mutants – on the basis that factors that are difficult to identify using X-ray crystallography (protonation states, hydrogen bonding, hydration) may be implicated in the varying TTR stabilities. High-resolution neutron crystallographic studies of two TTR mutants were carried out. These mutants were chosen for their strongly contrasting behaviour. The S52P mutant is highly amyloidogenic, whereas the T119M mutant is resistant to amyloid formation. As part of these studies, a unique way of measuring and exploiting H2O/D2O back-exchange was uncovered – allowing the stability of specific regions in the S52P structure to be probed. A wide range of mass spectrometry measurements were also carried out. These initially focused on the assessment of the impact of deuteration of the proteins produced for crystallographic work. However, in addition, the impact of different mutations on the subunit exchange kinetics of TTR tetramers was also studied. Finally, the impact of binding of a small-molecule ligand, tafamidis, to the S52P mutant was investigated through a combination of structural and biophysical studies. Collectively, these results have been placed in the context of the overall knowledge of TTR amyloidosis, and an exciting perspective for work is given.