Immunoglobulin A (IgA) is the most abundant and heterogenous human antibody class, being present in two major subclasses, IgAl and IgA2, and different oligomeric states. Secretory component (SC) binds to dimeric IgAl (dlgA) to form secretory IgA (SIgA). In this thesis, X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling were used to determine their structures in solution. Free SC consists of five Ig-variable domains, with seven glycosylation sites. SC was found to adopt a compact, J-shaped structure in solution, with the glycosylation sites probably located on one face of SC. In this model, one side of SC and SC Dl, containing known IgA-binding motifs, is free to interact with dlgA. Dimeric IgAl (dlgAl) is formed by two IgAl monomers covalently bound by the joining (J) chain. The modelling of the solution structure of dlgAl shows that the arrangement of the two monomers are near-planar in an extended arrangement. The two Fc regions form a slightly bent end-to-end contact. All four Fab antigen-binding sites are independent of one another. The model shows that the two Fc regions are accessible to the Fc alpha receptor (FcaRI) and the polymeric immunoglobulin receptor (plgR). This dlgAl structure suggests a mechanism for dlgAl aggregation in IgA nephropathy through oligomerisation caused by removal or movement of the Fab regions by proteolysis or changes in the glycosylation of the hinge region. SIgA is formed by the transcytosis of dlgA across epithelial cells, where after binding plgR (membrane-bound SC) it is secreted into the mucosa. At the mucosal surfaces SIgA acts as the first line of defence against pathogens. The solution structure of SIgAl suggests that the arrangement of the IgAl monomers are not altered when SC is binds to dlgAl, therefore retaining their near-planar structure. The five domains of SC are in an extended conformation along the convex Fc-Fc edge of the dimer. The SIgAl solution structure shows how SC and dlgAl confer mutual protection to one another from proteolysis. The modelling of the solution structure of SIgA2 shows that, although the IgA2 monomers are in a similar arrangment to that in SIgAl, SIgA2 has a non-planar structure in solution. SC is located in a similar extended arranagment on the Fc-Fc convex edge to that in SIgAl, yet the Fab regions are positioned out of the Fc-Fc plane. These structural differences between the SIgA isotypes may increase their efficiency in mucosal immunity but confer varied susceptibilities to mucosal proteases.

Magnetic Resonance Imaging (MRI) has been at the focus of medical research as its availability and fidelity has improved in the last thirty years. MRI offers both high spatial resolution and excellent soft tissue contrast compared to complimentary medical imaging techniques, without the need to expose patients to ionising radiation. Novel MRI methods that utilise the intrinsic body water signal are still being developed and refined. Arterial Spin Labelling (ASL) MRI provides a non-invasive method to measure tissue perfusion, which has been extensively applied in the brain, and demonstrated pre-clinically in the heart and kidneys. However, there is currently no literature reporting the development and use pre-clinical liver ASL – possibly due to complex methodology and quantification necessary in small animals. Clinical liver perfusion imaging is predominantly carried out using an injected Gadolinium-based contrast agent; this technique can be challenging to quantify, cannot be immediately re-administered and may have complications for patients with renal impairment. A methodology to measure liver perfusion without the need for a contrast agent would find utility in a number of different hepatic diseases; monitoring pathophysiology and therapy efficacy. This research investigates the feasibility of a pre-clinical measure of liver perfusion using ASL and its potential application to a pre-clinical model of hepatic disease. We aim to apply the method to monitor novel therapy efficacy in pre-clinical disease models, to eventually translate both therapy and hepatic ASL into the clinical environment.

Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are clinically established sensitive molecular imaging modalities. However, their use in clinical cardiology remains limited. Early or intermediate stages of vascular calcification are thought to enhance plaque vulnerability. Computed tomography (CT), the “gold standard” for clinical imaging of calcification, fails to detect the early stages of calcification. PET and SPECT imaging may be used to detect the early stages of calcification in vivo. The aim of the project was to evaluate novel and clinically established bone seeking SPECT and PET agents for imaging vascular calcification using experimental animal models. Technetium-99m labelled methylene diphosphonate (99mTc-MDP), fluorine-18 sodium fluoride (18F-NaF) and the novel bone seeking agents technetium-99m labelled dipicolylamine alendronate (99mTc-DPA Ale) and nitrido bis(dithiocarbamatebisphospho nate) (99mTc-N (DTCBP)2) were tested in vitro and for imaging in vivo using rat models of medial vascular calcification. In the initial in vitro studies the bone seeking radiotracers described here demonstrated their potential for avid binding with different minerals, both of biological and synthetic origin, with 99mTc-DPA-Ale and 18F-NaF binding most efficient. Rat models for vascular calcification were established by diet modification and sub-cutaneous injections. For optimising the in vivo imaging protocol, a robust rat model with extensive calcification was established by feeding a special warfarin diet and sub-cutaneous administration of vitamin D3. Another model, with slow and progressive calcification, was achieved by feeding rats with warfarin diet only, and was found to be an ideal candidate for the longitudinal imaging study. In vivo imaging with animal models for vascular calcification revealed that the radiotracers can detect vascular calcification earlier than computed tomography. Longitudinal studies with the calcified rat model showed that the radiotracers can also play a role in monitoring disease progression. However, the study also revealed several unexpected findings. SPECT and PET images showed radiotracer uptake in the abdominal aorta and mesenteric artery. Histological evidence of calcification was seen in the thoracic aorta which was not detected on SPECT and PET imaging. Histological sections also revealed that the calcified regions in the abdominal aorta are in proximity to the lumen whereas in the thoracic aorta they are deep seated, close to the adventitia which may account for the differential in uptake. The longitudinal imaging study showed that uptake of the radiotracers also reduced with maturity in calcification. These findings raise some intriguing questions, and answering them would help in a better understanding of the process of vascular calcification and developing strategies for in vivo imaging with SPECT-CT and PET-CT. In conclusion, bone seeking radiopharmaceuticals have the potential to be used as agents for early diagnosis of vascular calcification and further study needs to be done to address the interesting questions raised in this thesis.

Intracellular availability of the amino acid L-leucine in pancreatic β cells is thought to regulate cell growth and modulate D-glucose-induced insulin secretion (Data published in Cheng et al. 2016). System L amino acid transporters (LATs) control transport of L-leucine across the plasma membrane and may therefore influence intracellular L-leucine concentration. The aim of this project was to test the hypothesis that D-glucose supply to the cultured rat β cell line INS1E regulates leucine transport by acting on LATs. Incubation of cells in medium containing 0 versus 5 mM (control) D-glucose for 6 hours increased L-[3H]-leucine transport influx by 70% and increased intracellular L-leucine concentration by 150% in INS1E. In contrast, very variable responses were seen on exposure of INS1E to 25 mM D-glucose. Treating INS1E with the AMP-activated kinase (AMPK) agonist AICAR also increased L-[3H]-leucine influx by 70% suggesting that D-glucose deprivation regulates transport by lowering energy status which then activates the energy status sensor AMPK. D-glucose deprivation increased mRNA expression for the LAT1 (SLC7A5) isoform of LAT transporter about 3.5-fold when assessed by RT-Q-PCR. The effect showed very variable magnitude and was not observed for LAT2 (SLC7A8) or LAT4 (SLC43A2) mRNA, which were also strongly expressed in INS1E. In spite of strong LAT2/4 expression, siRNA silencing of LAT1 mRNA by 85% was sufficient to decrease basal L-[3H]-leucine influx by 35%. However, blockade of the LAT1 mRNA increase on D-glucose deprivation by treating with transcription inhibitor actinomycin-D failed to block the accompanying increase in L-[3H]-leucine influx, suggesting that activation of LAT transporter proteins by low energy status was largely occurring through an additional non-transcriptional mechanism. To test this, a human LAT1-eGFP-tagged cDNA construct completely lacking the normal LAT1 promoter sequence was transfected into HEK293A cells and successfully expressed eGFP fluorescence. Even though wild-type HEK293A showed high basal L-[3H]-leucine influx and no stimulation of this flux by AICAR, and LAT1-eGFP transfection alone failed to give statistically significant stimulation of L-[3H]-leucine influx, a significant 24% increase in transport was obtained in cells subjected to combined LAT1-eGFP transfection + AICAR. These results suggest that D-glucose starvation and AMPK activation in INS1E cells lead to a previously undescribed stimulation of L-leucine transport, which is similar in magnitude to that previously described for glucose transport in response to AMPK activation in muscle. It is concluded that declining energy status sensed through AMPK activates LAT1 (and maybe other isoforms of LAT L-leucine transporters), possibly through an AMPK-dependent translocation of LAT proteins between sub-cellular pools analogous to that previously described for GLUT monosaccharide transporters in muscle.

Medical ultrasound systems are undergoing continuous development. Five areas of particular recent interest are shear wave elastography, interventional ultrasound, microultrasound, and therapeutic ultrasound. These new developments motivate demand for new tissue models. The work discussed in this thesis presents different tissue models for these areas of contemporary ultrasound systems development. Acoustic test objects, known as phantoms, are the most widely used basis for testing medical ultrasound systems, but they have many limitations. Hence, the recent advancement in medical simulation, soft-embalmed Thiel human cadaver models being considered for their suitability as a model for testing shear wave elastography and interventional ultrasound systems. The results indicate significant similarities between the Thiel-embalmed cadavers, and human's in-vivo tissues in relation to their tissue stiffness measured with SWE. The use of these cadaveric models has also shown to bring benefits for interventional ultrasound systems testing and research. This is demonstrated with two specific case studies on ultrasound guided regional anaesthesia. Another aspect considered in this work is the suitability of Thiel-embalmed human breast tissues for testing therapeutic ultrasound systems. The results indicate that these models are unsuitable for therapeutic ultrasound systems testing because of their inability to produce lesions during sonication which can be seen visually or under histopathological examination. For testing microultrasound systems, mice preserved with the Thiel method have been assessed and shown to be a suitable model because of its small size, reusability and easier accessibility. This thesis has assessed the use of preserved human and animal tissue models for testing contemporary ultrasound systems.

A novel experimental conguration is developed combining a highintensity focused ultrasound source and a pulsed-laser, for the study of cavitation in a eld typical of those used for therapeutic ultrasound. The sonoptic chamber is specically designed to avoid the formation of acoustic standing waves, known to have a critical in uence on cavitation behaviour. A new technique of laser-nucleated acous- tic cavitation is presented, whereby a laser-pulse of energy below the breakdown threshold for the host medium, acts to nucleate acoustic cavitation in a pre-established eld. This facilitates the incorporation of high-speed cameras for interrogation at unprecedented temporal and spatial resolution, combined with acoustic detection directly correlated to the observed cavitation activity. A number of cavitation phenomena are investigated, including bubble-ensemble oscillations at a very early stage of development, in response to the acoustic driving. The frequency of oscillation, which bifurcates with increasing intensity, is also detected in the acoustic emissions. The application of a single-bubble model predicts a source for the acoustic emissions of quiescent radius equivalent to the bubble-ensemble observed, for each intensity investigated. The physical translation of the ensemble, due to the radiation force imposed by the primary eld, is also analysed. For laser-pulses of energy above the breakdown threshold, applying focused ultrasound to the cavity promotes and actuates jet-formation. The characteristics of the so formed jets depend on the intensity and location of the cavity relative to the ultrasound focus.

As blood vessel imaging techniques facilitate the fundamental understanding in vascular performance diagnosis and biomedical research improvement, we aimed to visualize and understand the blood vessels dynamics under human skin and their underlying mechanisms in real time. In this study, a noninvasive imaging system was selected to provide an investigation of the real time oscillation of blood vessels in vivo, using Spectral Radar Optical Coherence Tomography (SROCT). This main goal was achieved by evaluating the precision and confidence in recorded data by using a phantom made of Intralipid (IL) to mimic the physical properties of the skin. Then, we successfully managed to visualize for the first time the vasomotion under human skin using MatLab Image Processing Toolbox. After that, we explored mathematically the cyclic variations of the vascular area obtained from the images for a cohort of six participants. The Fourier and wavelet transforms were applied to identify the characteristic frequencies related to the oscillations in vascular cross sectional area. Finally, we investigated dynamical aspects of vasomotion, in response to temperature change, by using a Meleor Thermoelectric Temperature Controller (MTTC) to produce local heating in conjunction with Spectral Radar Optical Coherence Tomography (SROCT).

Functional Magnetic Resonance Imaging using the Blood Oxygenation Level Dependent (BOLD) contrast allows the brain's neural activity to be measured indirectly. This BOLD signal contains a wealth of information including changes in brain activity and functional connectivity (FC). FC is a measure of how correlated spatially separate brain regions are with each other. The work in this thesis is primarily concerned with novel methods of analysing the BOLD signal, in particular to give new measures of FC. A particular problem with typical measures of FC is that they assume that the networks are large scale and distributed, and that they originate from low frequency, static oscillations. It is clear from the way that we interact with the world that these assumptions are wrong, requiring a dynamic approach to investigate FC and the origins of what might be driving this. Here, a method combining short window correlation analysis and paradigm free mapping, a technique to detect spontaneous BOLD events without prior knowledge of their timings, is used to study the dynamic nature of these networks. It is further shown that these networks are at least in part driven by spontaneous activity, and that the rate of this spontaneous activity can be modulated by a task. These spontaneous events are then combined with network masks and temporal Independent Component Analysis to decompose these large scale networks into smaller sub-networks. Finally, the effects of spontaneous BOLD events on attention and task performance during a visual task is evaluated, highlighting how different brain regions that are not associated with a task can distract the subject's attention. It is shown that BOLD events that relate to a specific task use highly focal specific regions of the brain, confirming the spatial specificity of brain regions to a particular function.

Patient motion either internal (organ motion) or external (body movement) can produce artefacts that can adversely affect nuclear medicine imaging. Motion artefacts can impair diagnostic information and potentially affect the image findings and prognosis for patients. The goal of this work was to investigate the effect of motion on nuclear medicine imaging and to improve image quality, lesion detectability, and tumour volume delineation by applying motion correction techniques. To investigate the effects of motion under controlled simulated conditions, a three dimensional phantom drive system was designed and constructed suitable for use with planar, SPECT, PET and CT scanners. The system was used with a range of nuclear medicine phantoms for testing proof of principle with planar, SPECT and PET imaging prior to undertake further work involving patients. Planar phantom and patient 99mTc_DMSA studies demonstrated improvements in image quality by the application of motion correction techniques. A comparison between the motion correction software using dynamic frame and list mode data showed that "MOCO" software with the use of the list mode data produced the best quantification results with phantom data, whereas determining the best approach was more difficult with patient data. The potential of using list mode data as an improved method of combining data into frames for subsequent analysis was demonstrated. Motion correction techniques would appear to offer great potential in lung imaging. Respiratory gated SPECT phantom studies have been carried out to simulate the visualisation of small defects in the lung. The CNRs and alternative free response receiver operating characteristic (AFROC) analysis have demonstrated that summing the gated data after the application of motion correction software significantly improved image quality, observer confidence and small defect detectability (less than 20 mm, p=O.0002). The results of these studies have shown the promising role of "MCFLIRT" software as a motion correction tool with gated SPECT data. Tumour volume delineation was investigated on PET images both with and without motion. The accuracy and consistency of the gradient-based software method for segmentation in PET images, which is commercially available from Mimvista Ltd was investigated. The results of comparing the measured volumes to the true volumes indicated significant differences (p=O.0005). It was found that the Signal:Background ratio and registering the PET to the CT data have significant effects on volume measurements, whereas, the effect of using different grey scale and plane of orientation were not found to have significant effects on the volume measurement. Motion correction techniques also showed to be potentially beneficial in PET imaging. Improvement in volume measurement as a result of summing the motion corrected gated data was demonstrated. The results of these studies have also shown the promising role of "MCFLIRT" as a motion correction tool with gated PET data.

No description available.