Two major reasons for the failure of central nervous system axon regeneration are (i) lack of neurotrophic factors available to CNS neurones and (ii) the presence of molecules that inhibit the growth of axons. In this study a gene therapy approach using adeno-associated virus 8 (AAV8) was used to manipulate these two factors. The following major aims were addressed: (i) confirm the bioactivity of transgenes that would be packaged into the AAV8 vector; (ii) assess the cellular tropism of AAV8 in the dorsal root ganglion (DRG); (iii) evaluate the inflammatory responses of the nervous system to AAV8 after intra-DRG and intrathecal injection; (iv) determine the axon regenerative effect of AAV8-mediated delivery of nt-3 (a neurotrophic factor) and shRNA\(_{RhoA}\) (a disinhibitory therapy) to dorsal root ganglion neurones after spinal cord injury in the rat. Delivery of the nt-3 transgene in vitro resulted in production of high levels of NT-3 protein. Transfection of shRNA\(_{RhoA}\)-containing plasmids into cell lines resulted in a marked decrease in the amount of RhoA detectable in cell lysates. AAV8 was found to preferentially transduce large diameter, proprioceptive DRG neurones (DRGN) but in the context of a significant inflammatory response after intra-DRG injection 28d following intra-DRG injection. Axon regenerative effects of AAV8-mediated transgene delivery before lesioning were ambiguous and further work need to be undertaken to clarify this matter.

Long-haul optical fiber communication system is an important technology to support the latest broadband communication in the world, and there is strong competition in optical long-haul transmission to achieve high channel bit rates and large transmission capacity. Therefore, it is important to study a technology to improve the performance of such system. As we have already known, return-to-zero differential phase shift keying (RZ-DPSK) is an attractive solution to improve the long distance transmission system performance compared to the conventional on-off keying (OOK) in a 10-Gb/s system, because it has a high nonlinear tolerance. The dispersion flattened fiber (DFF) is attractive for its ability to improve the system performance. Therefore, it is possible to improve the transmission performance by a combination of the RZ-DPSK and the DFF, and one important technology of the current long-haul optical fiber communication system is the dispersion map. And it is widely deployed for already installed undersea optical fiber communication system in the world. A previous study reported that the blockless type dispersion map showed a superior performance than the block type dispersion map, and some efforts to improve the transmission performance of the block type map were conducted. Fundamental idea to improve the transmission performance of the block type map is to reduce the zero crossing points, and one idea is to shift the map toward the positive or the negative cumulative dispersion to reduce the zero crossing points within the map, but it was not so successful. The other idea is to tilt the dispersion map and it was more successful but not good enough. In this master thesis, I continued the study to improve the long-haul RZ-DPSK system performance using the block type dispersion map. One new idea of the dispersion map shifting, the split shifting, was tried, and another new idea of the dispersion map tilting, the split tilting, was examined. The performance with different repeater output power and different compensation scheme within the dispersion map was simulated by a numerical simulator .The goal is, following previous research, to clarify improved dispersion map design of the long-haul RZ-DPSK based transmission and find the effective method to improve the transmission performance. In addition, I also investigate tolerance of the effective area of the transmission fiber theoretically for the long-haul RZ-DPSK system based on the DFF.

The purpose of the work described in this dissertation is to better understand the role of Rz and Rz1 function with respect to phage lysis. We determined using both a genetic and biochemical approach that the Rz protein is an inner membrane protein containing a single N-terminal transmembrane domain (TMD) with an Nin/Cout topology. Consistent with previous work on Rz1, the Rz1 lipoprotein was found to be localized to the outer membrane (OM). Following localization, both Rz and Rz1 form homodimers in vivo due to intermolecular disulfide formation. Despite being localized to apposing membranes, the two proteins form a complex. A small number of phages encode a potential single protein equivalent of Rz-Rz1. This protein, termed a spanin, is predicted to tether the inner and outer membranes by a single polypeptide chain. Based on complementation, it was concluded that gp11 from the phage T1 is a functional equivalent of Rz-Rz1. Gp11, and by analogy the Rz-Rz1 two-component spanin complex, threads the meshwork of the PG layer. The presence of an Rz-Rz1 complex, which forms in the presence of peptidoglycan (PG), is supported by in vivo results. The soluble periplasmic domains of Rz and Rz1, which are dimeric and monomeric respectively, were purified. Circular dichroism analysis indicates that Rz is structured, with significant α-helical content, whereas Rz1, in which 10 out 39 residues are proline, is unstructured. Mixing the proteins results in the formation of a complex with significant new α-helical content. Negative-stain images reveal ~ 25 nm x ~ 4 nm rod-shaped structures. Holin independent activity of Rz and Rz1 is found to disrupt whole cells. Furthermore, time lapse microscopy of λ and λRzam lysis allows us to conclude that Rz and Rz1 are essential for lysis. These results suggest a model for Rz-Rz1 function which begins with Rz and Rz1 forming a complex through direct interaction prior to holin and endolysin function. Holin-mediated hole formation allows the endolysin to degrade PG which sterically hinders Rz-Rz1 activity. Removal of PG by endolysin degradation thus triggers Rz-Rz1 OM disruption via fusion of the inner and outer membranes.

With the essence of robustness toward fiber nonlinearity owing to the increasingly required high-speed data rate from the country to country or country to the state, many useful methods are proposed upon the long-haul optical fiber transmission, such as modulation format, dispersion map and repeater spacing, etc. Return-to-zero differential phase shift keying (RZ-DPSK) format was chosen in this master thesis because of its high tolerance toward nonlinear effect in the wavelength-division multiplexing (WDM) system transmission. It has been realized that the system performance is wavelength-dependent by the combination of the WDM technology and the RZ-DPSK system with the commonly used block-type dispersion map, especially for the significant performance difference between including or excluding the self-phase-modulation (SPM) effect. Therefore, it is quite significant to investigate the unwanted fiber nonlinearity. In this master thesis, the influence relating to the cross-phase modulation (XPM) effect and the SPM effect with the conventional dispersion map after long distance transmission is the mainly concerned issue to be discussed. In this master thesis, both experiment and theoretical simulation are investigated. On the theoretical simulation part, the Q-factor of the system zero dispersion wavelength at 1543.8nm, 1550nm, 1556.2nm were degraded and their value were less than 10dB. The Q-factor was around 1.5dB less than the averaged value. However, the performance of the long-haul RZ-DPSK system based on the block-type dispersion map shows no significant performance by shifting the system zero dispersion wavelength out of the WDM signal wavelength band. On the experimental part, the impact of the XPM effect and the SPM effect on the long-haul optical fiber communication system is investigated quantitatively. For the XPM experiment, the system performance was just only 0.1 dB difference after 6000km transmission. On the contrary, for the SPM experiment, the Q-factor between best and worst performance was up to 1.3dB difference. At last, the experiment and the simulation support each other successfully in this master thesis.

The aim of this thesis was to determine whether the blood flow velocities in the peripheral vascular system measured using phase contrast magnetic resonance imaging, PC-MRI, techniques could be used in the same way that blood flow velocities measured using spectral Doppler ultrasound are used to aid in the diagnosis of peripheral vascular disease. Specifically, we aimed to investigate the measurement of maximum velocities and the use of maximum velocity ratios; an area of investigation which has been neglected in studies of PC-MRI blood flow quantification to date. A series of optimisation and comparison studies were carried out using in-house developed test phantoms. Key to the in-vitro work was the establishment of a dual modality flow test system which would allow comparison of identical flow conditions measured using ultrasound and MRI. The work was complemented by in-vivo studies in healthy volunteers. A 4D PC-MRI commercial work-in-progress protocol and software package became available during the study and was evaluated in-vitro and in-vivo using similar methods as for the 2D PC-MRI studies. The main findings of the thesis were that 2D PC-MRI measurement of maximum velocities significantly underestimated those measured using spectral Doppler ultrasound. However, if corrections were applied to account for the overestimation of ultrasound maximum velocity due to spectral broadening, then the two methods were in agreement. In contrast, the use of maximum velocity ratios showed no difference between spectral Doppler ultrasound and 2D PC-MRI measurements. It was noted that one of the potential problems with the use of 2D PC-MRI in the measurement of the maximum velocity at a stenosis is the accurate positioning of the 2D velocity encoded slice in the stenotic jet. 4D PC-MRI, with a time resolved velocity encoded volume dataset, offers a potential solution to this. However, our evaluation of 4D PC-MRI showed that it can significantly underestimate both maximum velocities and maximum velocity ratios in comparison with 2D PC-MRI and spectral Doppler ultrasound and requires further development before it can be used for peripheral vascular applications.

A prospective randomised controlled clinical trial of treatment decisions informed by invasive functional testing of coronary artery disease severity compared with standard angiography-guided management was implemented in 350 patients with a recent non-ST elevation myocardial infarction (NSTEMI) admitted to 6 hospitals in the National Health Service. The main aims of this study were to examine the utility of both invasive fractional flow reserve (FFR) and non-invasive cardiac magnetic resonance imaging (MRI) amongst patients with a recent diagnosis of NSTEMI. In summary, the findings of this thesis are: (1) the use of FFR combined with intravenous adenosine was feasible and safe amongst patients with NSTEMI and has clinical utility; (2) there was discordance between the visual, angiographic estimation of lesion significance and FFR; (3). The use of FFR led to changes in treatment strategy and an increase in prescription of medical therapy in the short term compared with an angiographically guided strategy; (4) in the incidence of major adverse cardiac events (MACE) at 12 months follow up was similar in the two groups. Cardiac MRI was used in a subset of patients enrolled in two hospitals in the West of Scotland. T1 and T2 mapping methods were used to delineate territories of acute myocardial injury. T1 and T2 mapping were superior when compared with conventional T2-weighted dark blood imaging for estimation of the ischaemic area-at-risk (AAR) with less artifact in NSTEMI. There was poor correlation between the angiographic AAR and MRI methods of AAR estimation in patients with NSTEMI. FFR had a high accuracy at predicting inducible perfusion defects demonstrated on stress perfusion MRI. This thesis describes the largest randomized trial published to date specifically looking at the clinical utility of FFR in the NSTEMI population. We have provided evidence of the diagnostic and clinical utility of FFR in this group of patients and provide evidence to inform larger studies. This thesis also describes the largest ever MRI cohort, including with myocardial stress perfusion assessments, specifically looking at the NSTEMI population. We have demonstrated the diagnostic accuracy of FFR to predict reversible ischaemia as referenced to a non-invasive gold standard with MRI. This thesis has also shown the futility of using dark blood oedema imaging amongst all comer NSTEMI patients when compared to novel T1 and T2 mapping methods.

The aim of this thesis is ‘’Improving the Assessment of Exercise Capacity and Cardiorespiratory Fitness in Patients Attending Exercise-Based Cardiac Rehabilitation’’. Cardiorespiratory capacity is an important predictor of morbidity and mortality in cardiac patients, due to the prognostic power, is an essential outcome to measure in cardiac patients in clinical practice. In cardiac rehabilitation programmes the assessment of cardiorespiratory capacity (by field tests or treadmill test) is an essential practice supported by U.K., European and U.S.A. guidelines, which gives support to patients risk evaluation and stratification, setting individual patients goals, exercise prescription, and evaluation of the same. Overall, the findings of this thesis, which were generate by meta-analysis, crosssectional studies and laboratory research, provide an nsight into the factors associated with patients’ initial performance, and oxygen cost in functional capacity tests. Together, this data may improve the application, interpretation and patient understanding of these test results. One aim of CR is to improve patients’ functional capacity; we provide a standard value for ΔFitness, and information on factors which clinicians may need to consider when setting patient goals and interpreting changes in functional capacity, or ΔFitness due to CR.

Hepatitis C virus (HCV) infection is one of the major causes of liver morbidity and mortality worldwide. While effective therapies are now available, if eradication of this virus is to be achieved globally, an effective vaccine is still necessary. During hepatitis C virus (HCV) infection, broadly neutralizing antibody (bNAb) responses targeting E1E2 envelope glycoproteins are generated in many individuals. It is unclear if these antibodies play a protective or a pathogenic role during chronic infection or if they could prevent infection or reinfection with the virus. I investigated the presence and clinical associations of bNAb responses in three cohorts of individuals infected with or exposed to HCV infection. One with chronic HCV infection at differing disease states, one with chronic HCV infection at an early disease state and one group of individuals at high risk of HCV exposure who remained uninfected by conventional testing. I also studied bNAb responses in an individual from a HCV-HIV co-infected cohort who experienced spontaneous clearance of HCV after a post-therapy relapse (‘secondary spontaneous clearance’). I found a proportion of individuals when exposed to or infected with HCV produce a polyclonal bNAb response which may contribute to viral clearance in some cases. Host genetics and the ability to target multiple neutralising epitopes on the envelope protein are associated with such responses, although resistance mutations to bNAbs do exist in vivo. The presence of bNAbs is associated with lower levels of liver fibrosis. Using next generation sequencing technology in the study of B cell receptors in HCV infection revealed subtle changes in the B cell repertoire on HCV infection, this technology may be used in future to gain insight into the generation of bNAb responses.

Tissue engineering and regenerative medicine is a constantly evolving field of science that directs the use of cells to repair damaged or diseased tissue. Currently, stem cells are the most widely used source of cells due to their inherent characteristics of self-renewal and differentiation. However, stem cell regenerative therapies are still lacking, upon removal from the body, stem cell grow dysplastically towards unwanted lineages, compounded by their limited number makes the therapeutic potential of these cells difficult to obtain. These problems are due to the lack of knowledge of the underlying systems and mechanisms of phenotypical commitment through differentiation. In an attempt to circumvent these problems, scientists have begun the construction of dynamic surfaces, that is, surfaces that mimic the constantly evolving and changing environment in which stem cells reside in the body, known as the niche. These biomimetic strategies aim to reproduce the physical architecture, chemical composition and plasticity of the in vivo environment in vitro. This physical architecture can provide the cells with behavioural cues, mainly through transmembrane receptors known as integrins which link the extracellular matrix to the cytoskeleton; and therefore convey physical architecture of the environment to the cell. Scientists consciously design biomimetic systems to incorporate integrin binding ligands such as the adhesive tripeptide arginine-glycine-aspartic acid (RGD). The chemical composition of the niche depends on the in vivo milieu, and the needs of the body at this specified time. More specifically, mesenchymal stem cells reside in a variety of niches, with the bone marrow being a prime example. Upon osteogenesis, many chemical signals are delivered to this niche, with arguably the strongest osteogenic signal from bone morphogenetic protein 2 (BMP-2). This growth factor can activate osteogenic genes within the mesenchymal stem cell and predetermine differentiation of the cell towards an osteoblastic fate. In this work, we have developed a genetically engineered non-pathogenic bacteria, Lactococcus lactis to display the III7-10 fragment of fibronectin to allow mammalian cell integrin adhesion. This fragment contains two important sequences, the RGD adhesive tripeptide and the synergy sequence proline-histidine-serine-arginine-asparagine (PHSRN). RGD allows the mammalian cells to interact with the bacteria through promiscuous integrin attachment. The PHSRN sequence binds synergistically with RGD to some integrins, such as α5β1. These bacteria have also been modified to express the osteogenic growth factor, BMP-2 to direct mesenchymal stem cell differentiation towards an osteoblastic fate. These bacteria readily form spontaneous two-dimensional biofilms on a variety of surfaces, and can therefore act as a living interface between the synthetic surface below and the mammalian cells seeded above. The results of this thesis demonstrate that Lactococcus lactis can be used as a successful dynamic surface to control the adhesion, proliferation and differentiation of mesenchymal stem cells. Mesenchymal stem cells seeded over BMP-2 secreting Lactococcus lactis demonstrate decreased cell proliferation at short time points and increased osteoblastic markers at longer time points. Further to this, the interface has been made dynamic by making the bacteria inducible, that is, BMP-2 can be expressed in a temporal manner, and at different concentrations to finely tailor specific protein production. In the future, this system can be further exploited to express or deliver almost any protein or small molecule that can aid in the development of new tissues from their progenitor cells. As demonstrated, these proteins can both be secreted into the medium or displayed as cell wall bound proteins; and can also be constitutively or inducibly expressed. This interface, based on non-pathogenic bacteria establishes a new paradigm in surface functionalisation for regenerative medicine applications.

No description available.