Engineering the zinc finger recombinase for use in targeted genomic editing

No abstract available.

Conservation value, biodiversity value and methods of assessment in regenerating and human disturbed tropical forest

Although the value of primary forests for biodiversity conservation is well known, the potential biodiversity and conservation value of regenerating forests remains controversial. Many factors likely contribute to this, including: 1. the variable ages of regenerating forests being studied (often dominated by relatively young regenerating forests); 2. the potential for confounding on-going human disturbance (such as logging and hunting); 3. the relatively low number of multi-taxa studies; 4. the lack of studies that directly compare different historic disturbances within the same location; 5. contrasting patterns from different survey methodologies and the paucity of knowledge on the impacts across different vertical levels of rainforest biodiversity (often due to a lack of suitable methodologies available to assess them). We also know relatively little as to how biodiversity is affected by major current impacts, such as unmarked rainforest roads, which contribute to this degradation of habitat and fragmentation. This thesis explores the potential biodiversity value of regenerating rainforests under the best of scenarios and seeks to understand more about the impact of current human disturbance to biodiversity; data comes from case studies from the Manu and Sumaco Biosphere Reserves in the Western Amazon. Specifically, I compare overall biodiversity and conservation value of a best case regenerating rainforest site with a selection of well-studied primary forest sites and with predicted species lists for the region; including a focus on species of key conservation concern. I then investigate the biodiversity of the same study site in reference to different types of historic anthropogenic disturbance. Following this I investigate the impacts to biodiversity from an unmarked rainforest road. In order to understand more about the differential effects of habitat disturbance on arboreal diversity I directly assess how patterns of butterfly biodiversity vary between three vertical strata. Although assessments within the canopy have been made for birds, invertebrates and bats, very few studies have successfully targeted arboreal mammals. I therefore investigate the potential of camera traps for inventorying arboreal mammal species in comparison with traditional methodologies. Finally, in order to investigate the possibility that different survey methodologies might identify different biodiversity patterns in habitat disturbance assessments, I investigate whether two different but commonly used survey methodologies used to assess amphibians, indicate the same or different responses of amphibian biodiversity to historic habitat change by people. The regenerating rainforest study site contained high levels of species richness; both in terms of alpha diversity found in nearby primary forest areas (87% ±3.5) and in terms of predicted primary forest diversity from the region (83% ±6.7). This included 89% (39 out of 44) of the species of high conservation concern predicted for the Manu region. Faunal species richness in once completely cleared regenerating forest was on average 13% (±9.8) lower than historically selectively logged forest. The presence of the small unmarked road significantly altered levels of faunal biodiversity for three taxa, up to and potentially beyond 350m into the forest interior. Most notably, the impact on biodiversity extended to at least 32% of the whole reserve area. The assessment of butterflies across strata showed that different vertical zones within the same rainforest responded differently in areas with different historic human disturbance. A comparison between forest regenerating after selective logging and forest regenerating after complete clearance, showed that there was a 17% greater reduction in canopy species richness in the historically cleared forest compared with the terrestrial community. Comparing arboreal camera traps with traditional ground-based techniques suggests that camera traps are an effective tool for inventorying secretive arboreal rainforest mammal communities and detect a higher number of cryptic species. Finally, the two survey methodologies used to assess amphibian communities identified contrasting biodiversity patterns in a human modified rainforest; one indicated biodiversity differences between forests with different human disturbance histories, whereas the other suggested no differences between forest disturbance types. Overall, in this thesis I find that the conservation and biodiversity value of regenerating and human disturbed tropical forest can potentially contribute to rainforest biodiversity conservation, particularly in the best of circumstances. I also highlight the importance of utilising appropriate study methodologies that to investigate these three-dimensional habitats, and contribute to the development of methodologies to do so. However, care should be taken when using different survey methodologies, which can provide contrasting biodiversity patterns in response to human disturbance.

Methods matter: computational modelling in public health policy and planning

This work is aimed at understanding and unifying information on epidemiological modelling methods and how those methods relate to public policy addressing human health, specifically in the context of infectious disease prevention, pandemic planning, and health behaviour change. This thesis employs multiple qualitative and quantitative methods, and presents as a manuscript of several individual, data-driven projects that are combined in a narrative arc. The first chapter introduces the scope and complexity of this interdisciplinary undertaking, describing several topical intersections of importance. The second chapter begins the presentation of original data, and describes in detail two exercises in computational epidemiological modelling pertinent to pandemic influenza planning and policy, and progresses in the next chapter to present additional original data on how the confidence of the public in modelling methodology may have an effect on their planned health behaviour change as recommended in public health policy. The thesis narrative continues in the final data-driven chapter to describe how health policymakers use modelling methods and scientific evidence to inform and construct health policies for the prevention of infectious diseases, and concludes with a narrative chapter that evaluates the breadth of this data and recommends strategies for the optimal use of modelling methodologies when informing public health policy in applied public health scenarios.

Mindfulness-Based Cognitive Therapy for older people in a community setting: a mixed methods feasibility study

Rationale: In line with complex intervention development, this research takes a systematic approach to examining the feasibility and acceptability of delivering Mindfulness-Based Cognitive Therapy (MBCT) to older people who experience symptoms of depression. Methods: A mixed methods approach was adopted in line with recommendations made by the MRC Complex Intervention Development framework. Quantitative and qualitative methods were combined by administering questionnaires as well as conducting post intervention interviews. A number of trial feasibility factors were examined such as recruitment and attrition rates. Qualitative data was analysed using Braun and Clarke’s thematic analysis framework. Results: Nine participants started the MBCT intervention and six completed the 8-week programme. The results suggest that MBCT for older people is feasible and acceptable. Participants reported improved mindfulness skills. Participants responded positively to being asked to take part in research and appeared to particularly value the group delivery format of the intervention. Conclusions: MBCT is both feasible and acceptable for older people experiencing symptoms of depression. Further research is required with larger sample sizes to allow for more robust statistical exploration of outcome measures, including mechanisms of change.

Using an end-of-life care pathway in acute stroke: a mixed methods study of decision-making and care experiences

Background: The evidence base on end-of-life care in acute stroke is limited, particularly with regard to recognising dying and related decision-making. There is also limited evidence to support the use of end-of-life care pathways (standardised care plans) for patients who are dying after stroke. Aim: This study aimed to explore the clinical decision-making involved in placing patients on an end-of-life care pathway, evaluate predictors of care pathway use, and investigate the role of families in decision-making. The study also aimed to examine experiences of end-of-life care pathway use for stroke patients, their relatives and the multi-disciplinary health care team. Methods: A mixed methods design was adopted. Data were collected in four Scottish acute stroke units. Case-notes were identified prospectively from 100 consecutive stroke deaths and reviewed. Multivariate analysis was performed on case-note data. Semi-structured interviews were conducted with 17 relatives of stroke decedents and 23 healthcare professionals, using a modified grounded theory approach to collect and analyse data. The VOICES survey tool was also administered to the bereaved relatives and data were analysed using descriptive statistics and thematic analysis of free-text responses. Results: Relatives often played an important role in influencing aspects of end-of-life care, including decisions to use an end-of-life care pathway. Some relatives experienced enduring distress with their perceived responsibility for care decisions. Relatives felt unprepared for and were distressed by prolonged dying processes, which were often associated with severe dysphagia. Pro-active information-giving by staff was reported as supportive by relatives. Healthcare professionals generally avoided discussing place of care with families. Decisions to use an end-of-life care pathway were not predicted by patients’ demographic characteristics; decisions were generally made in consultation with families and the extended health care team, and were made within regular working hours. Conclusion: Distressing stroke-related issues were more prominent in participants’ accounts than concerns with the end-of-life care pathway used. Relatives sometimes perceived themselves as responsible for important clinical decisions. Witnessing prolonged dying processes was difficult for healthcare professionals and families, particularly in relation to the management of persistent major swallowing difficulties.

The role of cancer related inflammation, Src family kinases and matrix metalloproteinase 9 in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in the UK with 41,000 new cases diagnosed in 2011. Despite undergoing potentially curative resection, a significant amount of patients develop recurrence. Biomarkers that aid prognostication or identify patients who are suitable for adjuvant treatments are needed. The TNM staging system does a reasonably good job at offering prognostic information to the treating clinician, but it could be better and identifying methods of improving its accuracy are needed. Tumour progression is based on a complex relationship between tumour behaviour and the hosts’ inflammatory responses. Sustained tumour cell proliferation, evading growth suppressors, resisting apoptosis, replicative immortality, sustained angiogenesis, invasion & metastasis, avoiding immune destruction, deregulated cellular energetics, tumour promoting inflammation and genomic instability & mutation have been identified as hallmarks. These hallmarks are malignant behaviors are what makes the cell cancerous and the more extreme the behaviour the more aggressive the cancer the more likely the risk of a poor outcome. There are two primary genomic instability pathways: Microsatellite Instability (MSI) and Chromosomal Instability (CI) also referred to as Microsatellite Stability (MSS). Tumours arising by these pathways have a predilection for specific anatomical, histological and molecular biological features. It is possible that aberrant molecular expression of genes/proteins that promote malignant behaviors may also act as prognostic and predictive biomarkers, which may offer superior prognostic information to classical prognostic features. Cancer related inflammation has been described as a 7th hallmark of cancer. Despite the systemic inflammatory response (SIR) being associated with more aggressive malignant disease, infiltration by immune cells, particularly CD8+ lymphocytes, at the advancing edge of the tumour have been associated with improved outcome and tumour MSI. It remains unknown if the SIR is associated with tumour MSI and this requires further study. The mechanisms by which colorectal cancer cells locally invade through the bowel remain uncertain, but connective tissue degradation by matrix metalloproteinases (MMPs) such as MMP-9 have been implicated. MMP-9 has been found in the cancer cells, stromal cells and patient circulation. Although tumoural MMP-9 has been associated with poor survival, reports are conflicting and contain relatively small sample sizes. Furthermore, the influence of high serum MMP-9 on survival remains unknown. Src family kinases (SFKs) have been implicated in many adverse cancer cell behaviors. SFKs comprise 9 family members BLK, C-SRC, FGR, FYN, HCK, LCK, LYN, YES, YRK. C-SRC has been the most investigated of all SFKs, but the role of other SFKs in cellular behaviors and their prognostic value remains largely unknown. The development of Src inhibitors, such as Dasatinib, has identified SFKs as a potential therapeutic target for patients at higher risk of poor survival. Unfortunately, clinical trials so far have not been promising but this may reflect inadequate patient selection and SFKs may act as useful prognostic and predictive biomarkers. In chapter 3, the association between cancer related inflammation, tumour MSI, clinicopathological factors and survival was tested in two independent cohorts. A training cohort consisting of n=182 patients and a validation cohort of n=677 patients. MSI tumours were associated with a raised CRP (p=0.003). Hypoalbuminaemia was independently associated with poor overall survival in TNM stage II cancer (HR 3.04 (95% CI 1.44 – 6.43);p=0.004), poor recurrence free survival in TNM stage III cancer (HR 1.86 (95% 1.03 – 3.36);p=0.040) and poor overall survival in CI colorectal cancer (HR 1.49 (95% CI 1.06 – 2.10);p=0.022). Interestingly, MSI tumours were associated with poor overall survival in TNM stage III cancer (HR 2.20 (95% CI 1.10 – 4.37);p=0.025). In chapter 4, the role of MMP-9 in colorectal cancer progression and survival was examined. MMP-9 in the tissue was assessed using IHC and serum expression quantified using ELISA. Serum MMP-9 was associated with cancer cell expression (Spearman’s Correlation Coefficient (SCC) 0.393, p<0.001)) and stromal expression (SCC 0.319, p=0.002). Serum MMP-9 was associated with poor recurrence-free (HR 3.37 (95% CI 1.20 – 9.48);p=0.021) and overall survival (HR 3.16 (95% CI 1.22 – 8.15);p=0.018), but tumour MMP-9 was not survival or MSI status. In chapter 5, the role of SFK expression and activation in colorectal cancer progression and survival was studied. On PCR analysis, although LYN, C-SRC and YES were the most highly expressed, FGR and HCK had higher expression profiles as tumours progressed. Using IHC, raised cytoplasmic FAK (tyr 861) was independently associated with poor recurrence free survival in all cancers (HR 1.48 (95% CI 1.02 – 2.16);p=0.040) and CI cancers (HR 1.50 (95% CI 1.02 – 2.21);p=0.040). However, raised cytoplasmic HCK (HR 2.04 (95% CI 1.11 – 3.76);p=0.022) was independently associated with poor recurrence-free survival in TNM stage II cancers. T84 and HT29 cell lines were used to examine the cellular effects of Dasatinib. Cell viability was assessed using WST-1 assay and apoptosis assessed using an ELISA cell death detection assay. Dasatinib increased T84 tumour cell apoptosis in a dose dependent manner and resulted in reduced expression of nuclear (p=0.008) and cytoplasmic (p=0.016) FAK (tyr 861) expression and increased nuclear FGR expression (p=0.004). The results of this thesis confirm that colorectal cancer is a complex disease that represents several subtypes of cancer based on molecular biological behaviors. This thesis concentrated on features of the disease related to inflammation in terms of genetic and molecular characterisation. MSI cancers are closely associated with systemic inflammation but despite this observation, they retain their relatively improved survival. MMP-9 is a feature of tissue remodeling during inflammation and is also associated with degradation of connective tissue, advanced T-stage and poor outcome when measured in the serum. The lack of stromal quantification due to TMA use rather than full sections makes the value of tumoural MMP-9 immunoreactivity in the prognostication and its association with MSI unknown and requires further study. Finally, SFK activation was also associated with SIR, however, only cytoplasmic HCK was independently associated with poor survival in patients with TNM stage II disease, the group of patients where identifying a novel biomarker is most needed. There is still some way to go before these biomarkers are translated into clinical practice and future work needs to focus on obtaining a reliable and robust scientific technique with validation in an adequately powered independent cohort.

Autonomous formation flying: unified control and collision avoidance methods for close manoeuvring spacecraft

The idea of spacecraft formations, flying in tight configurations with maximum baselines of a few hundred meters in low-Earth orbits, has generated widespread interest over the last several years. Nevertheless, controlling the movement of spacecraft in formation poses difficulties, such as in-orbit high-computing demand and collision avoidance capabilities, which escalate as the number of units in the formation is increased and complicated nonlinear effects are imposed to the dynamics, together with uncertainty which may arise from the lack of knowledge of system parameters. These requirements have led to the need of reliable linear and nonlinear controllers in terms of relative and absolute dynamics. The objective of this thesis is, therefore, to introduce new control methods to allow spacecraft in formation, with circular/elliptical reference orbits, to efficiently execute safe autonomous manoeuvres. These controllers distinguish from the bulk of literature in that they merge guidance laws never applied before to spacecraft formation flying and collision avoidance capacities into a single control strategy. For this purpose, three control schemes are presented: linear optimal regulation, linear optimal estimation and adaptive nonlinear control. In general terms, the proposed control approaches command the dynamical performance of one or several followers with respect to a leader to asymptotically track a time-varying nominal trajectory (TVNT), while the threat of collision between the followers is reduced by repelling accelerations obtained from the collision avoidance scheme during the periods of closest proximity. Linear optimal regulation is achieved through a Riccati-based tracking controller. Within this control strategy, the controller provides guidance and tracking toward a desired TVNT, optimizing fuel consumption by Riccati procedure using a non-infinite cost function defined in terms of the desired TVNT, while repelling accelerations generated from the CAS will ensure evasive actions between the elements of the formation. The relative dynamics model, suitable for circular and eccentric low-Earth reference orbits, is based on the Tschauner and Hempel equations, and includes a control input and a nonlinear term corresponding to the CAS repelling accelerations. Linear optimal estimation is built on the forward-in-time separation principle. This controller encompasses two stages: regulation and estimation. The first stage requires the design of a full state feedback controller using the state vector reconstructed by means of the estimator. The second stage requires the design of an additional dynamical system, the estimator, to obtain the states which cannot be measured in order to approximately reconstruct the full state vector. Then, the separation principle states that an observer built for a known input can also be used to estimate the state of the system and to generate the control input. This allows the design of the observer and the feedback independently, by exploiting the advantages of linear quadratic regulator theory, in order to estimate the states of a dynamical system with model and sensor uncertainty. The relative dynamics is described with the linear system used in the previous controller, with a control input and nonlinearities entering via the repelling accelerations from the CAS during collision avoidance events. Moreover, sensor uncertainty is added to the control process by considering carrier-phase differential GPS (CDGPS) velocity measurement error. An adaptive control law capable of delivering superior closed-loop performance when compared to the certainty-equivalence (CE) adaptive controllers is finally presented. A novel noncertainty-equivalence controller based on the Immersion and Invariance paradigm for close-manoeuvring spacecraft formation flying in both circular and elliptical low-Earth reference orbits is introduced. The proposed control scheme achieves stabilization by immersing the plant dynamics into a target dynamical system (or manifold) that captures the desired dynamical behaviour. They key feature of this methodology is the addition of a new term to the classical certainty-equivalence control approach that, in conjunction with the parameter update law, is designed to achieve adaptive stabilization. This parameter has the ultimate task of shaping the manifold into which the adaptive system is immersed. The performance of the controller is proven stable via a Lyapunov-based analysis and Barbalat’s lemma. In order to evaluate the design of the controllers, test cases based on the physical and orbital features of the Prototype Research Instruments and Space Mission Technology Advancement (PRISMA) are implemented, extending the number of elements in the formation into scenarios with reconfigurations and on-orbit position switching in elliptical low-Earth reference orbits. An extensive analysis and comparison of the performance of the controllers in terms of total Δv and fuel consumption, with and without the effects of the CAS, is presented. These results show that the three proposed controllers allow the followers to asymptotically track the desired nominal trajectory and, additionally, those simulations including CAS show an effective decrease of collision risk during the performance of the manoeuvre.

The teaching of reading English in a foreign language in Libyan universities: methods and models

This study focuses on the learning and teaching of Reading in English as a Foreign Language (REFL), in Libya. The study draws on an action research process in which I sought to look critically at students and teachers of English as a Foreign Language (EFL) in Libya as they learned and taught REFL in four Libyan research sites. The Libyan EFL educational system is influenced by two main factors: the method of teaching the Holy-Quran and the long-time ban on teaching EFL by the former Libyan regime under Muammar Gaddafi. Both of these factors have affected the learning and teaching of REFL and I outline these contextual factors in the first chapter of the thesis. This investigation, and the exploration of the challenges that Libyan university students encounter in their REFL, is supported by attention to reading models. These models helped to provide an analytical framework and starting point for understanding the many processes involved in reading for meaning and in reading to satisfy teacher instructions. The theoretical framework I adopted was based, mainly and initially, on top-down, bottom-up, interactive and compensatory interactive models. I drew on these models with a view to understanding whether and how the processes of reading described in the models could be applied to the reading of EFL students and whether these models could help me to better understand what was going on in REFL. The diagnosis stage of the study provided initial data collected from four Libyan research sites with research tools including video-recorded classroom observations, semi-structured interviews with teachers before and after lesson observation, and think-aloud protocols (TAPs) with 24 students (six from each university) in which I examined their REFL reading behaviours and strategies. This stage indicated that the majority of students shared behaviours such as reading aloud, reading each word in the text, articulating the phonemes and syllables of words, or skipping words if they could not pronounce them. Overall this first stage indicated that alternative methods of teaching REFL were needed in order to encourage ‘reading for meaning’ that might be based on strategies related to eventual interactive reading models adapted for REFL. The second phase of this research project was an Intervention Phase involving two team-teaching sessions in one of the four stage one universities. In each session, I worked with the teacher of one group to introduce an alternative method of REFL. This method was based on teaching different reading strategies to encourage the students to work towards an eventual interactive way of reading for meaning. A focus group discussion and TAPs followed the lessons with six students in order to discuss the 'new' method. Next were two video-recorded classroom observations which were followed by an audio-recorded discussion with the teacher about these methods. Finally, I conducted a Skype interview with the class teacher at the end of the semester to discuss any changes he had made in his teaching or had observed in his students' reading with respect to reading behaviour strategies, and reactions and performance of the students as he continued to use the 'new' method. The results of the intervention stage indicate that the teacher, perhaps not surprisingly, can play an important role in adding to students’ knowledge and confidence and in improving their REFL strategies. For example, after the intervention stage, students began to think about the title, and to use their own background knowledge to comprehend the text. The students employed, also, linguistic strategies such as decoding and, above all, the students abandoned the behaviour of reading for pronunciation in favour of reading for meaning. Despite the apparent efficacy of the alternative method, there are, inevitably, limitations related to the small-scale nature of the study and the time I had available to conduct the research. There are challenges, too, related to the students’ first language, the idiosyncrasies of the English language, the teacher training and continuing professional development of teachers, and the continuing political instability of Libya. The students’ lack of vocabulary and their difficulties with grammatical functions such as phrasal and prepositional verbs, forms which do not exist in Arabic, mean that REFL will always be challenging. Given such constraints, the ‘new’ methods I trialled and propose for adoption can only go so far in addressing students’ difficulties in REFL. Overall, the study indicates that the Libyan educational system is underdeveloped and under resourced with respect to REFL. My data indicates that the teacher participants have received little to no professional developmental that could help them improve their teaching in REFL and skills in teaching EFL. These circumstances, along with the perennial problem of large but varying class sizes; student, teacher and assessment expectations; and limited and often poor quality resources, affect the way EFL students learn to read in English. Against this background, the thesis concludes by offering tentative conclusions; reflections on the study, including a discussion of its limitations, and possible recommendations designed to improve REFL learning and teaching in Libyan universities.

Mixed genotype Hepatitis C virus infections: incidence in Scotland and methods for detection

The diagnosis of mixed genotype hepatitis C virus (HCV) infection is rare and information on incidence in the UK, where genotypes 1a and 3 are the most prevalent, is sparse. Considerable variations in the efficacies of direct-acting antivirals (DAAs) for the HCV genotypes have been documented and the ability of DAAs to treat mixed genotype HCV infections remains unclear, with the possibility that genotype switching may occur. In order to estimate the prevalence of mixed genotype 1a/3 infections in Scotland, a cohort of 512 samples was compiled and then screened using a genotype-specific nested PCR assay. Mixed genotype 1a/3 infections were found in 3.8% of samples tested, with a significantly higher prevalence rate of 6.7% (p<0.05) observed in individuals diagnosed with genotype 3 infections than genotype 1a (0.8%). An analysis of the samples using genotypic-specific qPCR assays found that in two-thirds of samples tested, the minor strain contributed <1% of the total viral load. The potential of deep sequencing methods for the diagnosis of mixed genotype infections was assessed using two pan-genotypic PCR assays compatible with the Illumina MiSeq platform that were developed targeting the E1-E2 and NS5B regions of the virus. The E1-E2 assay detected 75% of the mixed genotype infections, proving to be more sensitive than the NS5B assay which identified only 25% of the mixed infections. Studies of sequence data and linked patient records also identified significantly more neurological disorders in genotype 3 patients. Evidence of distinctive dinucleotide expression within the genotypes was also uncovered. Taken together these findings raise interesting questions about the evolutionary history of the virus and indicate that there is still more to understand about the different genotypes. In an era where clinical medicine is frequently more personalised, the development of diagnostic methods for HCV providing increased patient stratification is increasingly important. This project has shown that sequence-based genotyping methods can be highly discriminatory and informative, and their use should be encouraged in diagnostic laboratories. Mixed genotype infections were challenging to identify and current deep sequencing methods were not as sensitive or cost-effective as Sanger-based approaches in this study. More research is needed to evaluate the clinical prognosis of patients with mixed genotype infection and to develop clinical guidelines on their treatment.