Characterisation of factors that regulate homologous recombination and antigenic variation in Trypanosoma brucei

Trypanosoma brucei is an evolutionarily divergent eukaryotic parasite of mammals in sub-Saharan Africa and is transmitted by the tsetse fly vector. To evade the mammalian immune response, T. brucei utilises antigenic variation, which involves switches in the Variant Surface Glycoprotein (VSG) expressed on the cell surface. Such reactions can occur at very high rates (~10-3 switches/cell/generation) and occur primarily by the recombination of VSG genes, selected from an enormous silent archive, into specialised expression sites. It has been previously shown that such VSG switching is a form of homologous recombination, as mutation of RAD51 and a related gene, RAD51-3, impairs the process. BRCA2 has emerged as a significant regulatory factor during RAD51-catalysed recombination. In humans, BRCA2 contains eight BRC repeats, six of which have been shown to bind RAD51. Similar repeats are present in BRCA2 from other organisms, though normally in smaller numbers. This thesis describes a T. brucei BRCA2 homologue that appears exceptional in that it contains up to 12 BRC repeats. Furthermore, the sequence degeneracy that is observed between the BRC repeats in most organisms is absent in T. brucei, with all but the C-terminal proximal repeat being identical. It was hypothesised that this unusual BRCA2 organisation is due to the high levels of RAD51-directed recombination needed during antigenic variation. To examine the function of the putative T. brucei BRCA2 homologue, mutants were generated and found to display impaired growth, sensitivity to induced DNA damage, impairment in the ability to form sub-nuclear RAD51 foci, a reduced ability to recombine DNA constructs into their genome and a reduction in frequency of VSG switching, all of which are consistent with roles for BRCA2 in DNA repair and recombination. Furthermore, genome instability in the mutants was observed through the loss of silent VSG gene copies and substantial reductions in the size of the mega-base chromosomes. Interestingly, other chromosome classes (the so-called mini- and intermediate-chromosomes) appear not to be susceptible to such instability. A potentially novel function for BRCA2 was identified through DNA content analysis of the T. brucei BRCA2 mutants. Mutation of BRCA2 was shown to result in an accumulation of cells with aberrant DNA content that is most readily explained by an increased number of cells that undergo cytokinesis without having completed nuclear division, phenotypes that are not observed in other T. brucei recombination mutants, such as RAD51. This result suggests that BRCA2 has a role in the regulation of cell division, with mutation causing impaired replication of T. brucei nuclear DNA, but without a cell cycle stall, leading to the accumulation of chromosomal aberrations. In order to investigate the potential role of T. brucei BRCA2 in DNA replication and the unusual BRC repeat organisation phenotypes further, various truncations of BRCA2 were expressed in a mutant background. Cell lines expressing BRCA2 with only 1 BRC repeat displayed reduced efficiency in recombination, DNA repair and RAD51 foci formation, indicating that the large BRC repeat expansion in T. brucei BRCA2 plays a critical role in the proteins function. Expression of a BRCA2 variant encompassing only the region of the protein, C-terminal to the BRC repeats appeared able to function, at least partially, in regulating cell cycle progression. Moreover, this DNA replication role appears not to be provided by conserved DNA binding motifs present within the C terminus of BRCA2 since a fusion of T. brucei BRCA2 and the parasites homologue of the replication protein A 70 kDa subunit was impaired in cell division, but was proficient in repair of DNA damage. Taken together, these data infer that T. brucei BRCA2 possesses a function that is distinct from BRCA2’s role as a regulator of RAD51, and acts in DNA replication or cell division. In addition to the above research on BRCA2, I sought to examine the factors that interact with RAD51 in T. brucei. This work demonstrated that it is possible to add an epitope tag for tandem affinity purification (TAP) to the N-terminus of RAD51 in both the bloodstream and procyclic stages of T. brucei without disrupting its function. Preliminary data suggest that TAP is potentially a feasible way of examining RAD51 interacting factors

Selenium supplementation for the primary prevention of cardiovascular disease

Background: Selenium is a key component of a number of selenoproteins which protect against oxidative stress and have the potential to prevent chronic diseases including cardiovascular disease (CVD). However, observational studies have shown inconsistent associations between selenium intake and CVD risk; in addition, there is concern around a possible increased risk of type 2 diabetes with high selenium exposure. Objectives: To determine the effectiveness of selenium only supplementation for the primary prevention of CVD and examine the potential adverse effect of type 2 diabetes. Search methods: The following electronic databases were searched: the Cochrane Central Register of Controlled Trials (CENTRAL) (Issue 10 of 12, October 2012) on The Cochrane Library; MEDLINE (Ovid) (1946 to week 2 October 2012); EMBASE Classic + EMBASE (Ovid) (1947 to 2012 Week 42); CINAHL (EBSCO) (to 24 October 2012); ISI Web of Science (1970 to 24 October 2012); PsycINFO (Ovid) (1806 to week 3 October 2012); Database of Abstracts of Reviews of Effects (DARE), Health Technology Assessment Database and Health Economics Evaluations Database (Issue 4 of 4, October 2012) on The Cochrane Library. Trial registers and reference lists of reviews and articles were searched and experts in the field were approached. No language restrictions were applied. Selection criteria: Randomised controlled trials on the effects of selenium only supplementation on major CVD end-points, mortality, changes in CVD risk factors, and type 2 diabetes were included both in adults of all ages from the general population and in those at high risk of CVD. Trials were only considered where the comparison group was placebo or no intervention. Only studies with at least three months follow-up were included in the meta-analyses, shorter term studies were dealt with descriptively. Data collection and analysis: Two review authors independently assessed trial quality and extracted data. Study authors were contacted for additional information. Main results: Twelve trials (seven with duration of at least three months) met the inclusion criteria, with 19,715 participants randomised. The two largest trials that were conducted in the USA (SELECT and NPC) reported clinical events. There were no statistically significant effects of selenium supplementation on all cause mortality (RR 0.97, 95% CI 0.88 to 1.08), CVD mortality (RR 0.97, 95% CI 0.79 to 1.2), non-fatal CVD events (RR 0.96, 95% CI 0.89 to 1.04) or all CVD events (fatal and non-fatal) (RR 1.03, 95% CI 0.95 to 1.11). There was a small increased risk of type 2 diabetes with selenium supplementation but this did not reach statistical significance (RR 1.06, 95% CI 0.97 to 1.15). Other adverse effects that increased with selenium supplementation, as reported in the SELECT trial, included alopecia (RR 1.28, 95% CI 1.01 to 1.62) and dermatitis grade 1 to 2 (RR 1.17, 95% CI 1.0 to 1.35). Selenium supplementation reduced total cholesterol but this did not reach statistical significance (WMD - 0.11 mmol/L, 95% CI - 0.3 to 0.07). Mean high density lipoprotein (HDL) levels were unchanged. There was a statistically significant reduction in non-HDL cholesterol (WMD - 0.2 mmol/ L, 95% CI - 0.41 to 0.00) in one trial of varying selenium dosage. None of the longer term trials examined effects on blood pressure. Overall, the included studies were regarded as at low risk of bias. Authors’ conclusions: The limited trial evidence that is available to date does not support the use of selenium supplements in the primary prevention of CVD

'Mediterranean' dietary pattern for the primary prevention of cardiovascular disease

It is well established that diet plays a major role in cardiovascular disease risk. The traditional Mediterranean dietary pattern is of particular interest because of observations from the 1960s that populations in countries of the Mediterranean region, such as Greece and Italy, had lower mortality from cardiovascular disease compared with northern European populations or the US, probably as a result of different eating habits. This review assessed the effects of providing dietary advice to follow a Mediterranean-style dietary pattern to healthy adults or people at increased risk of cardiovascular disease in order to prevent the occurrence of cardiovascular disease and reduce the risk factors associated with it. Definitions of a Mediterranean dietary pattern vary and we included only randomised controlled trials (RCTs) of interventions that reported at least two of the following components: (1) high monounsaturated/saturated fat ratio, (2) low to moderate red wine consumption, (3) high consumption of legumes, (4) high consumption of grains and cereals, (5) high consumption of fruits and vegetables, (6) low consumption of meat and meat products and increased consumption of fish, and (7) moderate consumption of milk and dairy products. The control group was no intervention or minimal intervention. We found 11 RCTs (15 papers) that met these criteria. The trials varied enormously in the participants recruited and the different dietary interventions. Four trials were conducted in women only, two trials were in men only and the remaining five were in both men and women. Five trials were conducted in healthy individuals and six trials were in people at increased risk of cardiovascular disease or cancer. The number of components relevant to a Mediterranean dietary pattern ranged from two to five and only seven trials described the intervention as a Mediterranean diet. The largest trial, which recruited only postmenopausal women and was not described as a Mediterranean diet meeting only two of the criteria described above, reported no difference in the occurrence of cardiovascular disease between the dietary advice group and the control group. The other trials measured risk factors for cardiovascular disease. As the studies were so different, it was not possible to combine studies for most of the outcomes. Where it was possible to combine studies, we found small reductions in total cholesterol levels as well as in the harmful low-density lipoprotein (LDL) cholesterol concentrations. The reductions in total cholesterol were greater in the studies that described themselves as providing a Mediterranean diet. None of the trials reported side effects. The review concludes that, from the limited evidence to date, a Mediterranean dietary pattern reduces some cardiovascular risk factors. However, more trials are needed to look at the effects of the different participants recruited and the different dietary interventions to see which interventions might work best in different populations

Green and black tea for the primary prevention of cardiovascular disease

Background: There is increasing evidence that both green and black tea are beneficial for cardiovascular disease (CVD) prevention. Objectives: To determine the effects of green and black tea on the primary prevention of CVD. Search methods: We searched the following databases on 12 October 2012 without language restrictions: CENTRAL in The Cochrane Library, MEDLINE (OVID), EMBASE (OVID) and Web of Science (Thomson Reuters). We also searched trial registers, screened reference lists and contacted authors for additional information where necessary. Selection criteria: Randomised controlled trials (RCTs) lasting at least three months involving healthy adults or those at high risk of CVD. Trials investigated the intake of green tea, black tea or tea extracts. The comparison group was no intervention, placebo or minimal intervention. The outcomes of interest were CVD clinical events and major CVD risk factors. Any trials involving multifactorial lifestyle interventions or focusing on weight loss were excluded to avoid confounding. Data collection and analysis: Two review authors independently selected trials for inclusion, abstracted data and assessed the risk of bias. Trials of green tea were analysed separately from trials of black tea. Main results: We identified 11 RCTs with a total of 821 participants, two trials awaiting classification and one ongoing trial. Seven trials examined a green tea intervention and four examined a black tea intervention. Dosage and form of both green and black tea differed between trials. The ongoing trial is examining the effects of green tea powder capsules. No studies reported cardiovascular events. Black tea was found to produce statistically significant reductions in low-density lipoprotein (LDL) cholesterol (mean difference (MD) -0.43 mmol/L, 95% confidence interval (CI) -0.56 to -0.31) and blood pressure (systolic blood pressure (SBP): MD -1.85 mmHg, 95% CI -3.21 to -0.48. Diastolic blood pressure (DBP): MD -1.27 mmHg, 95% CI -3.06 to 0.53) over six months, stable to sensitivity analysis, but only a small number of trials contributed to each analysis and studies were at risk of bias. Green tea was also found to produce statistically significant reductions in total cholesterol (MD -0.62 mmol/L, 95% CI -0.77 to - 0.46), LDL cholesterol (MD -0.64 mmol/L, 95% CI -0.77 to -0.52) and blood pressure (SBP: MD -3.18 mmHg, 95% CI -5.25 to - 1.11; DBP: MD -3.42, 95% CI -4.54 to -2.30), but only a small number of studies contributed to each analysis, and results were not stable to sensitivity analysis. When both tea types were analysed together they showed favourable effects on LDL cholesterol (MD - 0.48 mmol/L, 95% CI -0.61 to -0.35) and blood pressure (SBP: MD -2.25 mmHg, 95% CI -3.39 to -1.11; DBP: MD -2.81 mmHg, 95% CI -3.77 to -1.86). Adverse events were measured in five trials and included a diagnosis of prostate cancer, hospitalisation for influenza, appendicitis and retinal detachment but these are unlikely to be directly attributable to the intervention. Authors' conclusions: There are very few long-term studies to date examining green or black tea for the primary prevention of CVD. The limited evidence suggests that tea has favourable effects on CVD risk factors, but due to the small number of trials contributing to each analysis the results should be treated with some caution and further high quality trials with longer-term follow-up are needed to confirm this

Exploring links between early adversities and later outcomes for children adopted from care: Implications for planning post adoption support

This study explored how child maltreatment, alongside a range of other variables, predicted adverse outcomes for children adopted from the foster care system in England. The participants were 319 adoptive parents who completed an in-depth online survey about their most recently adopted child. The mean age of children at placement for adoption was 28 months (range 0–11 years) and their ages at the time of the survey ranged from 0 years to 17 years (mean¼7 years). Detailed information was collected about children’s backgrounds, including their experiences in the birth family and the care system before adoption. Adoptive parents also reported on how well children were getting on in a range of areas of functioning and how well they felt the adoption was going overall. Child maltreatment and child adverse outcomes were modeled as two factors in a latent factor structural equation model. The relationship between these two factors was explored alongside a range of covariates. Associated with worse outcomes for children were potentially heritable factors (parental learning disability), the pre-birth environment (exposure to drugs or alcohol in utero) and the period between birth and moving to the adoptive family (higher levels of maltreatment, spending more than a year in care, having two or more foster placements). The child’s distress on moving from the foster home to the adoptive family was also highly significant in linking to poorer outcomes, suggesting the detrimental effect of poorly managed transitions. Implications for child welfare practices before and after adoption are discussed

The molecular genetic investigation of paediatric liver disease

Liver disease in children is rare but often serious, life long, and in many cases leads to death. Advances in diagnosing and treating liver disease (including liver transplant) have improved the outlook for children in many cases however little is known about the molecular pathogenesis of the disease, an understanding of which may identify specific therapeutic options. The aim of this thesis is to investigate the molecular genetics of rare liver disorders as the first step in advancing the understanding of liver disease pathogenesis. As a paediatric hepatologist I have identified cohorts of children in whom there is paucity of knowledge about the disease pathogenesis. I have studied three conditions in detail to encompass different clinical presentations. Chapter 3 summarises the investigation of the multisystem disorder, phenotypic diarrhoea of infancy (PDI), which causes cirrhosis or liver failure. Autozygosity mapping was used to identify the gene TTC37 in which mutations are associated with the PDI disease phenotype. Further work is now required to characterise TTC37, and use knockdown studies to identify whether TTC37 mutations are causative of the PDI phenotype. Chapter 4 describes the molecular genetic investigation of Jeune asphyxiating thoracic dystrophy (JATD), a chondrodysplasia with extra skeletal manifestations including hepatic ductal plate malformation and renal cyst development. Using autozygosity mapping, IFT80 was dentified in which mutations are associated with the JATD disease phenotype in 4% of ases. The diverse linical phenotype of JATD limits the utility of utozygosity mapping s it suggests there is genetic heterogeneity. The identification of IFT80 has led to JATD eing classified as a ciliopathy. Chapter 5 is the first description of eonatal liver failure to be associated with variants in ABCB11 which previously have only been associated with chronic liver disease and liver disease in pregnancy. This thesis has described the identification of the molecular genetic basis of rare causes of paediatric liver disease which has provoked many additional research questions. Future work will be to extend our knowledge of molecular genetics to all aspects of paediatric liver physiology so to classify disease according to the molecular pathogenesis such as a ciliopathy or bile salt transport defect.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Understanding women’s weight-related health behaviours across pregnancy: A qualitative longitudinal study

Background: Women’s weight-related health behaviours (WRHBs) during pregnancy can contribute to excessive gestational weight gain, which can be retained throughout life. In this study, WRHBs are health behaviours which have the potential to impact on women’s gestational weight gain, but are not necessarily engaged in with the intention of weight change. Lacking from the evidence is an understanding of women’s WRHBs and their determinants across pregnancy and utilising psychological theory when designing antenatal behaviour change interventions. Aim: To understand women’s experiences of WRHBs across pregnancy, with a view to explore if this varies throughout the journey of pregnancy. Methods: Pregnant women were recruited at their antenatal appointment in two areas of a Northern UK city and interviewed at three timepoints (two antenatal, one postnatal), two of which are presented here: approximately 10-16 weeks and 28-32 weeks gestation. Interview data were first inductively analysed to generate a thematic framework. WRHBs were identified from the thematic framework and subsequently mapped to existing psychological theory. Findings: Eighteen women with differing demographic and obstetric characteristics (e.g., area of deprivation, body mass index and parity) participated in the study. Four themes with sub-themes were identified across both timepoints; which reflected similarities and nuanced differences in women’s WRHBs across pregnancy. Twenty-five WRHBs were identified; 8 of which were the same across both timepoints. Women’s WRHBs were disrupted in early pregnancy due to physiological and psychological factors, with women motivated to engage in WRHBs by changes in beliefs towards ‘health’. In later pregnancy women indicated passivity towards their WRHBs. Motivation primarily drove women’s WRHBs, whilst also interacting with other behavioural determinants. Conclusions: Engagement in pre-pregnancy WRHBs, changing physiological and psychological factors all interact with each other to impact on women’s WRHBs across pregnancy. This interaction is not the same for every WRHB, and each WRHB should be considered individually in future research

Belimumab : a technological advance for systemic lupus erythematosus patients? Report of a systematic review and meta-analysis

Objectives: To undertake a systematic review and meta-analysis to investigate clinical effectiveness of belimumab for patients with systemic lupus erythematosus (SLE) and antinuclear and/or anti-double-stranded DNA (dsDNA) autoantibodies. Methods: We searched eight electronic databases and reference lists for randomised controlled trials (RCTs) of belimumab against placebo or best supportive care. Quality assessment and random effects meta-analysis were undertaken. Design: A meta-analysis of RCTs. Participants: 2133 SLE patients. Primary and secondary outcome measures: SLE Responder Index (SRI) at week 52. Results: Three double-blind placebo-controlled RCTs (L02, BLISS-52 BLISS-76) investigated 2133 SLE patients. BLISS-52 and BLISS-76 trials recruited patients with antinuclear and/or anti-dsDNA autoantibodies and demonstrated belimumab effectiveness for the SRI at week 52. Ethnicity and geographical location of participants varied considerably between BLISS trials. Although tests for statistical heterogeneity were negative, BLISS-52 results were systematically more favourable for all measured outcomes. Meta-analysis of pooled 52-week SRI BLISS results showed benefit for belimumab (OR 1.63, 95% CI 1.27 to 2.09). By week 76, the primary SRI outcome in BLISS-76 was not statistically significant (OR 1.31, 95% CI 0.919 to 1.855)

The prevalence of constant supportive observations in a high, medium and low secure service

Aims and Method We explored the prevalence and use of constant supportive observations (CSO) in high, medium and low secure in-patient services in a single NHS mental health trust. From clinical records, we extracted data on the length of time on CSO, the reason for the initiation of CSO and associated adverse incidents for all individuals who were placed on CSO between July 2013 and June 2014. Results A small number of individuals accounted for a disproportionately large amount of CSO hours in each setting. Adverse incident rates were higher on CSO than when not on CSO. There was considerable variation between different settings in terms of CSO use and the reasons for commencing CSO. Clinical Implications The study describes the prevalence and nature of CSO in secure forensic mental health services and the associated organisational costs. The marked variation in CSO use between settings suggests that mental health services continue to face challenges in balancing risk management with minimising restrictive interventions