Binding Kinetics of Proteins at Immune-Cell Contacts

Protein-protein interactions are crucial in numerous cellular functions and biological processes that take place inside our body. It is therefore not surprising that these interactions also govern the response of our body´s defence mechanism, the so-called immune system, towards an infection. Understanding how proteins interact entails studying the binding affinity (strength) and the lifetime (duration) of the protein-protein interaction to better decompose how an immune response is initiated and how we can explore this knowledge to treat diseases. In this thesis, total internal fluorescence microscopy (TIRF) and single-molecule imaging were used to observe and characterize protein-functionalized supported lipid bilayers (SLBs) interacting with immune cells to obtain the binding kinetics of various protein-protein pairs.In the first part of this thesis, the interaction between the rat CD2 (rCD2) adhesion protein and its ligand rat CD48T92A (rCD48T92A), a high-affinity mutant of the wild type rat CD48, was used to establish a new method of obtaining single-cell binding affinities of T cells interacting with SLBs using imidazole titrations. The results showed a relatively small spread in the rCD2-rCD48T92A binding affinity values despite the considerable spread of receptor densities within the cell population. The lifetime of the rCD2/rCD48T92A interaction was also investigated using single-molecule imaging and tracking displaying a similarly small lifetime spread within the cell population. Using both these methods, the single-cell binding affinity and lifetime of the cell population can be investigated and their spread can provide information concealed withpopulation-average techniques.The second part of the thesis focused on the CD4 co-receptor whose role in initiating an immune response is ambiguous. Even though the CD4 co-receptor increases the sensitivity of T cell signalling manyfold, it binds to its ligand, peptide major histocompatibility complex II (pMHCII), with the lowest binding affinity known to this day. The CD4-MHC II interaction is so weak that adhesion molecules are needed to ensure a successful CD4-MHC II contact formation. For this reason, the influence of an adhesion molecule, rat CD2, on the obtained binding kinetics of the human CD4 co-receptor was initially examined showing that the accumulation of CD4 was influenced when having a high concentration of bound CD2 inside the cell-SLB contacts. Later, the studies focused on the CD4-TCR-MHC II ternary complex where it was demonstrated that the presence of L3-12 TCR strongly supported the CD4-MHC II interaction by increasing the local density of MHC II inside the cell-SLB contacts. However, the presence of TCR did not seem to significantly influence the specific affinity for CD4 to MHC II. Lastly, CD4 binding studies showed that the co-receptor did not noticeably affect the TCR-MHC II binding at physiological levels of hCD4 in the SLB

Three issues for mental health nurse educators preparing new preregistration programmes

Universities across the UK must develop and have in place new preregistration programmes by 2020. Unlike in the rest of the world, where initial nurse preparation is generic, preregistration education in the UK allows students to specialize in one of four specific fields of practice - adult, mental health, child or learning disability. To the relief of many , the new standards confirmed the continued survival of the specialist fields at undergraduate level. Educators across the UK now have an opportunity to fully review existing provision and address areas of significance. This paper explores three such issues. These are: •How best to respond to adverse life experiences or trauma.•The ethical tension which we believe is inherent in the use of coercive practice.•The potential to make a difference to the appalling mortality statistics which indicate that people with long standing mental health problems die much younger than their contemporaries In each case we make tentative suggestions for how they might be approached

Health beliefs and personality correlates of breast cancer: from screening to mastectomy adjustment - a cross-cultural study between Scotland and Greece

The present thesis consists of three thematic parts in relation to breast cancer: (a) practice of breast self - examination (BSE), (b) screening mammography attendance and (c) adjustment to breast cancer surgery / mastectomy. The aims of the respective studies are (a) to examine attitudes, beliefs and practices regarding BSE in younger (30 years old or under) and older women (over 30 years old), (b) to explore attitudes and beliefs regarding mammography and identify factors associated with screening mammography attendance and (c) to explore factors associated with adjustment to breast cancer surgery. The above aims are explored in two different cultural contexts, by comparing samples from Scotland and Greece. Samples consisted (a) of 205 younger and 258 older women, university staff and students in Scotland and 85 younger women, university students in Greece, (b) 283 women who attended and 72 women who did not attend the National Breast Screening in Scotland, and 72 women undergoing mammography in Greece and (c) 19 women in Scotland and 27 women in Greece, who have undergone surgery for breast cancer. All participants were assessed on a variety of measures. These included demographics, health history, health beliefs and health - related personality variables. Results indicated that: 1. BSE was predicted by different variables across age and cultural groups. 2. In particular, practice of BSE in younger women was predicted by knowledge about breast cancer, perceived barriers, health motivation and cues for action, whereas practice in older women was predicted by knowledge about BSE, perceived barriers and cues for action. 3. BSE rates in both countries were higher than previously reported but did not differ between women in Scotland and Greece. However, the two groups differed in knowledge and attitudes, regarding BSE, and in personality. Women in Scotland appeared more knowledgeable regarding BSE, felt less susceptible to breast cancer and were less active in coping with health issues than women in Greece. Women in Greece valued their health more and scored higher in chance health locus of control beliefs than women in Scotland. 4. The best predictor of breast screening mammography attendance in Scotland was knowledge about mammography. Attenders appeared to have more knowledge about breast cancer and about mammography and to focus more on emotional coping, in order to deal with health stresses. 5. The two cultural groups differed in health-related decision making and health - related coping styles. Attenders in Scotland were more knowledgeable about risk factors related to breast cancer and about mammograms and perceived significantly more pain/discomfort associated with the procedure, than attenders in Greece. Attenders in Greece resorted more to acceptance and denial and were more likely to seek emotional support, in order to cope with health stressors than attenders in Scotland. 6. Health beliefs of breast cancer patients, in relation to their condition, and their style of coping with threatening information, concerning their breast problem, are highlighted as important factors to their adjustment in both cultures. However, adjustment to breast cancer surgery appears culture-specific, as different factors seem to determine it in different cultural contexts. 7. Patients in Scotland and Greece did not differ in their overall adjustment. However, Greek patients were significantly less well adjusted sexually postoperatively than patients in Scotland. Findings are discussed in relation to theoretical and practical implications

‘It’s been a long haul, a big haul, but we’ve made it’: hepatitis C virus treatment in post-transplant patients with virus recurrence: An interpretative phenomenological analysis

The lived experience of both interferon-based and new interferon-free treatments in patients with hepatitis C virus remains understudied. To explore their journey through hepatitis C virus treatment, we interviewed seven post-transplant patients with recurrent hepatitis C virus. Three themes were identified using interpretative phenomenological analysis. Participants reported an ongoing sense of ontological uncertainty characterized by lack of control over their condition and treatment. Furthermore, an apposition of scepticism and hope accompanying each stage of hepatitis C virus treatment was described. A staged approach to psychological intervention tailored to the needs of the patient and their associated ‘stage’ of hepatitis C virus treatment was recommended

Suicide risk assessment in the emergency department:an investigation of current practice in Scotland

Background: Suicide is a global public health issue. Approximately one third of individuals who complete suicide have attended an emergency department in the year preceding their death. The aim of this study was to investigate current suicide risk assessment practices across emergency department clinicians in Scotland.Methods: A mixed‐methods design was employed. A total of 112 surveys for emergency department clinicians were posted to 23 emergency departments in Scotland between March and September 2016. Follow‐up semi‐structured interviews were also conducted exploring clinician's experiences of suicide risk assessment. Interviews were analysed using thematic analysis.Results: Fifty‐one emergency department clinicians across 17 emergency departments completed the survey. Thirty‐five (68.6%) participants were currently using a suicide risk assessment tool; with most using locally developed tools and proformas (n = 20, 62.5%) or the SAD PERSONS scale (n = 13, 40.6%). Remaining participants (n = 16, 31.4%) did not use suicide risk assessment tools during assessment. Variation in practice was found both across and within emergency departments. Six clinicians participated in follow‐up interviews, which identified four major themes: Clinician Experiences of Suicide Risk Assessment; Components of Suicide Risk Assessment; Clinical Decision‐Making; and Supporting Clinicians.Conclusions: There is substantial variation in current practice, with around two‐thirds of clinicians using a variety of empirically and locally developed tools, and a third using their judgement alone. Clinicians find suicide risk assessment a challenging part of their role and discuss the need for increased training, and appropriate and helpful guidelines to improve practice

Qualitative exploration of the benefits of group-based memory rehabilitation for people with neurological disabilities: implications for rehabilitation delivery and evaluation

Objective: To identify patient-perceived benefits of memory rehabilitation and draw transferrable lessons for the delivery and evaluation of similar interventions for people with neurological disabilities. Methods: A qualitative study was conducted as part of a pragmatic randomised controlled trial comparing 2 memory rehabilitation approaches with a self-help control group. Postintervention interviews were conducted with 20 participants with a diagnosis of traumatic brain injury, multiple sclerosis or stroke. Data were analysed using a qualitative content analysis approach. Results: Participants receiving memory rehabilitation reported that the sessions responded to previously unmet needs for information on brain injury and memory function and developed their insight along with a sense of self-efficacy and control over the management of their memory problems. Although they did not experience major improvements in their memory function per se, they reported that rehabilitation gave them the skills to effectively cope with the residual deficits. Respondents in the control groups did not report similar benefits. The opportunities for interaction offered by the group setting were greatly valued by all respondents. Mixed aetiology groups were received positively; however, marked differences in cognitive performance were frustrating for some participants. Conclusions: The study highlighted important patient-perceived outcomes that should be considered by researchers and rehabilitation professionals when evaluating the effects of memory rehabilitation. The use of domain-specific outcome measures which reflect these areas is recommended. Qualitative changes in the use of memory aids may be achieved which cannot be captured by frequency indices alone. The benefits of the group-based rehabilitation approach were stressed by participants, suggesting that a combination of group and individual sessions might be a good practice

The development of a questionnaire assessing the outcome of memory rehabilitation for people with acquired brain injury

Part A: Memory rehabilitation is a promising approach to address memory difficulties although its effectiveness with neurologically impaired individuals is yet to be established (Chapter 1). This thesis was conducted within the context of the pilot and main phase of a randomised controlled trial (ReMind) evaluating the effectiveness of memory rehabilitation for people experiencing memory problems following traumatic brain injury (TBI), stroke and multiple sclerosis (MS). The trial compared the effects of restitutive and compensatory memory rehabilitation strategies with a self-help control intervention on memory functioning, mood, activities of daily living and mental adjustment. The quantitative data obtained in this trial did not provide strong evidence to support the effectiveness of the intervention. The use of inappropriate outcome measures may account for the contradictory or inconclusive findings of the ReMind and other memory rehabilitation studies. Chapter 2 provides a review of measures that were used in the ReMind trial and/or were commonly used to evaluate outcome in memory rehabilitation studies. A lack of measures that considers the aims of memory rehabilitation and the needs of neurologically impaired individuals was observed. The post-intervention interviews of participants (N=19) in the pilot phase of the ReMind were analysed thematically (Chapter 3). Participants reported benefits in areas that were not covered by existing quantitative outcome measures such as insight into the nature and severity of their memory problems, confidence in their ability to manage these difficulties and qualitative improvements in the use of memory aids. The aim of the following studies was to develop and evaluate a questionnaire responsive to the effects of memory rehabilitation following acquired brain injury. The process included two stages: Part B: Identification of the content of AMEDO questionnaire: At this stage, studies were conducted within the main phase of the ReMind trial. The content areas of the questionnaire were identified based on the input and feedback of participants in each of the three memory rehabilitation programmes (Restitution, Compensation and Self-help groups). A mixed methods design was followed and information was drawn from two sources: 1)Real time observations of 43 sessions were performed (Chapter 4). The study introduced a new recording strategy by using a time sampling method to qualitatively record the content of conversations. Group activity was also evaluated. Following a quantitative content analysis method, observations were grouped into categories and their frequency was assessed in order to systematically describe and compare the content of the three programmes. 2)Semi-structured post-intervention interviews were conducted with 20 participants to explore their experience in the groups (Chapter 5). Recurrent patterns of data were identified inductively following a thematic analysis approach. Interviews from each programme were analysed separately and the emerging themes were compared and contrasted to highlight similarities and differences between the programmes. The majority of participants perceived the main benefits of memory rehabilitation to be: a) responding to their need for information on the cognitive effects of brain injury, b) enhancing their sense of self-efficacy and control over their memory difficulties, c) motivating them to adopt a more proactive attitude towards the management of these problems. The advantages of the group based approach to rehabilitation were also highlighted by most respondents. Questionnaire items were generated to cover the key content areas that were identified in both studies: memory knowledge, awareness, emotional adjustment, active coping, control beliefs, attention, significant others (comprised Part A of the questionnaire), the use of external memory aids (comprised Part B1) and the use of internal memory aids (Part B2). Part C: Evaluation of the psychometric properties of Adaptation to Memory Difficulties Outcome questionnaire (AMEDO): The final study of this thesis (Chapter 6) evaluated the psychometric properties of the new questionnaire. The first version of AMEDO included 45 items rated on a 4-point Likert scale (strongly disagree-strongly agree). Face validity was assessed by researchers and clinicians experienced in the area of neurological rehabilitation. The psychometric properties of AMEDO were evaluated by posting the questionnaire to a sample of people with MS and TBI identified through hospital records. It was returned by 110 people with MS and 34 people with TBI and 87 of these participants returned the second questionnaire that was sent to assess stability. After applying the criteria of face validity, response distribution, and construct validity 15 items were retained in Part A of the questionnaire, and four items in Parts B1 and B2 respectively. The distribution of responses was acceptable for all items except for the ones assessing the use of external memory aids which displayed negative skew (ceiling effects). Principal component analysis indicated that the questionnaire captured most of the content areas it was designed to cover. Part B1 and B2 formed two distinct subscales assessing effectiveness in the use of external and internal memory aids respectively. Part A comprised three components: “Memory knowledge”, “Control” and “Emotional adjustment”. Evaluation of item-convergent validity confirmed the three factor solution. Internal consistency estimates for all the subscales were found to be satisfactory (α=.74-.84). Correlations between the subscales indicated that the represented constructs were related in meaningful ways. Test–retest reliability coefficients for the subscales ranged between r=.60 to r=.82. The biggest discrepancies were found in the “Memory knowledge” and external memory aids (EMA) subscales whereas the “Control” and “Emotional adjustment” showed acceptable levels of stability. Differences between test-retest scores in the former subscales dropped to satisfactory levels when analyses were repeated excluding respondents who completed the 2nd questionnaire more than a month after completing the 1st questionnaire. This finding indicated that the observed differences between scores at Time 1 and Time 2 may be reflective of actual changes taking place during that interval. Finally, it was shown that scores were not significantly affected by age, diagnosis and time since injury and, therefore, the questionnaire may be relevant to a wider neurological population. In conclusion, AMEDO is a brief and simple measure tailored to the characteristics and needs of neurologically impaired individuals. The questionnaire shows promise as an outcome measure specific to the effects of memory rehabilitation, to complement memory batteries and established generic measures. Further evaluation of the scale should confirm the stability of the questionnaire and assess its responsiveness to changes following memory rehabilitation