Application of CRISPR/Cas9 screening to study cancer drivers and to identify novel cancer vulnerabilities

The development of targeted therapies has had a significant impact on cancer survival rates. However, targeting cancers that are driven by loss of tumour suppressor genes remains a major challenge. One promising approach to treat these cancers is the exploitation of synthetic lethal interactions. Synthetic lethality describes an interaction between two genes, where loss of one gene alone does not affect viability but loss of both genes induces cell death. Inhibiting the synthetic lethal partner of a tumour suppressor gene should specifically kill tumour cells, and so these represent potential therapeutic targets. However, very few synthetic lethal interactions have been well-established. The aim of this project was to systematically screen for synthetic lethal partners of known tumour suppressor genes. To do so, isogenic human induced pluripotent stem cell lines were generated, each carrying a loss-of-function mutation in a single tumour suppressor gene. These cells have a normal genetic background, thus making it simpler to accurately identify interactions. CRISPR/Cas9 technology was applied as it allows for large-scale, unbiased screening of genetic interactions. A genome-wide guide RNA library was prepared and implemented for knockout screening in the isogenic cell line panel. Analysis was performed to identify genes that were specifically essential for cell fitness/survival in the mutant lines. Particular focus was placed on four tumour suppressor genes that encode subunits of the PBAF/BAF complexes. Approximately 20% of human cancers harbour mutations in subunits of these complexes, so identifying dependencies associated with these could have broad therapeutic potential. Candidate synthetic lethal interactions with these genes were investigated using low-throughput assays in the stem cells and in a cancer cell line. The data obtained suggests that screening in stem cells produces highly variable results. Although potential vulnerabilities associated with all of the tumour suppressor genes were identified, further work is required to validate these and to assess the quality of the results. In addition to genome editing, CRISPR/Cas9 has been adapted as a tool for controlling gene regulation. In collaboration with Dr Louise van der Weyden, I applied this technology to address another challenging area of cancer biology. Metastasis is the main cause of cancer mortality, yet we still have a poor understanding of the genes that control this process. Considering this, an in vivo CRISPR activation screen was performed to identify novel drivers of metastatic colonisation. A mouse melanoma cell line was transduced in vitro with a library designed to up-regulate expression of membrane proteins, which represent ideal drug targets. These cells were then used in an in vivo experimental metastasis assay. Enrichment of guide RNAs in the lungs was assessed to identify genes that increased pulmonary metastatic colonisation when activated. Candidate genes were selected using three analysis strategies, and hits from each were tested. Several genes were successfully validated using the experimental metastasis assay. The most robust hit was studied further to explore its potential as a therapeutic target. Collectively, the work described in this thesis demonstrates how CRISPR/Cas9 screening can be applied in different model systems to study genes that drive cancer and to explore novel therapeutic strategies.I was funded by the Wellcome Sanger Institute and the MRC

The carer’s view: a new perspective on chronic illness and disability within the early modern family

Based on a close reading of Elizabeth Isham’s (b. 1609 – d. 1654) and Mary Rich’s (b. 1624 – d. 1678) writings, this thesis considers the neglected follow-up to Roy Porter’s statement: while “it takes two to make a medical encounter”, “it often takes many more because medical events have frequently been complex social rituals involving family and community as well as sufferers and physicians”. Using a case study approach, this thesis uniquely takes ‘the carer’s view’. It explores the experiences of two wealthy, early modern women who provided long-term care to a family member. It suggests that long-term caring was a deeply religious experience, which became entwined with the lives and spiritual identities of carers. Caring forced carers to grapple with difficult questions relating to love, time, and suffering. The religious significances of these concepts consequently became bound up with how carers could navigate and understand their roles. Because caring involved ‘immoderate’ quantities of love, time, and suffering, it was inherently spiritually problematic; contemporary religious discourses recommended moderation in these areas, to avoid sin. In lieu of ready-made, spiritually acceptable notions of long-term caring, carers had to personally find ways to make caring compatible with their spiritual aspirations. Resulting ideas of caring were highly particular and reliant on the carer’s personal circumstances. By examining the experiential and cultural content of early modern caring for the first time, this thesis fills a significant gap in the history of medicine and opens a rich seam for further research. It also offers a unique perspective on histories of family, love, time, lived religion, and salvation. Mary and Elizabeth show that carers experienced and negotiated with these concepts in unique ways. This work should be of interest more generally to historians of disability, sickness and health, personal identity, love, time, family, and ‘lived religion’

Self-management support intervention for parents of children with developmental disorders: The role of gratitude and hope

Objectives: Many parents of children with developmental disabilities, including autism spectrum disorders and attention deficit disorders, experience poor well-being and increased anxiety and depression. Very few interventions directly target parents’ needs. The peer-delivered HOPE Programme was designed to address this with six weekly group sessions focusing on self-management skills, including goal setting and expressing gratitude. Methods: This pre-post study aimed to examine changes in anxiety, depression, well-being, hope and gratitude, and to explore associations between changes in anxiety and depression and changes in gratitude and hope. Validated measures of depression, anxiety, positive well-being, gratitude and hope were used. Parents of children with a range of developmental disabilities, most commonly autism spectrum disorders, were recruited. Results: Of 137 (86.9% female) recruited, 108 parents completed the course and post-course data. Parents’ depression, anxiety, well-being, gratitude and hope all significantly improved between baseline and post-course. Hope and gratitude correlated significantly with depression, anxiety and well-being. Baseline depression, baseline gratitude, post-course hope and gratitude explained 50% of the variance in post-course depression. Reduced work hours, and baseline and post-course hope and gratitude explained 40% of the variance in post-course well-being. Anxiety was not associated to hope nor gratitude at either time point. Conclusions: This study provides initial support for feasibility and potential effect of the peer delivered self-management intervention on parental anxiety and depression. Changes in gratitude and hope account for some change in depression, but not anxiety. A randomised controlled trial is needed to establish efficacy and explore mechanisms of change in-depth

Direct Arylation of Thiazoles

An introduction to the thiazole ring system is presented together with a detailed, but non-exhaustive review of the rapidly emerging area of palladium-mediated directed arylation. The direct arylation of thiazole is also discussed together with our attempts to improve the established methods. A high-yielding, mild protocol has been developed for the functionalisation of the most electron-rich carbon-hydrogen bonds in a number of heterocyclic ring systems, this represents the first example of a C-H activation reaction being accomplished in aqueous media and allows access to a diverse range of functionalised aryl heterocycles. In addition, work towards functionalisation of the thiazole C4 position is described. A number of different approaches are discussed and our endeavors are recorded

Is the “end-of-study guess” a valid measure of sham blinding during transcranial direct current stimulation?

Studies using transcranial direct current stimulation (tDCS) typically incorporate a fade‐in, short‐stimulation, fade‐out sham (placebo) protocol, which is assumed to be indistinct from a 10‐30min active protocol on the scalp. However, many studies report that participants can dissociate active stimulation from sham, even during low‐intensity 1mA currents. We recently identified differences in the perception of an active (10min of 1mA) and a sham (20s of 1mA) protocol that lasted for 5 mins after the cessation of sham. In the present study we assessed whether delivery of a higher‐intensity 2mA current would exacerbate these differences. Two protocols were delivered to 32 adults in a double‐blinded, within‐subjects design (active: 10min of 2mA, and sham: 20s of 2mA), with the anode over the left primary motor cortex and the cathode on the right forehead. Participants were asked “Is the stimulation on?” and “How sure are you?” at 30s intervals during and after stimulation. The differences between active and sham were more consistent and sustained during 2mA than during 1mA. We then quantified how well participants were able to track the presence and absence of stimulation (i.e. their sensitivity) during the experiment using cross‐correlations. Current strength was a good classifier of sensitivity during active tDCS, but exhibited only moderate specificity during sham. The accuracy of the end‐of‐study guess was no better than chance at predicting sensitivity. Our results indicate that the traditional end‐of‐study guess poorly reflects the sensitivity of participants to stimulation, and may not be a valid method of assessing sham blinding

Help to Overcome Problems Effectively for Cancer Survivors: Development and Evaluation of a Digital Self-Management Program

Background People living with cancer face numerous psychosocial challenges, including cancer-related fatigue, fear of recurrence, and depression. There is a lack of digital interventions tailored to the needs of people living with all types of cancer. We developed a 6-week, digital, peer-delivered, self-management program: iHOPE (Help to Overcome Problems Effectively; where ‘i’ indicates the digital version of the program). The program is underpinned by positive psychology and cognitive behavioral therapy to meet these psychosocial challenges. Objective This study aimed to assess the feasibility of the iHOPE program among people living with cancer. Program adherence and satisfaction along with changes in psychological distress and positive well-being were measured. Methods A pre-post, acceptability, and feasibility design was used. People living with cancer (N=114) were recruited via a national cancer charity in the United Kingdom and were given access to the iHOPE program. Demographic and other participant characteristics were recorded. Participants completed digital measures at baseline and the end of the 6-week program for depression, anxiety, cancer-related fatigue, cancer worry or fear of cancer recurrence, positive mental well-being, hope, gratitude, and health status. The website’s system recorded data on the usage of the program. Satisfaction with the program was also measured. Results A total of 114 participants completed the baseline questionnaires. Of these, 70 people (61.4%) participated in all 6 sessions. The mean number of sessions undertaken was 5.0 (SD 1.5). Moreover, 44.7% (51/114) of participants completed at least three sessions and end-of-program outcome measures. A total of 59 participants completed the satisfaction questionnaire, where ≥90% (54/58) of participants reported that the program was easy to navigate and was well managed by the peer facilitators, and that they found the social networking tools useful. Preliminary efficacy testing among the 51 participants who completed baseline and postprogram outcome measures showed that postprogram scores decreased for depression, anxiety, cancer-related fatigue, and fear of recurrence (all P&lt;.001) and increased for positive mental well-being (P&lt;.001), hope (both P&lt;.001), and gratitude (P=.02). Conclusions The feasibility evidence is promising, showing that the peer-delivered digital iHOPE program is acceptable and practical. Implementation of the iHOPE program on a wider scale will incorporate further research and development to maximize the completion rates of the measures. Initial effectiveness data suggest positive impacts on important cancer-related quality of life and mental well-being outcomes. A randomized controlled trial design with a longer follow-up is needed to confirm the potential of the iHOPE program for improving mental and physical health outcomes for cancer survivors. </jats:sec

Single Crystal X-Ray Diffraction Study of Pressure and Temperature Induced Spin Trapping in a Bistable FeII Hofmann Framework

This is the peer reviewed version of the following article: Turner, G. F., Campbell, F., Moggach, S. A., Parsons, S., Goeta, A. E., Muñoz, M. C., & Real, J. A. (2020). Single-Crystal X-Ray Diffraction Study of Pressure and Temperature¿Induced Spin Trapping in a Bistable Iron (II) Hofmann Framework. Angewandte Chemie International Edition, 59(8), 3106-3111, which has been published in final form at https://doi.org/10.1002/anie.201914360. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."[EN] High-pressure single-crystal X-ray diffraction has been used to trap both the low-spin (LS) and high-spin (HS) states of the iron(II) Hofmann spin crossover framework, [FeII (pdm)(H2 O)[Ag(CN)2 ]2·H2 O, under identical experimental conditions, allowing the structural changes arising from the spin-transition to be deconvoluted from previously reported thermal effects.This work was supported by the Spanish Ministerio de Economia y Competitividad (MINECO), FEDER (CTQ2016-78341-P), Unidad de Excelencia Mar&a de Maeztu (MDM 2015-0538), the Generalitat Valenciana through PROMETEO/2016/147, and the EPSRC through EP/D503744 and GR/M81830. The authors acknowledge the facilities, and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, a facility funded by the University, State and Commonwealth Governments. G.F.T. acknowledges the Australian Government for the provision of an Australian Government Research Training Program (RTP) scholarship.Turner, GF.; Campbell, F.; Moggach, SA.; Parsons, S.; Goeta, AE.; Muñoz Roca, MDC.; Real, JA. (2020). Single-Crystal X-Ray Diffraction Study of Pressure and Temperature-Induced Spin Trapping in a Bistable Iron(II) Hofmann Framework. Angewandte Chemie International Edition. 59(8):3106-3111. https://doi.org/10.1002/anie.2019143603106311159

Crystalline phase discriminating neutron tomography using advanced reconstruction methods

Time-of-flight neutron imaging offers complementary attenuation contrast to X-ray computed tomography (CT), coupled with the ability to extract additional information from the variation in attenuation as a function of neutron energy (time of flight) at every point (voxel) in the image. In particular Bragg edge positions provide crystallographic information and therefore enable the identification of crystalline phases directly. Here we demonstrate Bragg edge tomography with high spatial and spectral resolution. We propose a new iterative tomographic reconstruction method with a tailored regularisation term to achieve high quality reconstruction from low-count data, where conventional filtered back-projection (FBP) fails. The regularisation acts in a separated mode for spatial and spectral dimensions and favours characteristic piece-wise constant and piece-wise smooth behaviour in the respective dimensions. The proposed method is compared against FBP and a state-of-the-art regulariser for multi-channel tomography on a multi-material phantom. The proposed new regulariser which accommodates specific image properties outperforms both conventional and state-of-the-art methods and therefore facilitates Bragg edge fitting at the voxel level. The proposed method requires significantly shorter exposure to retrieve features of interest. This in turn facilitates more efficient usage of expensive neutron beamline time and enables the full utilisation of state-of-the-art high resolution detectors

Understanding the experience of initiating community-based group physical activity by people with serious mental illness: a systematic review using a meta-ethnographic approach

Background People living with serious mental illness (SMI) experience debilitating symptoms that worsen their physical health and quality of life. Regular physical activity (PA) may bring symptomatic improvements and enhance wellbeing. When undertaken in community-based group settings, PA may yield additional benefits such as reduced isolation. Initiating PA can be difficult for people with SMI and so PA engagement is commonly low. Designing acceptable and effective PA programmes requires a better understanding of the lived experiences of PA initiation among people with SMI. Methods This systematic review of qualitative studies used the meta-ethnography approach by Noblit and Hare (1988). Electronic databases were searched from inception to November 2017. Eligible studies used qualitative methodology; involved adults (≥18 years) with schizophrenia, bipolar affective disorder, major depressive disorder or psychosis; reported community-based group PA; and captured the experience of PA initiation, including key features of social support. Study selection and quality assessment was performed by four reviewers. Results Sixteen studies were included in the review. We identified a ‘journey' that depicted a long sequence of phases involved in initiating PA. The journey demonstrated the thought processes, expectations, barriers and support needs of people with SMI. In particular, social support from a trusted source played an important role in getting people to the activity, both physically and emotionally. Discussion The journey illustrated that initiation of PA for people with SMI is a long complex transition. This complex process needs to be understood before ongoing participation in PA can be addressed