Investigating mechanisms and indicators of sensitivity to replication stress-targeting therapies in glioblastoma

Introduction Evidence suggests a subpopulation of treatment resistant glioblastoma (GBM) cancer stem cells (GSCs) is responsible for tumour recurrence, an almost universally deadly characteristic of this cancer of extreme unmet need. Current treatments fail to eradicate GSCs and novel GSC targeting therapies are a clinical priority. Elevated DNA replication stress (RS) in GSCs has been described, leading to constitutive DNA damage response activation and treatment resistance and targeting RS with combined ATR and PARP inhibition (CAiPi) has provided potent GSC cytotoxicity. Nevertheless, there are a relative lack of studies investigating the underlying mechanisms of response to CAiPi in GBM and a lack of robust transcriptional signatures or genomic biomarkers correlated with CAiPi response in GSCs. Aims This thesis aims to investigate RS as a targetable vulnerability of GSCs. It aims to achieve this by studying the mechanisms of sensitivity to inhibition of the RS response to inform transcriptional indicators of sensitivity. Lastly, it aims to investigate the feasibility of this therapeutic strategy in a preclinical model. Methods Paired GSC-enriched and GSC-depleted, differentiated (‘bulk’) populations, derived from resected GBM specimens, were maintained in serum-free, stemenriching conditions or differentiating conditions respectively. WGS and RNAseq were utilised to characterise the genomic and transcriptomic landscape of the cell line panel. Responses to CAiPi were assessed by clonogenic and cell viability assays and validated in a CD133 sorted population by neurosphere assay. Replication dynamics in paired GSC and bulk cells were investigated by a DNA fibre assay. Dysregulated S phase was analysed by quantification of 53BP1 nuclear bodies (53BP1NB), indicative of under-replication of the genome, and quantification of re-replicating cells by flow cytometry. Chromosomal instability was interrogated by quantification of chromatin bridges and micronuclei. Novel mechanistic discoveries prevalent in GSCs with potent CAiPi-sensitivity were used to curate a transcriptional marker of sensitivity for interrogation in GBM cell lines and in published clinical datasets. Lastly the feasibility of CAiPi was investigated in an in vivo preclinical model, assessing tolerability and tumour penetration. Results CAiPi was potently cytotoxic to a population of GSCs but highly heterogenous responses to CAiPi were observed across a panel of seven paired GSCs and bulk cells. Sensitivity was not predicted by elevated RS in GSCs or any previously defined biomarkers of RS or CAiPi sensitivity. Differential sensitivity was exploited for further investigations which identified transcriptional dysregulation of DNA replication, specifically in a CAiPi-responsive GSC line. Subsequent analysis of DNA replication identified PARPi-induced increase in origin firing, associated with PARP trapping. GSCs with this origin firing phenotype also exhibited an increase in both under-replicated DNA and re-replication in response to CAiPi, with an increase in chromosomal aberrations and instability. A curated transcriptional signature, based on mechanistic discoveries in CAiPisensitive GSCs, predicted GSC sensitivity and identified populations of GBM patients with poor survival who may respond to CAiPi treatment. In vivo studies demonstrated murine blood brain barrier (BBB) penetration of a PARPi and an ATRi with minimal toxicity, however optimal dosing and scheduling remains a challenge. Conclusions We propose that CAiPi-sensitivity is marked by loss of replication coordination leading to chromosomal damage as cells move through S phase. Additionally, we propose a model whereby under-replication and re-replication can occur due to spatial and temporal uncoupling during S phase. Targeting RS via CAiPi represents a promising therapeutic strategy for selectively targeting recurrence driving GSCs to improve clinical outcomes in GBM

Accompanying Survivors of Sexual Harm: A Toolkit for Churches

Accompanying Survivors of Sexual Harm is a trauma-informed resource that offers education and support of Christian clergy and lay leaders as they respond to sexual harm in their communities. It lays out plans for workshop-based sessions, which aim to educate clergy and lay leaders about • Understanding the nature of sexual harm and its prevalence in New Zealand society • Being alert to and responding in a pastorally sensitive manner to people within their community who have experienced/are experiencing sexual harm • Identifying and articulating some of the scriptural and theological foundations that work to justify/legitimise/enable sexual harm while silencing the voices of victims/survivors • Identifying and articulating some of the scriptural and theological foundations that work to challenge and resist sexual harm • Exploring how their church might work to create a safe space for victims/survivors of sexual harm. The toolkit will be of value to anyone in a church leadership position, including those training for Christian ministry and those who have extensive ministry/leadership experience. It is intentionally ecumenical in nature and does not require knowledge of any one denominational tradition. While some of the content relates specifically to the context of Aotearoa New Zealand, most of the material can be adapted and used further afield. There is space offered throughout the sessions for participants to discuss how issues pertaining to sexual harm relate to their own communities. Participants also have opportunities to consider how their own cultures, contexts, traditions, and languages will help shape their role of accompanying victims and survivors. The toolkit is free for anyone to download and use. If you have any queries about the use of the toolkit, please contact us at [email protected]. We hope this resource is a useful and meaningful tool for all those who accompany victims and survivors on their journey

Models of natural pest control : Towards predictions across agricultural landscapes

Natural control of invertebrate crop pests has the potential to complement or replace conventional insecticide based practices, but its mainstream application is hampered by predictive unreliability across agroecosystems. Inconsistent responses of natural pest control to changes in landscape characteristics have been attributed to ecological complexity and system-specific conditions. Here, we review agroecological models and their potential to provide predictions of natural pest control across agricultural landscapes. Existing models have used a multitude of techniques to represent specific crop-pest-enemy systems at various spatiotemporal scales, but less wealthy regions of the world are underrepresented. A realistic representation of natural pest control across systems appears to be hindered by a practical trade-off between generality and realism. Nonetheless, observations of context-sensitive, trait-mediated responses of natural pest control to land-use gradients indicate the potential of ecological models that explicitly represent the underlying mechanisms. We conclude that modelling natural pest control across agroecosystems should exploit existing mechanistic techniques towards a framework of contextually bound generalizations. Observed similarities in causal relationships can inform the functional grouping of diverse agroecosystems worldwide and the development of the respective models based on general, but context-sensitive, ecological mechanisms. The combined use of qualitative and quantitative techniques should allow the flexible integration of empirical evidence and ecological theory for robust predictions of natural pest control across a wide range of agroecological contexts and levels of knowledge availability. We highlight challenges and promising directions towards developing such a general modelling framework.Peer reviewe

Inhibition of ATR prevents macropinocytosis driven retraction of neurites and opposes invasion in GBM

Glioblastoma (GBM) is the most common and aggressive type of primary brain tumour and remains incurable despite decades of research. GBM are characterised by highly infiltrative growth patterns that contribute to the profound cognitive and neurological symptoms experienced by patients, and to inevitable recurrence following treatment. Novel treatments that reduce infiltration of the healthy brain have potential to ameliorate clinical symptoms and improve survival. Here, we report a novel role of the Ataxia telangiectasia and Rad 3 related kinase (ATR) in supporting the invasive properties of GBM cells through the regulation of macropinocytosis-driven internalisation of integrin adhesion receptors. We demonstrate that inhibition of ATR opposes GBM migration in vitro, and correspondingly reduces infiltrative behaviour in orthotopic mouse models. These results indicate that ATR inhibition, in addition to its use as a radiosensitiser, may be effective in reducing GBM infiltration and its associated symptoms

The interplay of landscape composition and configuration: new pathways to manage functional biodiversity and agroecosystem services across Europe

Managing agricultural landscapes to support biodiversity and ecosystem services is a key aim of a sustainable agriculture. However, how the spatial arrangement of crop fields and other habitats in landscapes impacts arthropods and their functions is poorly known. Synthesising data from 49 studies (1515 landscapes) across Europe, we examined effects of landscape composition (% habitats) and configuration (edge density) on arthropods in fields and their margins, pest control, pollination and yields. Configuration effects interacted with the proportions of crop and non‐crop habitats, and species’ dietary, dispersal and overwintering traits led to contrasting responses to landscape variables. Overall, however, in landscapes with high edge density, 70% of pollinator and 44% of natural enemy species reached highest abundances and pollination and pest control improved 1.7‐ and 1.4‐fold respectively. Arable‐dominated landscapes with high edge densities achieved high yields. This suggests that enhancing edge density in European agroecosystems can promote functional biodiversity and yield‐enhancing ecosystem services

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Strategy Construction in American Third Parties

(Statement of Responsibility) by Emily Clough(Thesis) Thesis (B.A.) -- New College of Florida, 1997(Electronic Access) RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE(Bibliography) Includes bibliographical references.(Source of Description) This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.(Local) Faculty Sponsor: Fitzgerald, Keit