A proteomics investigation into the role of zDHHC23 and MROH6 in neuroblastoma

Neuroblastoma (NB) is the most common malignant solid tumour diagnosed in infants, accounting for ~15% of all childhood cancer-related deaths. Current patient risk stratification criteria are heavily reliant on the presence of a MYCN amplification, albeit only accounting for ~25% of patients. The inadequate prognostic risk stratification of patients results in children receiving either inefficient or excessive treatment with a myriad of severe lifelong side effects for survivors. Therefore, the identification and characterisation of novel biomarkers could not only identify new therapeutic targets but could also improve risk stratification and treatment planning. A comparative transcriptomic analysis of NB tumours (obtained from the chick embryo model) grown under normal oxygen tensions (normoxia, 21% O2) or hypoxia (1% O2), a model for aggressive NB tumours that correlates with poor patient prognosis, identified multiple significantly upregulated genes in aggressive (hypoxic) tumours specifically, with Zinc Finger DHHC-Type Palmitoyltransferase 23 (zDHHC23) and Maestro Heat Like Repeat Family Member 6 (MROH6) exhibiting the best correlation with poor prognosis. This thesis sought to validate these expressed gene products as potential biomarkers in NB. I also investigated the molecular function of these two proteins under normoxic and hypoxic conditions, supplementing the currently limited available knowledge. Commercially available antibodies for these two proteins were unsuccessful for use in either immunostaining, a procedure currently used as the ‘gold-standard’ of clinical biomarker screening, or for immunoblotting of endogenous protein, with all of the antibodies evaluated lacking specificity. Although targeted mass spectrometry assays were successfully developed, they lacked the sensitivity to detect endogenous proteins, likely due to low levels of protein expression. Therefore, I focused on the biochemical characterisation of these two proteins, cloning dual reporter HA-mCherry-protein and protein-mCherry-HA plasmids to facilitate immunoprecipitation of exogenously expressed protein and evaluation of sub-cellular localisation. I developed and optimised a HA-tag based immunoprecipitation protocol for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis to allow identification of post-translational modifications (PTMs) and protein interaction networks. These experiments revealed extensive hypoxia-induced regulation of protein binding partners, with ~70% of the interactome (from a total of 262 and 253 co-immunoprecipitated proteins for zDHHC23 and MROH6 respectively) changing as a function of O2 tension. GOterm analysis of these interactomes suggests that zDHHC23 is a component of several potentially important malignancy pathways, including cytoskeletal reorganisation and adhesion. Label free quantification analysis of MROH6 identifies high stoichiometric binding to Breast Cancer Anti-oestrogen Resistance protein 1 (BCAR1), inferring potential roles in telomere maintenance and genetic stability. Additionally, PTM analysis identified one and three phosphorylation sites on MROH6 and zDHHC23 respectively, with zDHHC23 S252 predicted to be regulated by Cyclin dependent kinases. Finally, I developed, to my knowledge, the first reported click-chemistry based high-throughput LC-MS/MS pipeline for the unbiased identification of zDHHC23 palmitoylated substrates, concluding that the ‘palmitome’ is much more complex than currently understood and likely regulates localisation to membrane bound organelles and extracellular vesicles, as well as its established role in plasma membrane localisation. Overall, using LC-MS/MS approaches, I explore and discuss how zDHHC23 and MROH6 overexpression may contribute to aggressive NB development and poor patient prognosis

Phase One: June 2013 – September 2014

Food and nutrition security and gender equality are closely linked and mutually constitutive. The fact that women and girls are among the most undernourished in the world and are often hardest hit by food insecurity underlines this. Women’s productive labour and unpaid care work is central to the production, preparation and provision of food. Yet their ability to feed themselves and their families is persistently undermined by institutionalised gender biases in access to resources, markets, social services and social protection, as well as socio-cultural norms which prioritise the nutrition of men and boys and limit women’s decision-making power. Acknowledging this situation the WFP has, amongst other activities, entered into a learning partnership with the Institute of Development Studies (IDS). The premise for the ‘Innovations from the Field’ programme is that WFP staff and partners at the country level are often adopting innovative practices which respond to, and deal effectively with, local gender realities and priorities, but these are rarely shared. Taking a ‘bottom-up’ learning approach to gender mainstreaming will allow successful innovations to be captured, shared and embedded across the organisation. In this first phase of the programme, IDS has facilitated a process of ‘participatory action learning’ in five WFP Country Offices: Guatemala, Kenya, Lesotho, Malawi and Senegal. This has enabled staff to reflect on, explore, document and share good practices for gender-sensitive food security programming. It has also allowed wider reflection on current barriers to effective gender mainstreaming in WFP and how they could be overcome. This report summarises the learning so far

The midlife crisis

This paper documents a longitudinal crisis of midlife among the inhabitants of rich nations. Yet middle-aged citizens in our data sets are close to their peak earnings, have typically experienced little or no illness, reside in some of the safest countries in the world, and live in the most prosperous era in human history. This is paradoxical and troubling. The finding is consistent, however, with the prediction -- one little-known to economists of Elliott Jaques (1965). Our analysis does not rest on elementary cross-sectional analysis. Instead the paper uses panel and through-time data on, in total, approximately 500,000 individuals. It checks that the key results are not due to cohort effects. Nor do we rely on simple life-satisfaction measures. The paper shows that there are approximately quadratic hill-shaped patterns in data on midlife suicide, sleeping problems, alcohol dependence, concentration difficulties, memory problems, intense job strain, disabling headaches, suicidal feelings, and extreme depression. We believe the seriousness of this societal problem has not been grasped by the affluent world’s policy-makers

Design and implementation of the NUV/optical widefield Star Formation Camera for the Theia Observatory

The Star Formation Camera (SFC) is a wide-field (~19'×~15', >280 arcmin^2), high-resolution (18 mas pixels) UV/optical dichroic camera designed for the Theia 4-m space-borne space telescope concept. SFC will deliver diffraction-limited images at λ > 300 nm in both a blue (190-517nm) and a red (517-1075nm) channel simultaneously. The goal is to conduct a comprehensive and systematic study of the astrophysical processes and environments relevant for the births and life cycles of stars and their planetary systems, and to investigate the range of environments, feedback mechanisms, and other factors that most affect the outcome of star and planet formation

Targeted Development of Registries of Biological Parts

BACKGROUND: The design and construction of novel biological systems by combining basic building blocks represents a dominant paradigm in synthetic biology. Creating and maintaining a database of these building blocks is a way to streamline the fabrication of complex constructs. The Registry of Standard Biological Parts (Registry) is the most advanced implementation of this idea. METHODS/PRINCIPAL FINDINGS: By analyzing inclusion relationships between the sequences of the Registry entries, we build a network that can be related to the Registry abstraction hierarchy. The distribution of entry reuse and complexity was extracted from this network. The collection of clones associated with the database entries was also analyzed. The plasmid inserts were amplified and sequenced. The sequences of 162 inserts could be confirmed experimentally but unexpected discrepancies have also been identified. CONCLUSIONS/SIGNIFICANCE: Organizational guidelines are proposed to help design and manage this new type of scientific resources. In particular, it appears necessary to compare the cost of ensuring the integrity of database entries and associated biological samples with their value to the users. The initial strategy that permits including any combination of parts irrespective of its potential value leads to an exponential and economically unsustainable growth that may be detrimental to the quality and long-term value of the resource to its users

The Star Formation Observatory (SFO) mission to study cosmic origins near and far

The Star Formation Observatory (SFO) is a 1.65m space telescope that addresses pivotal components in the 2007 NASA Science Plan, with a primary focus on Cosmic Origins. The design under consideration provides 100 times greater imaging efficiency and >10 times greater spectroscopic efficiency below 115 nm than existed on previous missions. The mission has a well-defined Origins scientific program at its heart: a statistically significant survey of local, intermediate, and high-redshift sites and indicators of star formation, to investigate and understand the range of environments, feedback mechanisms, and other factors that most affect the outcome of the star and planet formation process. This program relies on focused capabilities unique to space and that no other planned NASA mission will provide: near- UV/visible (20-1100 nm) wide-field, diffraction-limited imaging; and high-efficiency, low- and high- resolution (R~40,000) UV (100-175 nm) spectroscopy using far-UV optimized coatings and recent advances in Micro-Channel Plate (MCP) detector technology. The Observatory imager has a field of view in excess of 17' × 17' (>250 arcmin²) and uses a dichroic to create optimized UV/blue and red/near-IR channels for simultaneous observations, employing detectors that offer substantial quantum efficiency gains and that suffer lower losses due to cosmic rays

Clinical outcomes and response to treatment of patients receiving topical treatments for pyoderma gangrenosum: a prospective cohort study

Background: pyoderma gangrenosum (PG) is an uncommon dermatosis with a limited evidence base for treatment. Objective: to estimate the effectiveness of topical therapies in the treatment of PG. Methods: prospective cohort study of UK secondary care patients with a clinical diagnosis of PG suitable for topical treatment (recruited July 2009 to June 2012). Participants received topical therapy following normal clinical practice (mainly Class I-III topical corticosteroids, tacrolimus 0.03% or 0.1%). Primary outcome: speed of healing at 6 weeks. Secondary outcomes: proportion healed by 6 months; time to healing; global assessment; inflammation; pain; quality-of-life; treatment failure and recurrence. Results: Sixty-six patients (22 to 85 years) were enrolled. Clobetasol propionate 0.05% was the most commonly prescribed therapy. Overall, 28/66 (43.8%) of ulcers healed by 6 months. Median time-to-healing was 145 days (95% CI: 96 days, ∞). Initial ulcer size was a significant predictor of time-to-healing (hazard ratio 0.94 (0.88;80 1.00); p = 0.043). Four patients (15%) had a recurrence. Limitations: No randomised comparator Conclusion: Topical therapy is potentially an effective first-line treatment for PG that avoids possible side effects associated with systemic therapy. It remains unclear whether more severe disease will respond adequately to topical therapy alone

Engaging and training students in the development of inclusive learning materials for their peers

With equal access requirements and increasing rates of enrolment of students with disabilities in higher education, universities must find appropriate and efficient ways to create accessible materials which benefit and support all students. In response to cuts to disability funding, issues relating to the provision of an inclusive curriculum are now dominating institutional policy and educational discourses. This paper reports on a trans-Atlantic project which utilized student employees to convert and develop inclusive learning materials for their peers, with the expressed purpose of piloting a sustainable intervention method generalizable to meeting similar needs of diverse universities for inclusive material provision and a future workforce aware of disability issues and accommodations. Qualitative in-depth interviews with ten students (eight UK and two US) find that involving student employees in the delivery of inclusive materials improves partnerships and attitudes around disability and accessibility measures

The midlife crisis

This paper documents a longitudinal crisis of midlife among the inhabitants of rich nations. Yet middle-aged citizens in our data sets are close to their peak earnings, have typically experienced little or no illness, reside in some of the safest countries in the world, and live in the most prosperous era in human history. This is paradoxical and troubling. The finding is consistent, however, with the prediction -- one little-known to economists of Elliott Jaques (1965). Our analysis does not rest on elementary cross-sectional analysis. Instead the paper uses panel and through-time data on, in total, approximately 500,000 individuals. It checks that the key results are not due to cohort effects. Nor do we rely on simple life-satisfaction measures. The paper shows that there are approximately quadratic hill-shaped patterns in data on midlife suicide, sleeping problems, alcohol dependence, concentration difficulties, memory problems, intense job strain, disabling headaches, suicidal feelings, and extreme depression. We believe the seriousness of this societal problem has not been grasped by the affluent world’s policy-makers