Analysis of protein phosphorylation sites using affinity enrichment and mass spectrometry

Reversible modification of proteins by phosphorylation of serine, threonine and tyrosine residues is the most common post-translational modification, which is estimated to occur in 30-90% of the cellular expressed protein component at any one time. Phosphorylation can alter proteins' subcellular distribution, enzymatic activity and specificity. Altered protein phosphorylation may correlate with disease states such as cellular transformation. The analysis of phosphorylated proteins is therefore of vital importance to the field of biology and in particular signal transduction. Protein phosphorylation sites are increasingly investigated using mass spectrometric methods, exploiting the inherent accuracy and sensitivity of these methods. However, the presence of unphosphorylated peptides in enzymatic digests of proteins causes ion suppression of phosphopeptides, reducing the effective sensitivity of detection; this sensitivity is further decreased by the relative lability of the phosphate moiety in the mass spectrometer and the occurrence of sub-stoichiometric modification, which together further reduce the achievable sensitivity. This study has examined techniques for the analysis of protein phosphorylation sites, with particular emphasis upon mass spectrometry. The technique of immobilised metal ion affinity chromatography (IMAC) was investigated in detail as a method suited to phosphorylation site analysis. IMAC exploits the relatively specific affinity of phosphorylated peptides for metal ions, particularly Fe(III), to isolate phosphopeptides upon a solid-phase affinity matrix, separating the suppressing non- phosphorylated component and allowing improved detection of phosphorylated peptides. Conditions for the application of IMAC to phosphopeptide segregation have been established and applied. Using IMAC, protein phosphorylation site identification of both standards and signal transduction mediators has been carried out. Apparent sequence-specific binding of phosphorylated and non-phosphorylated peptides to IMAC resins has been found and investigated. IMAC methodology has been further improved to optimise phosphopeptide analysis using mass spectrometry. The developed methods have clear utility for phosphorylation site analysis, which is vital to the understanding of signal transduction

Dissecting the genotype to phenotype relationships of genomic disorders

Over the last decade, major advances in the development and application of microarray-based comparative genomic hybridisation (aCGH) technology have significantly contributed to our understanding of Genomic Disorders. My aims here were to provide insight into the genotype to phenotype relationships of three Genomic Disorders; CUL4B-deleted X-Linked Mental Retardation (XLMR), Wolf-Hirschhorn Syndrome (WHS) and 16p11.2 Copy Number Variant Disorder. CUL4B encodes a structural component of the Cullin-RING-ligase 4-containing class of E3 ubiquitin ligases. CUL4B-deleted XLMR represents a syndromal form of mental retardation whereby patients exhibit other clinical features aside from the MR, such as seizures, growth retardation and disrupted sexual development. I used CUL4B-deleted patient-derived cell lines to investigate the impacts of CUL4B loss on mitochondrial function. I have shown that loss of CUL4B is associated with a distinct set of mitochondrial phenotypes, identifying CUL4B-deleted XLMR as a disorder associated with mitochondrial dysfunction. Furthermore, I have uncovered a reciprocal relationship between CUL4B and Cereblon, providing evidence of a potential role for the CUL4-CRBN E3 ligase complex in maintaining mitochondrial function. Deletion or duplication of the 16p11.2 region is associated with macro-/microcephaly respectively. Here, I have evaluated the cellular consequences of 16p11.2 CNV, specifically with regards KCTD13 expression, DNA replication and checkpoint activation. WHS is typically caused by a small hemizygous telomeric deletion of the 4p16.1 region. Haploinsufficiency of 4p16.1 is associated with microcephaly, growth retardation and complex developmental abnormalities. I investigated the impacts of LETM1 copy number change in WHS patient-derived cells. Here, I have shown that copy number change of LETM1 specifically segregates with mitochondrial dysfunction, likely underlying the seizure phenotype exhibited by the large subgroup of WHS patients whose deletions incorporate LETM1 as well as the rarer instances of the reciprocal duplication. In this thesis I use patient-derived cell lines from three Genomic Disorders as a fundamental tool providing new pathomechanistic insight into the clinical presentation of these conditions

A new paradigm for SpeckNets:inspiration from fungal colonies

In this position paper, we propose the development of a new biologically inspired paradigm based on fungal colonies, for the application to pervasive adaptive systems. Fungal colonies have a number of properties that make them an excellent candidate for inspiration for engineered systems. Here we propose the application of such inspiration to a speckled computing platform. We argue that properties from fungal colonies map well to properties and requirements for controlling SpeckNets and suggest that an existing mathematical model of a fungal colony can developed into a new computational paradigm

Polytechnical Institute for the Study of the Expanding field of Radical Urban Life

The Archway Polytechnic was a durational project by artist Ruth Maclennan in collaboration with Anna Hart, AIR. The polytechnic considered non-commercial everyday interactions with the city and with each other through a series of artworks by Maclennan and invited artists

Health-related decision-making in its personal, social and health service contexts: a critical review of relevant findings from seven publications and consideration of their contribution to understandings of decision-making and the wider field of applied health services research

Health-related decision-making, in particular patients’ involvement in decision-making about their treatment and care, has been an important and enduring concern for many practitioners and researchers working in applied health services research and allied fields. This is evidenced by the substantial (and still growing) body of work on ‘shared decision-making’ (SDM). With the aim of advancing understandings of decision-making, and the associated literature, this critical review seeks to situate, present, draw together, and critically consider, relevant findings from work (seven papers) I have first-authored. These papers arose from three applied (qualitative) health services research studies which directly or indirectly explored the experiences of different groups of patients confronted with decisions about their treatment and/or care. I begin my review with a short overview of relevant theoretical and empirical work pre-dating and informing my own research studies and publications, noting some emergent critiques, and highlighting where important gaps in evidence and understanding were said, at the time, to remain. Then, shifting focus to my own work, I introduce the three studies from which the submitted publications arose, detailing their backgrounds, aims, methods and my involvement in each. Next, I reflect on the findings of my submitted papers, noting how individually and collectively they indicate the importance of the context(s) in which health-related decisions are made. Using techniques of qualitative synthesis to identify a series of descriptive and analytic themes, I develop – and evidence – the proposition that health-related decision-making happens in, and is shaped by, its personal, social and health service contexts. This includes detailing the various ways in which different features of context may influence patients’ decision-making. I then consider, critically, how my findings fit with the wider literature. I proceed to argue that, in attending to, and highlighting, the role of context, my papers, synthesis and review provide insights that complement and extend the historic emphasis in SDM scholarship on what goes on within clinical encounters. Reflecting on the focus of more recent SDM literature (publications contemporaneous with or subsequent to work leading to the submitted papers) I note where other authors have similarly gone on to assert the importance of taking a more context-cognisant approach to understanding health-related decision-making. I also consider how other literatures (such as the classic literature of medical sociology and more recent work in psychology) support and might usefully inform this and future thinking about health-related decision-making. Moving the review towards a close, I offer an assessment of the strengths and limitations of my published work and, moreover, of my synthesis and review. I finish by reflecting upon the methodological and other learning I have accrued over the course of undertaking the contributing studies, including preparing the submitted publications, and from the process of producing this critical review

Youth Resilience to Drought:Learning from a Group of South African Adolescents

Exposure to drought is on the increase, also in sub-Saharan Africa. Even so, little attention has been paid to what supports youth resilience to the stressors associated with drought. In response, this article reports a secondary analysis of qualitative data generated in a phenomenological study with 25 South African adolescents (average age 15.6; majority Sepedi-speaking) from a drought-impacted and structurally disadvantaged community. The thematic findings show the importance of personal, relational, and structural resources that fit with youths’ sociocultural context. Essentially, proactive collaboration between adolescents and their social ecologies is necessary to co-advance socially just responses to the challenges associated with drought.UK Natural Environment Research Councilhttp://www.mdpi.com/journal/ijerphpm2021Educational Psycholog

CLINICIAN-RESEARCHERS AND CUSTODIANS OF SCARCE RESOURCES: A QUALITATIVE STUDY OF HEALTH PROFESSIONALS’ VIEWS ON BARRIERS TO TEENAGERS AND YOUNG ADULTS’ INVOLVEMENT IN CANCER TRIALS

Background: Equipoise and role conflict have been previously identified as important factors in professionals’ engagement with trials, inducing behaviours which can impact on recruitment. We explored these phenomena as potential explanations for the low levels of involvement of teenagers and young adults (TYA) with cancer in clinical trials in oncology. Methods: We report findings from interviews with 30 purposively sampled direct care professionals, involved in delivering cancer care and/or facilitating clinical trials in Scotland. We undertook qualitative descriptive analysis, focussed on identifying key issues and themes. Results: Interviewees largely identified as clinician-researchers and portrayed oncology as a specialty in which research was integral to care. They saw their primary responsibility as ensuring patients received the best treatment, but asserted that, in general, trials provided a vehicle for optimal care. Role conflict in its traditional form was little in evidence; however, other tensions were manifest. Professionals found the significant time costs of delivering trials difficult to reconcile with the increasing pressures on clinical services. They felt a responsibility to make prudent choices about which trials to engage with. Guided by utilitarian principles, these choices were oriented towards benefiting the largest number of patients. This favoured trials in high volume diseases; as TYA tend to have rarer forms of cancer, professionals’ support for – and TYA’s access to – relevant trials was by default more limited. Conclusions: Neither lack of individual equipoise nor experiences of traditional forms of role conflict accounted for low levels of involvement of TYA with cancer in clinical trials. However, prominent tensions around the management of scarce resources provided an alternative explanation for TYA’s limited access to cancer trials. The prevailing approach to decision-making about whether and which trials to support was recognised as contributing to inequalities in access and care. Professionals’ choices, however, were made in the context of scarcity, and structured by incentives and sanctions understood by them as signalling governmental priorities. A franker discussion of the extent and distribution of the costs and benefits of trials work is needed, for change to be achieved.</p