Regulation of Gap Junction Internalization and Degradation

Gap junctions (GJs) are channels that traverse the plasma membrane of neighboring cells and provide direct intercellular communication (GJIC) in multi-cellular organisms. GJIC is a prerequisite for coordinated development, differentiation and tissue function; disruption of which can cause disease. Post-translational modification is essential for regulation of GJ trafficking, gating, internalization, and degradation. In the dissertation work presented here (1) I show the role of clathrin-mediated endocytosis (CME) on acute Cx43 (a GJ protein) GJ internalization. I treated primary pulmonary artery endothelial cells (pPAECs) with CME inhibitors after vascular endothelial growth factor (VEGF) stimulation and analyzed internalization patterns of GJs. VEGF treatment of endothelial cells leads to rapid internalization of GJs. I found that pharmacological inhibition of CME during VEGF treatment leads to GJs remaining in the plasma membrane, suggesting CME as the acute GJ internalization pathway. (2) I elucidated the ubiquitin-mediated regulatory mechanism of Cx43 GJ internalization and degradation. I utilized mutational analysis to identify two lysine residues that are K63-polyubiquitinated in the Cx43 C-terminal domain that are necessary for GJ internalization. Mutating these residues results in loss of K63-polyubiquitination, accumulation of GJs in the plasma membrane, and longer Cx43 protein half-life. My analysis also revealed a link between phosphorylations known to decrease GJIC (pS368, pS279/pS282, and pS255) and K63-polyubiquitination that regulates GJ internalization. (3) Additionally, I used knockdowns of key autophagy proteins and immuno-colocalization analysis to identify autophagy as the pathway that degrades internalized Cx43 GJs. I found that Beclin-1 and p62/SQSTM1 knockdowns leads to increased AGJs and decreased colocalization of Cx43 AGJs with the autophagic membrane protein, LC3. p62 (a ubiquitin binding protein) colocalization of Cx43 at GJs and AGJs strongly suggests a link between ubiquitination of GJs and autophagic degradation. Additionally, I found that p62 and LC3 robustly colocalize with Cx43 in endogenous Cx43 expressing PAECs. Work during my Ph.D. contributed to four primary research articles (three published, one submitted), four review articles and one book chapter

Engaging students with assessment and feedback: improving assessment for learning with students as partners

Within the Higher Education sector in the UK, it is acknowledged that the area of ‘Assessment and Feedback’ receives consistently poor levels of satisfaction from students when they complete module level feedback, course level feedback and the National Student Survey (NSS). There is evidence to suggest that this problem is pronounced within Schools of Architecture, particularly with the assessment and feedback of design work. This case study describes reflective practice at Portsmouth School of Architecture, UK, where academics worked in consultation with students to identify the issues. The aim of the project was to evaluate assessment and feedback strategies from across the School resulting in the creation of a new and innovative set of ‘Assessment for Learning’ tools produced with students as partners. These tools include: a refined marking matrix, an improved ‘design review’ and a ‘lexicon’ for marking design projects to enhance understanding and autonomy. This case study also explores how alignment and enhancement of learning through assessment and feedback and the quality of assessment tools has the ability to increase students’ confidence and assessment literacy, their overall satisfaction and levels of autonomy

From JWH-018 to OXIZIDS: Structural evolution of synthetic cannabinoids in the European Union from 2008 to present day

With new synthetic cannabinoids (SCs) appearing on the European drug market every year, early warning systems are key to detect, monitor, and respond to threats posed by them. The European Union Early Warning System (EU EWS) implemented by the European Monitoring Centre for Drugs and Drug Addiction has monitored these substances since their first European detection in 2008. Since then, national and international responses have been implemented, aimed at tackling risks posed by SCs. Throughout this time, new SCs have emerged on the European market containing diverse structural moieties, appearing to be designed in a way that circumvents existing legal controls, contributing to a complex public health scenario. This study provides an inventory of the SCs detected in the EU from 2008 to 2022, describing their structural evolution by analysing separately four structural features: their core, tail, linker, and linked groups. The range of structural changes is analysed considering key milestones, including the year of first report by the European Union Early Warning System to the key legislative changes that have occurred since. The analysis shows that from June 2021 to July 2022, 20 out of 23 newly emerged SCs evade the generic SC legislation introduced in China in May 2021. This supports the hypothesis that the protection of public health benefits from timely information exchange and careful assessment of the risks associated with these substances. Additionally, the introduction of legal responses, albeit an important instrument to reduce the availability of dangerous substances on the market, may also be accompanied by unintended consequences

Shark Fishing in Ghana: What We Ought to Know

The main objective of the study was to assess the abundance and distribution of sharks in Ghana’s coastal area. Samples were collected daily from the three sampling sites from April – to December 2021. The data obtained from this study were assessed for species abundance and composition, ecological indices; species diversity, and exploitation status using PAST and Microsoft Excel. A total of eight species were recorded with Prionace glauca (68%) as the dominant species and Carcharhinus leucas (1%) as the least dominant species. The mean species richness index (SRI) of 0.9 indicated infinite diversity of the species. The mean species evenness index (SEI) of 0.5 showed an evenly distributed species. The mean species dominance index (SDI) of 0.4 implied that the habitat was not dominated by only one or two particular species. The mean Shannon Weiner index (SWI) was less than 3, which is suggestive of pollution and habitat degradation. The exploitation status indicated that the stock of the shark species is in healthy condition. The month with the higher index was October which is possibly the aftermath of the close season and the major upwelling season. Extending the period of the closed season and reducing fishing effort are recommended

Why asthma still kills: The national review of asthma deaths (NRAD)

Advancements in drug treatments, applied research and the development of evidence-based clinical guidelines have contributed to the reduction of deaths from asthma over the past 50 years.Previous confidential enquiries have suggested that avoidable factors play a part in as many as threequarters of cases of asthma death. These studies have often been small, conducted locally and undertaken at a considerable time after death. The National Review of Asthma Deaths (NRAD), reported here, is the first national investigation of asthma deaths in the UK and the largest study worldwide to date. Work on the NRAD was undertaken over a 3-year period and was one element of the Department of Health inEngland’s Respiratory Programme. The primary aim of the NRAD was to understand the circumstances surrounding asthma deaths in the UK in order to identify avoidable factors and make recommendations to improve care and reduce the number of deaths.Asthma deaths occurring between February 2012 and January 2013 were identified through the Office for National Statistics (ONS) for England and Wales, the Northern Ireland Statistics and Research Agency(NISRA) and the National Records of Scotland (NRS). Extensive information about each death was sought from multiple sources, including primary, secondary and tertiary care, as well as ambulance, paramedic and out-of-hours care providers. 374 local coordinators were appointed in 297 hospitals across the NHS to collect and submit information to the project team, and 174 expert clinical assessors were recruited from primary, secondary and tertiary care throughout the UK to join expert panels that reviewed data. Each assessor participated in one or more expert panels, during which all information gathered on each death, including post-mortem reports, was reviewed by two assessors in detail, and this was followed by discussion and a consensus agreement of avoidable factors and recommendations by the whole panel.Data were available for analysis on 195 people who were thought to have died from asthma during the review period and the key findings relate to this group. Denominators vary according to where data were missing

Viva Survivors – the effect of peer-mentoring on pre-viva anxiety in early-years students

Viva voce exams are used in many disciplines as a test of students’ knowledge and skills. Whilst acknowledged as a useful form of assessment, vivas commonly lead to a great deal of anxiety for students. This anxiety is also apparent for vivas in phonetics, where students must produce and recognise sounds drawn from across the world’s languages, and pervious work has shown that viewing a video of a mock-viva does not reduce this anxiety. To address anxiety prior to phonetics vivas, 63 students, across three cohorts, engaged in a brief, isolated, peer-mentoring session with previously successful students (‘viva survivors’). Anxiety about the viva was measured before and after the mentoring experience, using the short form of the State Trait Anxiety Inventory. There was a significant reduction in anxiety after mentoring, and a significant correlation between anxiety before mentoring and the decrease in anxiety after mentoring. Short-term mentoring is posited as a time- and cost-effective method to decrease viva-related anxiety across disciplines

Photochemical fingerprinting is a sensitive probe for the detection of synthetic cannabinoid receptor agonists; towards robust point-of-care detection

With synthetic cannabinoid receptor agonist (SCRA) use still prevalent across Europe and structurally advanced generations emerging, it is imperative that drug detection methods advance in parallel. SCRAs are a chemically diverse and evolving group, which makes rapid detection challenging. We have previously shown that fluorescence spectral fingerprinting (FSF) has the potential to provide rapid assessment of SCRA presence directly from street material with minimal processing and in saliva. Enhancing the sensitivity and discriminatory ability of this approach has high potential to accelerate the delivery of a point-of-care technology that can be used confidently by a range of stakeholders, from medical to prison staff. We demonstrate that a range of structurally distinct SCRAs are photochemically active and give rise to distinct FSFs after irradiation. To explore this in detail, we have synthesized a model series of compounds which mimic specific structural features of AM-694. Our data show that FSFs are sensitive to chemically conservative changes, with evidence that this relates to shifts in the electronic structure and cross-conjugation. Crucially, we find that the photochemical degradation rate is sensitive to individual structures and gives rise to a specific major product, the mechanism and identification of which we elucidate through density-functional theory (DFT) and time-dependent DFT. We test the potential of our hybrid “photochemical fingerprinting” approach to discriminate SCRAs by demonstrating SCRA detection from a simulated smoking apparatus in saliva. Our study shows the potential of tracking photochemical reactivity via FSFs for enhanced discrimination of SCRAs, with successful integration into a portable device

Photochemical Fingerprinting Is a Sensitive Probe for the Detection of Synthetic Cannabinoid Receptor Agonists; Toward Robust Point-of-Care Detection

With synthetic cannabinoid receptor agonist (SCRA) use still prevalent across Europe and structurally advanced generations emerging, it is imperative that drug detection methods advance in parallel. SCRAs are a chemically diverse and evolving group, which makes rapid detection challenging. We have previously shown that fluorescence spectral fingerprinting (FSF) has the potential to provide rapid assessment of SCRA presence directly from street material with minimal processing and in saliva. Enhancing the sensitivity and discriminatory ability of this approach has high potential to accelerate the delivery of a point-of-care technology that can be used confidently by a range of stakeholders, from medical to prison staff. We demonstrate that a range of structurally distinct SCRAs are photochemically active and give rise to distinct FSFs after irradiation. To explore this in detail, we have synthesized a model series of compounds which mimic specific structural features of AM-694. Our data show that FSFs are sensitive to chemically conservative changes, with evidence that this relates to shifts in the electronic structure and cross-conjugation. Crucially, we find that the photochemical degradation rate is sensitive to individual structures and gives rise to a specific major product, the mechanism and identification of which we elucidate through density-functional theory (DFT) and time-dependent DFT. We test the potential of our hybrid "photochemical fingerprinting"approach to discriminate SCRAs by demonstrating SCRA detection from a simulated smoking apparatus in saliva. Our study shows the potential of tracking photochemical reactivity via FSFs for enhanced discrimination of SCRAs, with successful integration into a portable device.</p

Radio continuum observations of Class I protostellar disks in Taurus: constraining the greybody tail at centimetre wavelengths

We present deep 1.8 cm (16 GHz) radio continuum imaging of seven young stellar objects in the Taurus molecular cloud. These objects have previously been extensively studied in the sub-mm to NIR range and their SEDs modelled to provide reliable physical and geometrical parametres.We use this new data to constrain the properties of the long-wavelength tail of the greybody spectrum, which is expected to be dominated by emission from large dust grains in the protostellar disk. We find spectra consistent with the opacity indices expected for such a population, with an average opacity index of beta = 0.26+/-0.22 indicating grain growth within the disks. We use spectra fitted jointly to radio and sub-mm data to separate the contributions from thermal dust and radio emission at 1.8 cm and derive disk masses directly from the cm-wave dust contribution. We find that disk masses derived from these flux densities under assumptions consistent with the literature are systematically higher than those calculated from sub-mm data, and meet the criteria for giant planet formation in a number of cases.Comment: submitted MNRA