Investigating the role of centrosome amplification in extracellular vesicle secretion in pancreatic ductal adenocarcinoma

PhD thesisPancreatic Ductal Adenocarcinoma (PDAC) is characterised by a dense desmoplastic reaction that is attributed to the activation of pancreatic stellate cells (PSCs) in the stroma. This alteration of the tumour microenvironment is thought to contribute to PDAC aggressiveness and resistance to therapy. Recent studies have shown that exosomes (a subgroup of secreted extracellular vesicles) secreted by cancer cells facilitate cross talk between tumour cells and the microenvironment. However, the mechanisms that lead to the secretion of these vesicles remains elusive. Here, we report for the first time, a novel role for centrosome amplification, a common feature of human tumours, in the secretion of small extracellular vesicles (sEVs). We show that centrosome amplification significantly correlates with and is sufficient to induce the elevated secretion of sEVs in PDAC cell lines. Furthermore, we demonstrate that oxidative stress in cells with supernumerary centrosomes is the driving force behind this altered sEV secretion. An analysis of centrosome amplification-associated increases in cellular reactive oxygen species (ROS) demonstrated an impaired lysosome function and the prevention of MVB/lysosome fusion events. The results indicate that centrosome amplification induced ROS induces sEV secretion by preventing MVB degradation by the lysosome, shifting their fate to fusion with the plasma membrane and subsequent secretion of their intraluminal vesicles (ILVs) as exosomes. To understand if exosomes secreted from cells with amplified centrosomes could impact the tumour microenvironment, we subsequently investigated the role of these sEVs on the activation of PSCs, as measured by the formation of fibres containing alpha-smooth muscle actin (α-SMA). We found that sEVs isolated from cells with supernumerary centrosomes elicit significantly stronger activation of PSCs compared to sEVs isolated from cells with a normal centrosome number, suggesting a difference in their biological cargo. SILAC based-proteomic analysis revealed the gain or loss of 6 EV protein in sEVs isolated from cells upon the induction of centrosome amplification, that may have a role in the activation of PSCs. We hypothesise, that further understanding the role of centrosome amplification in sEV-mediated PSC activation may help us to identify innovative ways to block PSC activation and prevent the progression of PDAC, which could have major clinical implications for patients with this devastating disease

Environmental Enrichment in the ISS Rodent Habitat Hardware System

Responses of animals exposed to microgravity during in-space experiments were reviewed from NASAs and ESA available video recording archives. These documented observation of animal behavior, as well as the range and level of activities during spaceflight, clearly demonstrate that weightlessness conditions and the extreme novelty of the surroundings exert damaging psychological stresses on the inhabitants. In response to a recognized need for in-flight animals to improve their wellbeing we propose to reduce such stresses by shaping and interrelating structures and surroundings to satisfying vital physiological needs of inhabitants. Rodent Habitat Hardware System based housing facility incorporating a tubing network system, to maintain and monitor rodent health environment with advanced accessories has been proposed. The new tubing configuration was found suitable for further incorporation of innovative monitoring technology and accessories in the animal holding habitat unit which allow to monitor in real-time the most valuable health related biological parameter under weightlessness environment of spaceflight

Multi-gene panel testing for hereditary cancer predisposition in unsolved high-risk breast and ovarian cancer patients.

PurposeMany women with an elevated risk of hereditary breast and ovarian cancer have previously tested negative for pathogenic mutations in BRCA1 and BRCA2. Among them, a subset has hereditary susceptibility to cancer and requires further testing. We sought to identify specific groups who remain at high risk and evaluate whether they should be offered multi-gene panel testing.MethodsWe tested 300 women on a multi-gene panel who were previously enrolled in a long-term study at UCSF. As part of their long-term care, all previously tested negative for mutations in BRCA1 and BRCA2 either by limited or comprehensive sequencing. Additionally, they met one of the following criteria: (i) personal history of bilateral breast cancer, (ii) personal history of breast cancer and a first or second degree relative with ovarian cancer, and (iii) personal history of ovarian, fallopian tube, or peritoneal carcinoma.ResultsAcross the three groups, 26 women (9%) had a total of 28 pathogenic mutations associated with hereditary cancer susceptibility, and 23 women (8%) had mutations in genes other than BRCA1 and BRCA2. Ashkenazi Jewish and Hispanic women had elevated pathogenic mutation rates. In addition, two women harbored pathogenic mutations in more than one hereditary predisposition gene.ConclusionsAmong women at high risk of breast and ovarian cancer who have previously tested negative for pathogenic BRCA1 and BRCA2 mutations, we identified three groups of women who should be considered for subsequent multi-gene panel testing. The identification of women with multiple pathogenic mutations has important implications for family testing

Environmental Enrichment in the ISS Rodent Habitat Hardware System

Responses of animals exposed to microgravity during in-space experiments were observed via available video recording stored in the NASA Ames Life Sciences Data Archive. These documented observations of animal behavior, as well as the range and level of activities during spaceflight, demonstrate that weightlessness conditions and the extreme novelty of the surroundings may exert damaging psychological stresses on the inhabitants. In response to a recognized need for in-flight animals to improve their wellbeing we propose to reduce such stresses by shaping and interrelating structures and surroundings to satisfying vital physiological needs of inhabitants. A Rodent Habitat Hardware System (RHHS) based housing facility incorporating a tubing network system, to maintain and monitor rodent health environment with advanced accessories has been proposed. Placing mice in a tubing-configured environment creates more natural space-restricted nesting environment for rodents, thereby facilitating a more comfortable transition to living in microgravity. A sectional tubing structure of the RHHS environment will be more beneficial under microgravity conditions than the provision of a larger space area that is currently utilized. The new tubing configuration was found suitable for further incorporation of innovative monitoring technology and accessories in the animal holding habitat unit which allow to monitor in real-time monitoring of valuable health related biological parameters under weightlessness environment of spaceflight

Undoing elimination: Modelling Australia's way out of the COVID-19 pandemic

In the middle of 2020, with its borders tightly closed to the rest of the world, Australia almost achieved the local elimination of COVID-19 and subsequently maintained 'COVID-zero' in most parts of the country for the following year. Australia has since faced the relatively unique challenge of deliberately 'undoing' these achievements by progressively easing restrictions and reopening. Exploring the role of mathematical modelling in navigating a course through the pandemic through qualitative interviews with modellers and others working closely with modelling, we argue that each of these two significant phases of Australia's COVID-19 experience can be understood as distinct forms of 'model society'. This refers at once to the society enacted through the governance of risk, and to the visions of societal outcomes - whether to be sought or to be avoided - that are offered up by models. Each of the two model societies came about through a reflexive engagement with risk facilitated by models, and the iterative relationship between the representations of society enacted within models and the possibilities that these representations generate in the material world beyond them

In search of a 'good number': knowledge controversy and population estimates in the endgame of hepatitis C elimination.

We explore the contentious life of a metric used to assess a country's progress in relation to global disease elimination targets. Our topic is hepatitis C elimination, and our context is Australia. A fundamental metric in the calculation of progress toward hepatitis C elimination targets, as set by the WHO, is the population prevalence of people living with hepatitis C. In Australia, this modelled estimate has generated some controversy, largely through its repeated downsizing as an effect of calculus. The 2015 baseline population estimate in Australia, from which measures of current elimination progress are assessed, has reduced, over time, by around 30%. Informed by a social study of science approach, we used qualitative interviews with 32 experts to explore the knowledge controversy. The controversy is narrated through the core concerns of 'scale' and 'care', with narratives aligning differently to imaginaries of 'science' and 'community'. We trace how constitutions of 'estimate' and 'number' circulate in relation to 'population' and 'people', and as affective values. We show how enactments of estimates and numbers materialise hepatitis elimination in different ways, with policy implications. The event of the knowledge controversy opens up the social and political life of enumerations-for science and community-inviting deliberation on how to make 'good numbers' in the race to eliminate hepatitis C

Transferrin-modified chitosan nanoparticles for targeted nose-to-brain delivery of proteins

Nose-to-brain delivery presents a promising alternative route compared to classical blood-brain barrier passage, especially for the delivery of high molecular weight drugs. In general, macromolecules are rapidly degraded in physiological environment. Therefore, nanoparticulate systems can be used to protect biomolecules from premature degradation. Furthermore, targeting ligands on the surface of nanoparticles are able to improve bioavailability by enhancing cellular uptake due to specific binding and longer residence time. In this work, transferrin-decorated chitosan nanoparticles are used to evaluate the passage of a model protein through the nasal epithelial barrier in vitro. It was demonstrated that strain-promoted azide-alkyne cycloaddition reaction can be utilized to attach a functional group to both transferrin and chitosan enabling a rapid covalent surface-conjugation under mild reaction conditions after chitosan nanoparticle preparation. The intactness of transferrin and its binding efficiency were confirmed via SDS-PAGE and SPR measurements. Resulting transferrin-decorated nanoparticles exhibited a size of about 110-150 nm with a positive surface potential. Nanoparticles with the highest amount of surface bound targeting ligand also displayed the highest cellular uptake into a human nasal epithelial cell line (RPMI 2650). In an air-liquid interface co-culture model with glioblastoma cells (U87), transferrin-decorated nanoparticles showed a faster passage through the epithelial cell layer as well as increased cellular uptake into glioblastoma cells. These findings demonstrate the beneficial characteristics of a specific targeting ligand. With this chemical and technological formulation concept, a variety of targeting ligands can be attached to the surface after nanoparticle formation while maintaining cargo integrity

Supporting primary teachers to address loss and death in the classroom: a case study of an interdisciplinary, creative pedagogical intervention using education, children’s literature, architecture/design and the arts

Schools inevitably face difficult discussions with children about loss and death, irrespective of whether it is factored into formal teaching. A range of societal factors in many countries, not least a lack of training, compound to leave many teachers unprepared to manage these sensitive issues. Whilst resources and guidance are widely available, there is a gap in generating pedagogical tools which are underpinned by solid theoretical grounding. This article addresses that gap, reporting on a case study of staff (n = 12) working in an infant school in England, where an interdisciplinary project was implemented to explore whether creative pedagogies could empower staff to address these difficult topics with young children. Contextualised within thanatology, (the study of death and its social practices), an intervention was designed and co-created with teachers synthesising education, literature, architecture/design and the arts. The paper details its theoretical and practical development, and the participants’ responses to it. The article concludes that despite initial ambivalence among some school staff about using creative pedagogies, responses at the end of the study were positive and confidence had increased. The need for theoretically sound approaches is supported as part of the long-advocated calls for mandatory death/life education, although the potential dissonance between academics and practitioners’ views about the value of theory is acknowledged. However, the paper argues that co-created interdisciplinary projects such as this can help bridge theory-practice divides. Furthermore, they can offer practical spaces for reflection and confidence building in time-effective ways to benefit teachers and children

Tuning the antimicrobial activity of low molecular weight hydrogels using dopamine autoxidation

We present a method to trigger the formation of dipeptide-based hydrogels by the simple addition of dopamine. Dopamine undergoes oxidation in air, reducing the pH to induce gelation. The production of polydopamine and release of reactive oxygen species such as hydrogen peroxide confers antimicrobial activity. Gel stiffness can be controlled by modulating the initial starting pH of the gelator solution. We can use this method to tune the antimicrobial activity of the gels, with gels that are less stiff demonstrating increased bactericidal efficacy against Gram-positive bacteria