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Revolting hillbillies: exploring cracks in the neoliberal order through the prism of Appalachian activism.
No full textThis thesis studies Appalachian activism in the Trump era, arguing that it constitutes an instructive and encouraging counterpoint to contemporaneous mainstream, metropolitan anti-Trump dissent. The thesis considers the Appalachian region’s media-assigned role as “Trump Country” before complicating this narrative through detailed case studies of progressive and leftist organising in the region that was both more radical and class conscious than the anti-Trump liberalism that dominated major urban centres elsewhere in the United States. The twenty-first-century forms of Appalachian organising and activism studied here—the teachers’ strike of 2018 and the mobilisations for LGBTQ and women’s rights—are not marginal or anomalous but should be seen as clear continuations of a lineage of radicalism in the region. Drawing on archival material such as unpublished memoirs, oral histories and movement ephemera, I show how the region’s millennial activists are informed and bolstered by a distinctive local cultural and political heritage. This heritage includes strong anti-capitalist elements and an innate affinity with the language of unionism and labour, despite a steep decline in industrial action and organising beginning in the 1990s. It is this class-conscious legacy that differentiates Trump-era Appalachian activists from their liberal metropolitan counterparts, whose commitment to an elite-captured version of identity politics reached its apex in the mid to late 2010s. My case studies show that Appalachian activism managed to organise around facets of identity—queer liberation and feminism—whilst maintaining a leftist structural critique of capitalist social relations and conceiving of class “equiprimordially” as both an identity, in an Appalachian context, and as a relationship to power
Targeting DNA damage repair pathways in breast and ovarian cancers
Get PDFBackground
DNA damage could be due to many endogenous and exogenous agents producing DNA lesions which block the transcription process affecting the gene expression, cell growth and survival. The DNA damage response network detects the damage and any flaw in the network including defects in DNA repair systems can lead to cancer. Thus, drugs targeting DNA repair systems relying on this concept are under development with various drug monotherapy or combination therapies. Targeting DNA repair genes of nucleotide excision repair system (XAB2) and Fanconi anaemia repair system (FANCD2 and FANCA) for synthetic lethality is a novel strategy for treating breast and ovarian cancers. Hence, the synthetic lethality concept is an exciting area for future research.
Methods
In this study, XAB2 expression in the breast cancer cohort was investigated. Cisplatin and olaparib sensitivity was evaluated in a panel of gene XAB2, FANCD2, FANCA deficient and proficient breast and ovarian cancer cell lines. XAB2 expression and the clinicopathological outcomes were investigated in the ovarian cancer cohort. The gene knockdown and cisplatin sensitivity were tested in platinum-sensitive and platinum-resistant ovarian and breast cancer cell lines. Functional studies for wild and knockdown genes such as PI FACS-cell cycle and Annexin V-apoptosis assay were performed.
Results
In breast cancer cases, results showed that a low XAB2 nuclear expression was associated with a high tumour grade and poor breast cancer-specific survival (BCSS) in patients. In invasive breast cancer, low XAB2 expression had clinicopathological associations with aggressive forms of breast cancer.
In gene knockdown, cisplatin and olaparib were synthetically lethal in gene-deficient breast cancer cell lines. In ovarian cancers, XAB2 was significantly overexpressed in serous adenocarcinoma. The low XAB2 expression was significantly linked to good overall survival. The cisplatin was selectively toxic in gene-deficient platinum-sensitive ovarian cancer cell lines. The drug cytotoxicity was associated with double-strand breaks (DSBs) formation, cell cycle arrest and apoptosis in gene-deficient cells. The gene expression was a predictor of platinum sensitivity in ovarian cancer patients. The gene knockdown not only increased platinum sensitivity but also reduced invasion and migration in breast and ovarian cancer cell lines. The cisplatin drug was selectively toxic in gene-deficient breast and ovarian cancer cells.
Conclusion
Targeting DNA repair genes was an attractive synthetic lethality strategy and the chemoprevention in gene-deficient breast cancers. In ovarian cancers, gene deficiency was a biomarker for drug inhibitor sensitivity. It provided alternative synthetic lethality approaches for inhibitors in clinics. The gene depletion can re-sensitize ovarian cancer patients to platinum. Therefore, studying the expression of DNA repair proteins assisted in new drug therapies development and opened alternative treatment options
Investigating the role of ascorbic acid and its transporters in medulloblastoma
Get PDFMedulloblastoma is a highly malignant paediatric brain tumour, accounting for 20% of cases. Current treatment achieves a 70% 5-year-survival rate but can result in long-term neurological side effects. Treatment is further complicated by the disease’s heterogeneity, comprising four molecular subgroups: Group 3, Group 4, SHH and WNT. Novel therapeutic strategies are urgently needed to improve outcomes while addressing both treatment toxicity and the diverse molecular landscape. Adjuvant therapies that enhance treatment efficacy could reduce therapeutic doses and minimise side effects. A unique feature of the SHH subgroup is a collagen-rich extracellular matrix (ECM) shell, which is associated with improved survival, possibly by limiting tumour growth and invasiveness. This makes it a potential target for therapeutic manipulation in SHH tumours. Ascorbic acid (Vitamin C) plays a critical role as a co-factor in collagen production and could influence the collagen-rich ECM shell encapsulating SHH tumours. In addition, at high doses, ascorbic acid generates reactive oxygen species (ROS) via the Fenton reaction, leading to enhanced oxidative stress. Therefore, high-dose ascorbic acid could be a compelling candidate as an adjuvant treatment of SHH tumours, by promoting a tumour-restrictive microenvironment and increasing oxidative stress to enhance current treatment efficacy.
This thesis investigated the expression of ascorbic acid transporter genes in medulloblastoma patients, with a focus on SHH tumours. Analysis identified GLUT10 and SVCT2 as potential players in medulloblastoma biology, with differential expression linked to survival outcomes in SHH patients. These transporters were also significantly correlated with genes involved in antioxidant activity and collagen metabolism, including those contributing to the ECM shell in SHH tumours. To explore the functional effects of ascorbic acid, SHH spheroids, a 3D cell culture model that mimics tumour architecture, were treated with increasing concentrations of ascorbic acid. Physiological concentrations promoted spheroid growth, while high-dose ascorbic acid, when combined with the chemotherapeutic agent cisplatin, reduced spheroid size, suggesting an enhancement of cisplatin’s anti-proliferative effects. To investigate the underlying mechanism, acute ROS production in spheroids was measured following treatment. Pre-treatment with physiological ascorbic acid protected SHH spheroids from cisplatin-induced oxidative stress, whereas high-dose ascorbic acid exacerbated ROS production in pre-stressed spheroids. These findings suggest that high-dose ascorbic acid could enhance ROS generation achieved by a chemotherapeutic alone. Future research should further explore the potential synergy between ascorbic acid and cisplatin, as well as determine whether ascorbic acid-mediated spheroid growth is due to increased collagen deposition. This would help establish high-dose ascorbic acid as a potential adjuvant therapy in SHH medulloblastoma
Assessing the influence of fibre type and biofilms on the ingestion and retention of microfibres by freshwater invertebrates
Get PDFMicrofibre pollution is an escalating environmental issue, with natural fibres found in greater abundance than their synthetic counterparts in the environment. This study examines the ingestion and retention of microfibres by two freshwater macroinvertebrates, Gammarus pulex and Corbicula fluminea, under varying conditions of fibre type, biofilm presence, water turbidity, and exposure time. Ecotoxicity tests revealed significant ingestion of both polyester and cotton microfibres, with species-specific retention and ingestion patterns that differed with fibre type. For G. pulex, polyester fibres exhibited longer retention, while cotton fibres were ingested in larger quantities. The presence of biofilms enhanced fibre ingestion, indicating a key role in retention. Notably, G. pulex continued ingesting microfibres over one- and two-week exposure periods, suggesting no learning avoidance of microfibre consumption. C. fluminea ingested consistently low number of microfibres throughout all experiments, suggesting that selective feeding mechanism of suspension feeding bivalves may limit the risk of microfibre ingestion. These findings underscore the environmental risks posed by both natural and synthetic microfibres and highlight the need for further research, particularly on natural fibres, to assess their ecological impacts at environmentally relevant concentrations
Understanding the ZEB1 interactome during lymphangiogenesis
No full textEndothelial cells (ECs) constitute the crucial lining of blood and lymphatic vessels, controlling vascular integrity and function. During quiescence, ECs regulate junctional integrity and barrier stability. Recently, ZEB1 was hypothesised to contribute to endothelial quiescence. Integrating mass spectrometric and transcriptomic analyses in lymphatic ECs uncovered that ZEB1 contributes to junctional reorganization. Silencing ZEB1 significantly reduced VE-cadherin phosphorylation at Y731 and Y685 (59%±5.39, 71%±15,p<0.05, respectively), consequently compromising barrier integrity. Silencing ZEB1 downregulated YES and YAP1 suggesting that altered VE-cadherin signalling was a consequence of ZEB1 mediated-YES/YAP1 signalling. In LECs, silencing YAP1 induced cytoskeletal remodelling, and functionally compromised EC barrier phenocopying ZEB1 loss. Consistently, YAP1 loss showed dephosphorylation of Y685 and Y731
The impact of step count reduction and remobilisation on body composition, blood glucose disposal and muscle protein turnover in healthy, active young and older volunteers
Get PDFEstablishment of horse blood-brain barrier model for the study of drug delivery to the brain
Get PDFThe lack of a benchmark standard in vitro model of blood-brain barrier (BBB) is the principal reason behind high failure rates of central nervous system drugs in clinical trials during their development phase. In the last few decades, in vitro models of BBB have been developed using brain endothelial cells from various species like rat, mice, pig, and cattle However, no in vitro BBB model till date has been developed using horse brain endothelial cells. Hence, the main aim of this study was to establish an in vitro primary horse brain endothelial cell (HBEC) transwell model of BBB for determining the how much drugs are transported and handled by the BBB by measuring various pharmacokinetic parameters.
Phylogenetic analysis of multi-drug efflux molecules, evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model. Species having only 1:1 orthologue genes to these multi-drug efflux molecules were included. This study revealed that these molecules are phylogenetically closest to similar molecules of donkey and there the horse was divergent from other species.
The next step established and standardised a method to isolate HBECs from horse brains. Different conditions were optimised to ensure that near-pure population of HBECs were being cultured. For this, addition of 4µg/ml puromycin for first 3 days of culture yielded the highest purity. These HBECs were then characterised to confirm retention of BBB phenotype by using PCR, immunofluorescence and Western blot for various cell type biomarkers and key protein. It was determined that these HBECs had similar key features to the BBB. In addition, the application of scanning electron microscopy brought to light, for the first time, the detailed structure of cell-cell junctions in HBECs. Further characterisation revealed that the presence of astrocyte conditioned medium (ACM) and puromycin affected the expression of multi-drug efflux transporter protein, P-Glycoprotein (P-gp). Furthermore, presence of both ACM and puromycin significantly enhanced P-gp expression.
Transwell model of horse BBB was standardised for drug transport studies by culturing HBECs in presence of transport medium (in essence control) or ACM or astrocyte co-culture or in a combination of transport medium and ACM. The transwell model with ACM performed most optimally in terms of highest transendothelial electrical resistance assay (TEER) and FITC-dextran permeability assay. Drug transport studies were undertaken on this transwell model of the horse BBB. Eight drugs were selected which were substrate for three important multi-drug efflux molecules. The results of drug studies using the HBEC Transwell model did reveal that paracellular movement of drugs was likely to the main transport process taking place for several drug. This could be suggestive of leaky cell-cell junctions. This finding is also corroborated by low-to-moderate TEER values observed in these studies.
Therefore, the project has established and standardised a method for isolation and culture of primary HBECs. Also, for the first time this project has established methodology of novel transwell model of Horse BBB. Nevertheless, additional improvements will be required to establish this model as a candidate for use in drug development for central nervous system
Genomic exploration of Cepaea species: from phylogeography to phenotypic variation
No full textThe land snail genus Cepaea has long served as a model system for understanding the evolutionary forces shaping phenotypic diversity. This thesis builds on that rich history by integrating genomic, mitochondrial, and phenotypic analyses to uncover new insights into the evolutionary history, genetic architecture, and phenotypic variation of Cepaea hortensis and Cepaea nemoralis.
Leveraging whole-genome sequencing, I reconstructed the phylogeography of C. hortensis, revealing that North American populations cluster with Icelandic populations, indicating a complex historical connection rather than direct descent from a single closely related European population. Further analysis uncovered a geographically isolated North American lineage shaped by historical bottlenecks and genetic drift.
In parallel, I assembled, annotated and then explored the first complete mitochondrial genome of C. hortensis, highlighting significant intraspecific variation, particularly in tRNA-Thr and COX3 copy numbers. These results suggest that heteroplasmy may play a key role in mitochondrial genome evolution, advancing our understanding of gastropod mitochondrial biology and adaptation.
Focusing on C. nemoralis, I investigated the genetic basis of shell colour polymorphism, emphasizing the role of supergenes. My findings show that continuous variation in shell colour is primarily driven by variation at the supergene locus, with additional contributions from unlinked loci. These findings highlight the importance of supergenes and unlinked loci in generating phenotypic diversity.
The thesis makes significant contributions to understanding evolutionary processes in the Cepaea genus, offering insights into adaptation, speciation, and the role of genetic and genomic architecture in phenotypic diversity.
This work lays a foundation for further exploration into the evolutionary genetics of molluscs
Investigation of host innate antiviral mechanisms that block replication of influenza A virus and coronaviruses
No full textInfluenza A virus (IAV) and coronaviruses, including OC43 and SARS-CoV-2 variants, are
RNA respiratory viruses that pose a substantial global health challenge due to their high
transmissibility, potential for severe illness, and risk of co-infections. Despite the
availability of vaccines and antiviral treatments, the continuous emergence of viral
variants and resistance mechanisms underscores the urgent need for innovative host
targeted antiviral therapies, which are less likely to induce viral revertants. Increasing
evidence supports the antiviral activity of thapsigargin (TG), an inhibitor of the
sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump and an inducer of ER stress,
against a variety of viruses, including tick-borne encephalitis virus (TBEV), foot-and-mouth
disease virus (FMDV), and IAV. However, the precise antiviral mechanisms of TG remain
to be fully elucidated.
The aim of this thesis was to investigate the potential antiviral mechanisms of TG against
IAV, OC43, and SARS-CoV-2 variants. The first objective was to assess how TG influences
the replication of these viruses. TG was found to inhibit these viruses at various stages of
their life cycles; with post-translational inhibition observed for IAV, and transcriptional
inhibition for OC43 and SARS-CoV-2 variants. The second objective focused on evaluating
the induction of ER stress-related genes, both under basal conditions and during IAV,
OC43, and SARS-CoV-2 infections. qPCR analysis revealed that TG increased the
expression of ER stress-related genes both basally and during IAV, OC43, and SARS-CoV
2 infections. Furthermore, TG demonstrated extended antiviral activity accompanied with
activation of ER stress response during OC43 and SARS-Cov-2 infections. The third
objective was to examine the activation of the host innate immune response both basally
and during IAV, OC43, SARS-CoV-2 infection using qPCR. TG enhanced early but also
regulated activation of innate immune response. Moreover, in a preliminary study to
evaluate if TG induces paracrine antiviral effect between macrophages and epithelial
cells, supernatants from TG-treated macrophages, when used to prime epithelial cells,
effectively inhibited SARS-CoV-2 infection.
The final objective was to investigate the impact of TG on ER-synthesized IAV proteins—
HA, NA, and M2—in both pig and human cells, and to elucidate TG's antiviral mechanisms
on cellular processes exploited by these IAV proteins, including ubiquitination,
glycosylation, autophagy, and their influence on virus morphology and budding. This
objective was achieved using various techniques such as western blotting,
immunofluorescence, a ubiquitin enrichment kit, a glycoprotein isolation kit, and
transmission electron microscopy (TEM). It was found that TG induces cell-type specific
effects on ER-synthesized IAV HA, NA, and M2 proteins. In human cells (A549), TG reduced
the production of HA, NA, and M2 viral proteins and caused their accumulation in the
perinuclear region compared to controls. In contrast, in pig cells (NPTr), TG did not affect
the production or subcellular localization of HA, NA, and M2 viral proteins.
In A549 cells, TG-induced M2 reduction was accompanied by a reduction in LC3 II levels,
i.e. a decrease in autophagy, and absence of M2 ubiquitination. While, in NPTr cells, TG
did not influence M2 protein production, which exhibited a notable double-band pattern.
This TG-induced double banding of M2 was associated with (1) reduced co-localisation
between M2 and LC3, (2) interference with M2 ubiquitination, (3) reduced M2 levels in
the supernatant, and (4) a reduction in the length of the filamentous virus produced.
Similarly, TG induced a double banded pattern for NP protein, a cytoplasmic-synthetized
IAV protein, in both A549 and NPTr cells. Moreover, HA2 of HA and NP glycosylation was
reduced by TG in NPTr cells. Consequently, in both A549 and NPTr cells, TG disrupted the
assembly of viral proteins into new virions, as evidenced by a reduction in the quantity of
IAV proteins (NA, HA, NP, M1, M2) present in the supernatant. This was accompanied by
a decrease in viral RNA and infectious virus particles released, without affecting virus
budding or causing significant changes to virus morphology.
In conclusion, the findings demonstrate TG’s potential as a broad-spectrum antiviral
against these viruses, acting through multiple mechanisms that target critical host
pathways. TG induces several antiviral mechanisms, thus it offers a promising strategy for
targeting other RNA viruses that similarly exploit these pathways during replication. However, further research is required to explore TG's efficacy as a host-targeted antiviral
across a wider range of RNA viral pathogens and to assess its therapeutic potential in vivo
The development of guidance on mouth care for dysphagia in hospital patients with dementia (GUMS-D)
No full textBackground
The oral health of older adults (people over 65 years of age) often deteriorates when they are admitted to hospital. This is a cause for concern as poor oral health has been linked to an increase in hospital-acquired infections, reduced nutritional intake, longer hospital stays and increased care costs. Patients with dysphagia (swallowing difficulties) who are living with dementia are especially at risk of poor oral health. Patients with dementia are often dependent on hospital staff to help them clean their teeth and mouths, but may have difficulty asking for help. The presence of dysphagia increases the risk of aspiration which may result in chest infections from saliva, food or drink entering the lungs. With poor mouth care, there is also a greater risk of developing mouth ulcers and infections such as oral thrush. All of this can lead to pain, distress and a reduced quality of life.
Currently there is limited research on the best way of providing mouth care for people with swallowing difficulties living with dementia. There are few evidence-based mouth care guidelines and clinicians therefore implement local practices. The research presented in this thesis explored published mouth care evidence and policies, which led to the development of an intervention called Guidance on Mouth care with Swallowing difficulties for hospital patients with Dementia (GUMS-D). This intervention was co-created together with carers and healthcare staff to ensure that it was appropriate, safe and implementable for use with patients in hospital.
Methods
There were three stages of intervention development, broadly focussing on synthesis, validation, and implementation. Research ethics committee approval was received prior to the start of recruitment and data collection for the interviews and the focus groups. The approach was informed by critical realism, and qualitative mixed methods were adopted using the Medical Research Council framework for developing and evaluating complex interventions. Behaviour change techniques and frameworks were used to support data collection and analysis. Causation was explored through proposed generative mechanisms.
During the first stage, a systematic review was conducted to identify existing, evidence-based interventions. This was followed by semi-structured 1:1 qualitative interviews, using a purposive sampling design, to recruit healthcare professionals and carers to explore their experiences of mouth care with this patient group in an acute hospital setting. Findings were analysed using reflexive thematic analysis. A first version of the intervention was developed from these two sets of findings using the template for intervention description and replication (TIDieR) to organise the data and provide subheadings to support synthesis. There was also review and refinement from additional clinical experts.
During the second stage, a scoping review of clinical practice guidelines was conducted. Triangulation and synthesis of this third data set was used for validation of the intervention, which was subsequently modified to produce a second version of the intervention. During the final stage, focus group discussions with stakeholders took place to explore the challenges and facilitators for implementation. A purposive sampling strategy was adopted for recruitment, and thematic analysis was used to analyse the findings. Two versions of the intervention were produced, scientific and plain English versions.
There was patient and public involvement throughout all stages of the study: during the design, analysis and interpretation of empirical findings, and formatting of the final version of the GUMS-D.
Results
The systematic review identified seven studies of mixed design with little consensus about how best to deliver mouth care, and no protocol or intervention specifically developed for this patient group. The papers came from Japan and USA, and had been published between 2008 and 2018. This lack of published evidence supported the development of a mouth care intervention.
A total of 14 participants were recruited for the qualitative interviews, and five main themes were produced from the findings: interacting with patients with dementia, mouth care needs for good oral hygiene, accountability, products and tools for cleaning the mouth, time demands and limitations. Each theme had sub-themes. The first iteration of the GUMS-D intervention was developed based on the interviews and literature. It included components on resources, activities, causation, outcomes and impacts to individuals and organisations.
The scoping review of clinical practice guidelines identified eight guidelines from across the UK, the USA and Canada. Data were extracted and synthesised using a triangulation protocol to facilitate consensus about key components and to modify the programme theory. There was limited information available about how such an intervention would be implemented.
Three focus groups, which included speech and language therapists, nursing and dental staff, and carers, with a total of 12 participants, were completed. Eight main themes relating to implementation were produced: leadership, organisation structure and systems, funding, education and training, assessment, documentation systems, communication, mindset. The findings were used to modify the GUMS-D to produce a final version. The results identified that in order to effect changes in mouth care practice in acute NHS settings, there was a need for changes from top to bottom at both organisation and individual levels, and for investment in resources and training. The data highlighted that changes may be impacted by competing priorities within organisations, as well as economic and political factors.
Conclusion
The GUMS-D intervention was developed using systematic methods underpinned by theory. It includes practical guidance for use by clinicians in acute hospitals and policy recommendations for health care services. The next stage of research would aim to empirically evaluate the feasibility of implementing the GUMS-D in acute hospitals to patients with dysphagia who are living with dementia
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