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Generation of CRISPR engineered prostate cancer cell line models to study androgen receptor signalling in advanced prostate cancer
Get PDFPhD ThesisProstate cancer resistance to AR targeted therapies due to the emergence of AR point mutations and AR splice variants that cannot be targeted by the currently available agents comprise a major clinical challenge.
There is paucity of models that accurately reflect the mechanisms of AR regulation in advanced disease. This highlights the high demand of generating novel disease relevant models. A CRISPR pipeline was developed to generate cell line models which harbour specific point mutations in the LBD of AR as well as stop codons in AR exon 5 which resulted in AR-FL knock-out so that the remaining endogenous AR-Vs could be studied discriminately of interfering AR-FL.
Using a streptavidin-tagged Cas9 in conjugation with a biotinylated donor template resulted in high donor template knock-in efficiencies and yielded (i) an ARW741L CWR22Rv1 cell line derivative and (ii) an AR-FL knock-out cell line derivative called CWR22Rv1-AR-EK (Exon Knock-out). CWR22Rv1-AR-EK cells retained all endogenous AR-Vs following AR gene editing. AR-Vs acted unhindered following AR-FL deletion to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrated that AR-Vs drive expression of a cohort of cell cycle and DNA damage response genes and depletion of AR-Vs sensitised cells to ionising radiation.
To date, elimination of AR-Vs by pharmacological inhibition remains challenging. However, disruption of AR pre-mRNA splicing is nowadays a highly attractive option. A CRISPR-based approach, called CRIME (Cas9-directed Rapid Immunoprecipitation Mass Spectrometry of Endogenous proteins) was developed to isolate and identify the AR-V7-specific spliceosome, in association with a nuclease deficient Cas9 at AR cryptic exon 3. Mass spec-derived hits were screened for their ability to alter AR-V7 mRNA levels in CWR22Rv1 cells. SRSF3 was identified as a potential AR-V7 splicer which promotes cryptic exon 3 skipping in normal prostate. In CRPC, SRSF3 is significantly downregulated and hence cryptic exon 3 inclusion in the mature AR-V7 mRNA transcript is permitted leading to generation of AR-V7
The Inclusion of Refugee and Asylum-Seeking Children: Exploring Educational Professionals’ Perspectives through Meta-Study and Appreciative Inquiry
Get PDFD. App. Ed. Psy. ThesisWith data indicating around 126,720 refugees and 45,244 pending asylum cases in the UK, inclusion of refugee and asylum-seeking children in UK schools is highly important. The Asylum and Immigration Act 1999 has meant refugees and asylum-seekers have been dispersed around the country into the care of local authorities, some with little prior experience supporting their inclusion into communities and schools.
Seeking the perspective of educational professionals, a qualitative synthesis of four research articles was undertaken using a meta-study method. The review of the literature, presented in Chapter one, revealed three key themes related to inclusion: how inclusion is conceptualised, how others are valued, and how the needs of children are conceptualised. The review concludes with a reflective framework built on the premise that conceptualisation of inclusion influences the practice of inclusion.
Arising from the systematic literature review, Chapter three presents the five stage process of an Appreciative Inquiry in which a group of eight professionals from a local authority and two primary schools in the North East of England considered: their previous and current successes in inclusive practice, and their dreams and wishes for the future. Analysed using abbreviated realist grounded theory, findings revealed several factors related to supporting the structural and relational inclusion of refugee and asylum-seeking children in schools: being human, proactive working together/sharing, professional qualities which empower and enable others, and access and opportunities.
Chapter one and three are linked by a bridging document outlining the philosophical and methodological stance underpinning the thesis. The hoped contribution of the thesis is to add to the understanding of factors and conditions that support the inclusion of refugee and asylum-seeking children in UK schools, and to emphasise the implications for policy.
Chapters one and three are written for considered for publication in The Journal of Refugee Studies
An evaluation of a local authority addiction recovery provision : the impact of change on service users
Get PDFDurham County CouncilThe character and focus of drug and alcohol services has seen major changes in the last decade. The period 2005-8 saw a shift away from dominant harm minimisation approaches to those advocating a recovery focus. By 2011 a drive for abstinence was fundamental to service delivery. The commissioning of drug and alcohol services also changed with the implementation of the Health and Social Care Act in 2012; local authorities now have more powers to determine how health services should be commissioned and re-commissioned to provide „population focused‟ health provision. This research investigated how concepts of recovery were implemented within addictions recovery services in one locality in the North East of England and how the changes to service through regular re-commissioning affected the recovery journeys of those attending the services and the staff delivering recovery provision.
Sequential qualitative design was adopted, comprising of a systematic literature review of qualitative evidence of facilitators and barriers to recovery from addiction and semi-structured interviews and thematic analysis of data from service users, service staff, service managers and a service commissioner. Normalisation Process Theory (NPT) was used to frame interviews and interrogate the qualitative data.
Multiple factors facilitate or hamper recovery success, including ability to identify with others, creation of a non-addict identity, access to positive peer support and meaningful activities and avoiding the „cliff edge‟ of treatment services. The use of NPT indicates that changes in service delivery, although inevitable, must consider certain criteria (including supportive routines / staff changes / consultation with service users / service location and layout) to prevent detrimental effects on recovery.
Recovery is a complex and non-linear process that can be impacted by numerous domains. Maintenance of routine, support of bonds with other service users and staff and encouragement to have ownership of their own future are all aspects important in creating a sustainable recovery
The closet, encounters and lived religion : reflections on the experiences of LGBT and British Muslims
Get PDFPhD ThesisThis thesis addresses the paucity of research at the intersections of geographies of
religion, ethnicity and sexuality by exploring the lived experiences of LGBT+ British
Muslims. Discourses concerning British Muslim sexualities all too often frame British
Muslims as antithetical to the celebration and progression of sexual minorities, while
rarely providing the space for them to share their own accounts and practices. Drawing
on in-depth, semi-structured interviews with 19 LGBT+ British Muslims, I use an
intersectional approach informed by black and postcolonial feminism to analyse how
they experience and negotiate what are often framed and judged as oppositional
minority religious, ethnic and sexual identities and communities. I contend that LGBT+
British Muslims understand and respond to the conflicts and relations of power across
different spatial contexts and their scalar relationships in ways that are meaningful to
them without subscribing to linear, teleological constructions. I highlight the discursive,
spatial and embodied contexts that regulate their sexualities and develop
understandings of closet geographies by attending to the multi-scalar, intersectional
and intergenerational relations of LGBT+ British Muslim experiences that disrupt binary
and individualised notions of the closet. By paying attention to how power operates
across different domains and social and political contexts, I show how urban
encounters make difference, aggregate temporalities and condition senses of
belonging and their assertions. Further, I problematise secularist representations of
LGBT+ identities and institutionalised and cultural logics of religious homophobia by
analysing how LGBT+ British Muslims maintain affiliation with Islam through everyday
practices of faith that may not be theologically ordained but nevertheless develop
capacities for developing religious agency and affirmation of their different identities.
By highlighting experiences of LGBT+ British Muslims, I address their extant absence
within geographic research and demonstrate their negotiations of different everyday
contexts that contour exclusion
Novel Catalytic Transformations of Organophosphorus Compounds
Get PDFPhD ThesisThe overall aim of this project was to investigate whether it would be possible to develop methods for the successful synthesis of biaryl monophosphonates by using a variety of cross-coupling reactions enhanced via palladium catalysis in which the phosphonate substituent is present on the nucleophilic component to produce potentially advantageous compounds. Three separate, quite distinct approaches to the synthesis of these nucleophilic components have been attempted in this thesis to find the optimal method : firstly, the synthesis of phosphonate-substituted aryl boronate esters by palladium-catalysed borylation of the corresponding aryl bromide; secondly the conversion of bromoaryl phosphonates into the corresponding organozinc pivalates by metal-halogen exchange; and finally, the synthesis of phosphonate-substituted aryl boronates by iridium-catalysed C-H borylation of the aromatic ring. The potential uses of the resulting biaryl monophosphonates are as precursors to phosphorus-containing ligands, in fine chemical synthesis and as leads in the search for new pharmaceuticals that counteract antibiotic resistance or in treatment of heart conditions.
A short two-step synthetic route was successfully carried out to make the desired diethyl (2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)phosphonate from 1-bromo-2-iodobenzene. In a similar way, the desired naphthyl analogue was synthesised from 1-bromo-2-naphthol following an initial conversion to the corresponding triflate.
Diethyl (2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)phosphonate was successfully used in a range of Suzuki-Miyaura cross-coupling reactions, utilising the designer monophosphine KITPHOS ligands made by the Knight/Doherty group with low catalyst loadings.
The potassium trifluoroborate salt of the ortho-phosphonate phenyl boronate ester was successfully synthesised from diethyl (2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)phosphonate using non-glassware etching conditions. However this method did not work when the same conditions were trialled on the corresponding naphthalene derivative.
Transmetalation of the ortho-bromo phosphonate (used in the synthesis of the ortho-phosphonate phenyl boronate ester) was accomplished with iPrMgCl and the resulting Grignard reagent was subsequently converted into the corresponding pivalate by treatment with Zn(OPiv)2. Negishi cross-coupling of this species with a range of functionalised aryl bromides gave the biaryl phosphonates in moderate yield. Separation of the products from diethyl phenylphosphonate, produced by competing protonation of one of the organometallic intermediates, proved troublesome in some cases.
Regioselective iridium-catalysed C-H borylation of three meta-substituted aryl phosphonates (methyl/methyl ester and trifluoromethyl substituents were used) gave the corresponding pinacol boronate esters in high conversions. Suzuki-Miyaura cross-coupling with a range of aryl bromides gave the corresponding 5-substituted biaryl phosphonates.
All the above methods displayed success in synthesising the desired nucleophilic phosphonate coupling partners and in turn, these coupling partners have been successfully incorporated into the relevant cross coupling reactions, producing desired biaryl monophosphonates in good yield. These biaryl motifs can then be further developed in the synthesis of pharmaceuticals or as precursors to phosphine ligands. In the case of the ortho-biaryls made, their potential use as a ligand for asymmetric catalysis in the fine chemicals industry is an attractive proposition.
An Empirical Analysis of Labour,Education and Health Related Policies inIndonesia
Get PDFPhD ThesisThe thesis investigates the impact of the Indonesian Government’s policies related
to labour, education and health. The thesis consists of three empirical analyses:
first, an examination of the impact of a household conditional cash transfer programme
on female labour participation; secondly, an investigation of the effects
of the nine-year compulsory schooling law enactment on educational and work
outcomes; and finally an examination of the impact of the national programme of
community empowerment on community participation and health facilities utilisation
and health outcomes. The research employs econometric methods including
difference-in-differences, instrumental variable and regression discontinuity design:
it uses data from two large-scale randomised controlled trials (RCT), data from
the household cash transfer programme and community empowerment programme
and Indonesia Family Life Survey Data (IFLS).
The thesis reveals that the household conditional cash transfer programme does
not have an impact on female labour participation. The CCT programme did not
motivate the women to work, and two years after its implementation it had not
discouraged working in the short run. Six years after its implementation there was a
significant reduction in female labour participation using IV estimation. The effects
of nine-year compulsory schooling law were investigated by employing a regression
discontinuity design. The results show that the compulsory schooling law has an
impact on educational attainment in the fraction of people completing at least nine
years of schooling. In terms of work outcomes, the policy has a significant effect
on the likelihood of working in the formal sectors, but no effect on monthly wages.
Lastly, this thesis evaluates the effects of the Generasi, a national programme
of community empowerment, on community participation and health outcomes.
The results from Generasi community empowerment programme suggest that the
programme does not have an impact on the village decision-making process for
community self-help activities. The programme does not have an impact on the
quality of health facilities as measured by the availability of health personnel in a
village. The programme suggests a marginally significant positive effect on mothers’
health knowledge. Despite the effect on mothers’ health knowledge, the programme
does not have an impact on health facilities use for mothers. The empirical results
for children suggest that the programme had significant positive effects on child
visits to the Posyandu, the monthly health clinic, and whether nutritious meals
were served during Posyandu sessions. However, the programme does not suggest
any effect on children health outcomes.Indonesia Endowment Fund for Education
(LPDP) managed by the Indonesian Ministry of Finance
Simulation and Analysis of Stimulus Evoked and Seizure-like Activity in an Acute Rat Neocortical Brain Slice Preparation
Get PDFPh. D. ThesisThis thesis aims to provide tools for the simulation and analysis of acute brain slice
experiments that have recorded spontaneous seizure-like activity and activity evoked
using electric eld stimulation. The Virtual Electrode Recording Tool for Extracellular
Potentials (VERTEX) (Tomsett et al., 2015) is a simulation framework that can act
as a sca old for anatomical and physiological knowledge and can be used to test how
interventions a ect the dynamics observed in the extracellular potentials. We extend
VERTEX so that one can model a greater range of experimental setups, in particular
those that involve electric eld stimulation. We also devise a software pipeline for the
identi cation and analysis of epileptiform neuronal activity, which we apply to in vitro
recordings from acute neocortical slices from a chronic model of epilepsy in rat.
In Chapter 1 we look at the intersection of electric eld stimulation, synaptic plasticity
and epilepsy.
In Chapter 2 we look at the implementation of electric eld stimulation that we
have added to the VERTEX simulator. We show that our simulation compares well
with simulations using detailed neuron models, and with previously published in vitro
data. We also describe our implementations of short term plasticity and spike-timing
dependent plasticity.
In Chapter 3 we describe some example simulations of focal electric eld stimulation
in neocortex, investigating a single pulse of stimulation, a paired pulse of stimulation
and the role of short term plasticity in the response. We also use theta burst stimulation
to provoke a potentiation of the response when we apply spike-timing dependent
plasticity to the synapses of the network.
We then look to the experimental context of our framework. Chapter 4 describes
an analysis tool for identifying and evaluating epileptiform activity recorded in vitro
and outlines a method devised to measure the abruptness of seizure build up.
Chapter 5 uses this analysis pipeline to analyse seizure-like events recorded in vitro
from slices of motor cortex prepared from rats with chronic seizures induced by injection
of tetanus toxin. In this chapter we also describe two VERTEX simulations; one that
uses short term plasticity as the vehicle for the breakdown in inhibition and the build
up of activity during a seizure-like event, and another of stimulus evoked activity in a
seizure-prone neocortical slice. We compare the latter with stimulus evoked potential
in an example in vitro multi-electrode array recording from the chronic epilepsy model.
In Chapter 6 we discuss the future uses of VERTEX in modelling stimulus evoked
activity and epileptiform activity
Bioinformatic analysis of human dendritic cell development
Get PDFPh. D. ThesisDendritic cells (DCs) are professional antigen presenting cells of the mammalian
immune system, and constitute a vital link between the adaptive and the innate
immune systems. These cells are phenotypically and functionally highly
heterogeneous, comprising at least 3 subsets in human: classical/conventional DC
types 1 and 2 (cDC1, cDC2), and plasmacytoid DC (pDC). Additional heterogeneity
has been described within the cDC2 compartment which can be divided into two
populations, termed DC2 and DC3, more closely related to cDC1s or monocytes,
respectively. DCs develop in the bone marrow (BM) under the control of lineagespecific transcription factors (TFs). However, the cellular pathways and genetic
factors that govern the development of human DC subpopulations from
haematopoietic stem cells are not well known, in part due to their rarity in vivo.
First, this work addressed the rarity of DCs via a novel in vitro culture system that
favoured the production of large numbers of DCs from primary human CD34+
stem/progenitor cells. Two transcriptomic approaches were employed to verify the
culture output: the NanoString assay and bulk RNA-Seq. The transcriptomic analyses
confirmed that all DC subsets produced in culture exhibited appropriate transcription
profiles and bore close resemblance their ex vivo-derived counterparts.
Furthermore, these methods attested that Notch stimulation predisposed the culture
output toward the production of cDC1, the rarest of the DC subsets.
The culture system, confirmed to produce bona fide DC subsets, facilitated the
interrogation of DC haematopoiesis to establish the phenotypic identities of putative
progenitor and precursor populations. These early populations, derived from human
BM, along with mature DCs, were subjected to single cell transcriptomics. Pseudotemporal ordering and lineage branching reconstruction analyses revealed two
pathways of DC development, marked by differential expression of the TF IRF8 and
explaining the origin of cDC2 heterogeneity. The IRF8high pathway generated pDC,
cDC1 and DC2, while DC3 and monocytes developed along an IRF8low trajectory.
Mass cytometry analysis validated the link between the two pathways in BM and DC
populations found in peripheral blood.
III
Finally, the project focused on determining the role of IRF8 in the homeostasis of
human cDC1s and pDCs, both of which develop through the IRF8high pathway and
retain IRF8 expression as they mature. The previously established in vitro culture
techniques were employed to generate sufficient DCs for low-input IRF8 chromatin
immunoprecipitation, followed by high-throughput DNA sequencing (ChIP-Seq). The
analysis of the ChIP-Seq data revealed that IRF8 maintains both the function and
surface phenotype of cDC1s, while in pDCs it controls important functional modules.
During this work, a wide variety of transcriptomic and genomic bioinformatic
techniques and analyses enabled the verification of a novel human DC culture
system (Kirkling and Cytlak et al., 2018), the identification of two pathways of human
DC development (Cytlak and Resteu et al., 2020), and have generated new insights
into the role of IRF8 in human DC
Discordia, hegemony, and popular subjectivities: towards a model for the analysis of social conflict in ancient Rome
No full textPh.D ThesisThis study is a methodological and historiographical discussion of four key theoretical concepts for the analysis of social conflict in the long phase of transition from Gaius Gracchus’ death (121 BCE) to the Tiberian age. It aims to lay the foundations for a model for the investigation of the production of political discourse concerning social conflict in Late Republican and Early Imperial Rome, which is here understood as the result of a dialectic between the elites and the masses. The study of ideology has long been a crucial component of the research on Roman political culture. Nevertheless, theorisation and explicit discussions of how ideology works have only recently found place in the scholarly literature. Similarly, over the last three decades, the role of the crowd in Roman political has also become an increasingly prominent question.
However, scholarship has struggled to agree on categories and definitions for popular
subjectivities. This thesis proposes a study of three categories to think with when reflecting upon the agency of the masses in political life and their dialectical relationship with the political, cultural, and economic elites. In the first part, Gramsci’s ‘constellation’ of ideology/hegemony, testing the validity of his categories of religione, folklore, filosofia, and senso comune will be discussed, aiming to enable a sharper understand of the different roles played by different actors in shaping ideology. The two categories for popular subjectivities discussed in the second part – the People and multitude- aim to offer a new taxonomy of the masses’ intervention on the public scene, which can help distinguish different phenomena until now lumped under the generic label of ‘collective actions’. To test the viability of the categories, a range of case studies drawn from the history of the Late Republic and Early Principate underpin the theoretical and methodological discussion in each chapter
Energy recovery from a high strength waste stream using pilot-scale bioelectrochemical systems
Get PDFPh. D. Thesis.Bioelectrochemical systems (BESs) have the potential to produce energy from wastewater.
However, they are far from ready to be applied into industry. This study attempts to
reduce the gap between pilot-scale and commercial application.
First, we present a systematic review on the published semi-pilot and pilot-scale MECs,
and benchmark their performance against existing wastewater treatment. We find that
factors which are perceived to be problematic, such as low conductivities and temperatures,
have been overcome by BESs at pilot-scale, and that these systems have met the regulatory
requirements for discharge standards. We identify reactor depth and volumetric treatment
rates (VTRs) as the areas that require further research. It was hypothesised that the use of
high strength return sludge liquor (RSL), rather than raw domestic wastewater, may boost
BES performance. At a laboratory scale, it was seen that MFCs fed RSL performed as
well as the pure substrate when comparing wastewater treatment performance, and COD
saturation was reached with respect to VTRs. The use of RSL with a high soluble COD
appears to reduce the effect of the slow breakdown of complex substrates in wastewater.
Building on this success, a pilot-scale microbial electrolysis cell (MEC) was then operated
in continuous flow for 6 months. The reactor was fed RSL, and successful optimisation of
the hydraulic retention time (HRT) resulted in the highest VTR achieved by a pilot-scale
MEC treating real wastewater. Peak HRT was 0.5-days, resulting in an average VTR
of 3.82 kgCOD/m3
·day and a 55% COD removal efficiency. Using the data obtained, a
direct analysis of the potential savings from the reduced loading on AS was then made.
Theoretical calculation of the required tank size with the estimated costs and savings
indicate that the use of an MEC as a RSL pre-treatment technique could result in an
industrially viable system.
Throughout the thesis, there was a distinct variability between identical reactors when
using wastewater. Variability reduces the overall performance and therefore increases
costs, but more importantly, it highlights our lack of understanding of, and our inability to
control and engineer these systems. Analysis in the previous chapters saw that variability
was most obvious during inoculation with fresh wastewater, and appears to be caused by
the total number of electrogens able to establish onto the anode. Artificially seeding the
reactors enabled the creation of higher performing biofilms; however, this was only with
sterile wastewater. Periods of stable current when running the pilot reactor in continuous
flow give hope that modification to the design and operational conditions could reduce the
impact of this variability.
Assuming that other dimensions will be overcome by the use of modular electrodes, depth
remains a major challenge, and even if this is accomplished, the natural variability from
using wastewater may prevent this technology from ever being implemented into industry. However, if these issues can be solved, the switch to a more sustainable wastewater
treatment method would be both economically and environmentally beneficial.EPSRC, Newcastle University School of Engineerin