VictorJuiz/thesis huggingface dataset:
Mechanism of action studies of the utrophin modulator ezutromid for the treatment of Duchenne muscular dystrophy
Keywords: Proteomics
Target identification
Affinity-based Protein Profiling
Mechanism of action
Photoaffinity labelling
Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscle wasting disease arising from mutations in the dystrophin gene, affecting about 1 in 5000 boys. Whilst there is currently no available cure for DMD, a number of therapeutic strategies are under development. One such therapy aims to promote expression of utrophin, an autosomal paralogue of dystrophin. Utrophin upregulation rescues the dystrophic phenotype in disease model mice. A novel small molecule utrophin modulator, ezutromid (Summit Therapeutics; formerly SMT C1100), which was identified using a phenotypic screen, progressed to Phase II clinical trials in DMD patients. Interim 24-week data demonstrated reduced muscle fibre damage and increased levels of utrophin, providing the first evidence of ezutromid target engagement and proof of mechanism. However, these effects were not seen after the full 48 weeks of the trial. This work aims to define the mechanism of ezutromid in order to help understand the trial results, and to aid development of new generations of utrophin modulators.
Target identification through affinity based protein profiling (AfBPP) has been carried out in this work alongside phenotypic profiling studies. AfBPP requires bioactive analogues suitable for affinity purification. Biotinylated and photoaffinity-labelled analogues of ezutromid designed to be positive and negative control probes have been synthesised and bioactivity confirmed. These probes were used in AfBPP experiments with disease model mouse myoblasts followed by LC-MS/MS. The results from this experiment and phenotype profiling converged on the aryl hydrocarbon receptor (AhR) as a target of ezutromid. Target validation studies demonstrated that ezutromid binds AhR potently, behaving as an AhR antagonist. Moreover, reported AhR antagonists also upregulate utrophin, showing that this pathway could be exploited in future DMD therapies.
Investigating the links between N2 fixation and metabolism in rhizobia
Keywords: Metabolism
Rhizobia

In this thesis the metabolic flow of carbon and electrons enabling N2 fixation in the agriculturally relevant model organisms Rhizobium leguminosarum and Azorhizobium caulinodans was investigated. Building upon functional genomic data-sets including transcriptomics, metabolomics and mutagenic screens, the core metabolic reactions needed for dicarboxylate ORS571 and RL3841. Isocitrate dehydrogenase was found to be essential for N2-fixation and operation of the TCA-cycle in RL3841, contradicting previously published INSeq data. This was due to TA insertions in gene termini misleading the Hidden Markov Model used to asses INSeq data. As a result of this, new bioinformatic pipelines have been developed to avoid such errors. The redox sink poly-3-hydroxy butyrate (PHB) was shown to be non-essential for N2 fixation in ORS571. Previously published results showing PHB synthesis to be essential are actually due to missregulation of phaR translation in phaC mutants used to block PHB synthesis, rather than the loss of PHB as a redox store. This bring ORS571 in line with other species of rhizobia, where PHB defective mutants retained their ability to fix N2. Two alternative metabolic pathways, the malonate shunt in ORS571 and the isocitrate shunt in RL3841 have both been investigated and shown to be inoperative at biologically relevant levels in their respective organisms. In ORS571, the entirety of the carbon flux between succinate and acetyl-CoA has been demonstrated to proceed via NAD+ dependent malic enzyme (DME) and phosphoenolpyruvate carboxykinase, allowing existing models of carbon flux to be revised to remove the erroneous malonate shunt. In addition, DME has been demonstrated to be essential for N2 fixation, but dispensable for growth on succinate, indicating a disconnect between the requirements of N2 fixation and dicarboxylate metabolism.
Improving the measurement and treatment of infant pain
Keywords: brain imaging
paracetamol
electroencephalography
touch
neonate
CT fibre
pain relief
pain
newborn infant

Infants undergo numerous medically necessary painful procedures while in hospital, and this early life pain can lead to adverse effects. A range of pain relief interventions are available for use in neonatal units, including pharmacological analgesics, and non- pharmacological comfort measures. However, the literature regarding the efficacy of many of these pain relief interventions is inconclusive and inconsistent, and infant pain is consequently undertreated.
To assess infant pain, behavioural and physiological surrogate measures are utilised in the absence of verbal report. However, as these measures are not direct measures of pain, and can be influenced by other factors such as procedure-related distress, their use can lead to inconsistent conclusions regarding the efficacy of pain relief interventions. The perception of pain manifests in the brain, therefore it is plausible that measures of brain activity can provide specific and objective indications of the infant pain experience, and that these measures can be used to assess the efficacy of pain relief interventions. In this thesis, noxious-evoked brain activity is measured and used to investigate the effect of a pharmacological analgesic, paracetamol, and a non- pharmacological pain relief intervention, gentle touch targeted towards C-tactile (CT) fibres.
Noxious-evoked brain activity has been well characterised following medically required heel lances and experimental noxious stimuli, but brain activity evoked by alternative clinically relevant stimuli must also be characterised to assess the efficacy of a wider range of pain relief interventions for these procedures. In this thesis, brain activity evoked by immunisation is characterised, enabling the analgesic efficacy of paracetamol to be tested during routine infant immunisations.
Between-subject comparisons are often necessary when investigating the efficacy of pain relief interventions for medical procedures. However, there is great individual variability in nociceptive sensitivity, and controlling for this variability could enable better between-group comparisons. In the last section of this thesis, infant nociceptive sensitivity is measured and accounted for, in order to explore how this approach can be used to assess intervention efficacy, using the example of gentle touch targeted towards CT fibres.
Overall, the aims of the research described in this thesis are to advance knowledge regarding how infants respond to noxious clinical procedures, how pain relief interventions can modulate these responses, and how individual variability can be accounted for to enable better assessment of intervention efficacy. Research in the area of infant pain management is critical to inform clinical practise and improve the treatment of infant pain.
Vascular disease in multiple sclerosis: impact on pathology and radiologic outcomes
Keywords: Vascular disease
Brain Magnetic Resonance Imaging
Neuropathology
Multiple Sclerosis

Shared risk factors, immobility, and inflammation may predispose to systemic- and cerebro-vascular disease in Multiple Sclerosis (MS). On the other hand, vascular risk factors (VRF) and comorbidities may have a direct impact on MS pathology. A post mortem and an in vivo magnetic resonance imaging (MRI) study were set up as complementary approaches to test these hypotheses. In the first, objective measures of systemic and cerebro-vascular disease were obtained from a unique cohort of MS and non-MS cases in whom whole body autopsy reports and brain tissue were available for study. This work revealed that cardiovascular disease is less common, but has a stronger impact on cerebral small vessel disease (cSVD) in MS compared with non-MS, and associates with a lower burden of active plaques. Histological analysis showed an increased cSVD burden in those who die young with highly active MS and that periarteriolar hemosiderin deposition, enlarged spaces and inflammation are key features of MS pathology. In the in vivo brain MRI study imaging markers were compared between MS cases with and without VRF and non-MS cases with VRF. By doing so, imaging discriminators between MS and cSVD were identified. While MS does not seem to influence cSVD imaging markers, the different VRF have distinct effects on MS imaging markers: smoking and dyslipidaemia associating with a higher burden of these markers and diabetes having the opposite effect. Using this combined approach, this thesis advanced the knowledge of the contribution of vascular disease/VRF into MS radiological and pathological heterogeneity, identified markers that can be useful on MS differential diagnosis, and has evidenced that the cerebral microvasculature is structurally abnormal in MS and may be an important therapeutic target.
Interpretations of marks from draughting tools in some Italian renaissance drawings
Keywords: harmonic numbers
design process
geometric design
Leonardo
architectural drawings
Palladio

The focus of this thesis is the examination of evidence left by draughting tools in Italian Renaissance drawings. Because of the archaeological evidence of tool marks it is possible to investigate artists’ use of design systems in a scien-tific manner, with almost forensic certainty. When evidence is sufficient and in-terpreted in the context of design traditions, the sequence of steps that the de-signer employed can be recreated, shedding light on the artists’ methods and the decisions that informed the design process.
The historical survey in Part I describes the origins and subsequent man-ifestations of two primary design systems that, I argue, were traditional among designers up to the Renaissance. These are the numerical and geometrical sys-tems of designs. Numerical systems involve simple ratios that derive from re-lationships found in nature, e.g. musical harmonies. Geometrical systems in-volve using a compass and straightedge to locate points and intersections that aid in building compositions. Part II contains analyses of physical evidence in drawings by Leonardo, Antonio da Sangallo, Palladio, Uccello, Michelangelo and Peruzzi.
My conclusion is that evidence from draughting tools can be brought to bear on the question of artists’ design procedures; that information on artists’ design procedures, thus obtained, cannot usually be found anywhere else; and that revealing the information requires the implementation of an investigative methodology. This methodology involves examining and recording the tool marks and determining the sequence of their creation by reference to the de-sign traditions. The results of the drawing analyses provide examples of artists design procedures revealed by this methodology.
As a draughtsman and designer myself, I have a special appreciation for drawings, compositions and the process of making them. It is my hope that others who share this appreciation and desire to investigate further, can benefit from the methodology and investigatory results of this thesis.
How restorative justice ‘works’: psychological changes expected and experienced by victims who communicate with offenders
Keywords: justice
crime
victim
perceptions of justice
restorative justice
victims of crime

There is evidence that some victims benefit from communication with offenders, at least some of the time, but little is known about how these benefits occur. Many have theorised about the mechanisms by which restorative justice produces outcomes for victims, but few have empirically tested these theories. This thesis proposes five main types of psychological change generated for victims by a specific restorative justice practice (communication with the offender), which in turn lead to satisfaction and wellbeing. Three studies were designed to test whether victims expect and experience changes in their perceptions of 1) procedural justice, 2) punishment, 3) prevention, 4) the offender and 5) themselves. Analysis of quantitative data from a national victimisation survey and a complementary smaller-scale online survey demonstrated that four of the five types of victim perception predicted willingness to meet the offender. Victims were particularly motivated to meet the offender when they believed the criminal justice system had failed to fulfil their justice objectives. Analysis of qualitative data from interviews with forty people demonstrated that victims expected and experienced each of the five types of psychological change, and attributed improvements in their wellbeing and satisfaction to having experienced these five changes. This constitutes preliminary but promising evidence that together the five mechanisms explain how communication with the offender benefits victims. The thesis also provides conceptual clarity on the precise nature of the changes that take place during victim-offender communication, and a theoretical framework upon which future studies can build. Knowledge of the mechanisms by which victim-offender communication ‘works’ can help victims make informed choices about participating, can enhance preparation and facilitation, and can contribute to the wider conversation about how to deliver meaningful justice for victims.
Public opinion and seditious language from the Wars of the Roses to the Pilgrimage of Grace, c.1461-1537
Keywords: public opinion
political culture
seditious language

Although the traditional divide between the late medieval and the early modern periods has increasingly been addressed by scholars, this arbitrary division has long dictated historical research and it has inevitably affected the way we study public opinion and seditious language. While late medievalists have increasingly acknowledged that public opinion was indeed significant in the late medieval period, they do not always agree about its workings, fluctuations, intensity, and most importantly about who mattered in the public arena. Early modernists have increasingly argued against the Habermasian ‘public sphere’ in order to establish that England experienced a dynamic public debate in the sixteenth century. This study interacts with both groups of scholars and approaches public opinion and seditious language by surveying a variety of sources that combines legal records, private and official correspondence, chronicles, proclamations, legislation, and other texts that were directly or indirectly related to the contemporary public opinion. To illustrate the workings of public opinion, the popular attacks against the hegemonic voices of the ruling elites, and the interaction of these two features, this thesis resorts to a combined theoretical framework to examine five broad features: the rhetorical references to ‘the people’ and ‘the commons’, and the stereotypes about them; the profound and instinctive nature of rumour-mongering; the patterns of seditious language; the continuities and shifts in popular discourses across the period; and the government’s reactions and attempts to control all these different variants in the public realm. In other words, this study explores the workings of the communication system that underlay the interaction between rulers and ruled to explain how the tropes and discourses of the Wars of the Roses mostly remained in use and powerful during the early Reformation – and the extent to which some of these discourses shifted or intensified. Overall, this thesis argues that the continuities in how people criticised and reacted to crises throughout the period partly explain the peaks in records of complaint and rumour as well as provide significant insights into how government policy was more frequently reactive than proactive.
Understanding the structure and interactions of polycystin-2 through structural and simulation studies
Keywords: Molecular Simulations
Cryo EM

Polycystin-2 (PC2) is a member of the transient receptor potential (TRP) superfamily of non-selective cation channels. It has a systemic distribution with relatively high expression in kidney tubules. PC2 shares a common transmembrane fold with other TRP channels, in addition to having a extracellular/luminal domain (TOP domain) unique to TRPP and TRPML channels. Mutations in PC2 are associated with autosomal dominant polycystic kidney disease (ADPKD), which is one of the most prevalent genetic disorders in human. Despite that the genetic link between PC2 and ADPKD is well established, the molecular basis of the disease is still elusive. It has been proposed that dysfunction of ciliary PC2 is the main mechanism of pathology of PC2-associated ADPKD. Given the complex organisation of ciliary membrane, it is important to establish how PC2 interacts with specific lipids in its membrane environment. Lipid binding assay results suggest that detergent-solubilised PC2 can bind a range of phosphatidylinositol phosphates but not other simple anionic (phosphatidylserine, phosphatidic acid) or zwitterionic (phosphatidylethanolamine, phosphatidylcholine) lipids. Combining cryo-electron microscopy (cyro-EM) and multiscale molecular dynamics (MD) simulations, we identified a hydrophobic pocket in PC2 formed between S3, S4 transmembrane helices and S4-S5 linker, which showed a preference for phosphatidylinositol bisphosphate (PIP2) binding. Simulations of other members of the TRP channel family suggest this lipid-binding site may be shared amongst a number of TRP channels. In addition, our cryo-EM maps revealed a binding site for cholesterol on PC2, which is further characterised with MD simulations. These results help to position PC2 within an emerging model of the complex roles of lipids in the regulation and organisation of ciliary membranes. Moreover, we used MD simulations to probe the ion selectivity of PC2. Our results suggest that PC2 is selective for Na+ over Ca2+, and not only the selectivity filter residues but also the TOP domain contribute to the selectivity of the channel.
Genomic investigations of bacteriocins and bacteriophages in Streptococcus pneumoniae and other streptococci
Keywords: Phages
Genome Mining
Streptococcal bacteriocins
In silico Drug Discovery
Bacteriocins
Streptococcus
Prophages
PhageMiner
Peptide antibiotics
Phage discovery
Bioinformatics Tools
Streptococcal phages
Bacteriophages
Bioinformatics
Streptococcus pneumoniae

Streptococcus pneumoniae (the “pneumococcus”) is a major public health problem, leading to significant morbidity and mortality worldwide. The global increase in antimicrobial-resistant pneumococci is of serious concern, which necessitates investigations into novel antimicrobial strategies. Two promising areas of research are the exploitation of phages (viruses that infect bacteria) and bacteriocins (antimicrobial peptides produced by bacterial species to kill other bacteria). This thesis uses genomic approaches to address questions related to the prevalence, diversity and molecular epidemiology of bacteriocins and prophages (phage genomes that are integrated into bacterial chromosomes).
The first part analysed >6,200 pneumococcal genomes and discovered 14 novel bacteriocin gene clusters. The molecular epidemiology of the bacteriocin clusters was investigated in the context of the pneumococcal population structure. The results revealed extraordinary bacteriocin diversity among pneumococci and the majority of bacteriocin clusters were also present in other streptococcal species. Genomic hotspots for the integration of different bacteriocin gene clusters were discovered. Bacteriocin genes were found to be transcriptionally active when the pneumococcus was under stress and when two different strains were co-cultured in broth.
The second part describes the molecular epidemiology of satellite prophages (prophages that rely on the host and an additional helper phage for survival) in a large global and historical pneumococcal dataset. Forty-four unique satellite prophages were newly identified, which had persistent associations with specific widely-circulating pneumococcal clonal complexes over many decades. Collaborative experimental work demonstrated that one of these satellite prophages was associated with virulence in a murine model of infection. RNA sequencing revealed that satellite prophage genes were overexpressed when pneumococci were grown planktonically versus in in vitro biofilm experimental conditions.
The final part analysed >1,300 genomes of 70 different Streptococcus species and identified nearly 800 prophages and satellite prophages. The data showed that prophages and satellite prophages were widely distributed among streptococci and constituted two distinct entities with little effective genetic exchange between them. Contrary to the current dogma that suggests prophages are bacterial species-specific, there was convincing evidence that transmission of prophages occurred between genetically different streptococcal species.
These results broaden our understanding of bacteriocins and phages among streptococci and uncover many areas for future studies.
Impact of host restriction factors and capsid evolution on HIV disease progression
Keywords: TRIM
Capsid
AIDS

HIV-1 is responsible for the global HIV pandemic whereas HIV-2 has mostly been limited to West Africa. The key to their successful spread is their unparalleled ability to adapt to the host immune system. The contemporary HIV pandemics are the latest iteration in a millennia-long evolutionary arms race between retroviruses and their hosts. Two host restriction factors which have been important actors in this arms race are TRIM5α and its paralogue TRIM22. TRIM5α targets the retroviral capsid, whereas TRIM22 suppresses retroviral transcription indirectly.
The central hypothesis for the thesis is that the HIV capsid and TRIM restriction are associated with HIV disease progression. In this thesis we present findings from four projects. In chapter three we discuss the effect of TRIM genotype on disease outcomes in children and adolescents infected perinatally with HIV-1. We characterised TRIM genetic diversity in this cohort and found that TRIM22 genotype is associated with CD4+ T cell counts and, potentially, disease progression rates. Chapter four presents the outcomes of using CD4+ T cell kinetic data to stratify HIV-2 infected adults into disease progressor groups. Forty-six percent of patients were classified as fast progressors and 54% as slow progressors. Slow progressors advanced to AIDS at approximately half the rate of fast progressors, had higher CD4+ T cell levels and a slower CD4+ T cell decline rate. In chapter five we discuss the effect of TRIM genotype on HIV-2 disease outcomes in adults. TRIM22 genotype associated with significant effects on long-term CD4+ levels, though these effects were modest. Finally, we report that intrahost evolutionary rates of HIV-2 gag are significantly higher in fast progressors, that the p24 region evolves under negative selection and that evolutionary rates correlate negatively with CD4+ T cell percentage