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DNA damage can pose an irreversible steric block to RNA polymerase II (RNAPII), preventing transcription. RNAPII becomes stalled at DNA lesions, blocking normal repair. As a ‘last-resort’ mechanism to clear the stalled polymerase - and repair the damage - the largest subunit of RNAPII is poly-ubiquitylated and degraded. In yeast, this process is dependent on the Def1 protein, through a previously unresolved mechanism. Using a combination of yeast genetic, biochemical and cell biological techniques this thesis reports the molecular mechanism of Def1 in this process. Upon DNA damage induced RNAPII stall, Def1 becomes ubiquitylated and partially proteolytically processed by the 26S proteasome. This creates a biologically active, shorter form of Def1, termed pr-Def1. Removal of the C-terminus of Def1, which usually promotes nuclear export, results in nuclear accumulation of the N-terminal processed fragment. Nuclear pr-Def1 binds to stalled, mono-ubiquitylated RNAPII and recruits the Elongin-Cullin ubiquitin ligase complex, promoting RNAPII polyubiquitylation and degradation. Interestingly, Def1’s ubiquitin-binding CUE domain and a novel ubiquitin homology domain in the Elongin complex mediate this interaction. These results outline the multi-step mechanism of RNAPII poly-ubiquitylation, elucidate Def1 activation and function, and identify an atypical ubiquitin-like domain in the yeast Elongin complex.

Through a multi-method approach, this thesis uncovered the nature and characteristics of a hitherto little understood crime, internal child sex trafficking (ICST). This original large-scale study, the first of its kind in the UK, addressed an important knowledge gap in the literature and publications from this thesis have been used to support new investigations, policy development and to guide further academic research. Through access to sensitive and restricted documents and case files, this thesis explored the group-based element to ICST offending and identified patterns and typologies. Through identified commonalities among ICST cases, the thesis focused on supporting future investigations and prosecutions through four main approaches. First, an exploration of different group-based factors to ICST was conducted. This included identifying methods of recruitment, such as introduction via another offender, and benefits to joining a group, such as additional access to children or abuse locations. Second, an assessment of investigative and prosecution strategies was conducted which showed a wide range of tactics being used with no consistent approach across force areas. Third, a forensic experimental study was conducted aimed at improving understanding around persistence of semen on stained then laundered clothing, a common scenario seen in ICST cases. The findings from this study showed that the tested samples contained enough DNA to produce a full profile for use in UK and international courts, an important development for supporting future ICST cases. Finally, an eight theme framework for understanding a group’s structure and function was developed and examined using SWOT analysis. Suggestions such as the use of undercover police officers or targeted awareness raising were presented as possible options for tackling ICST groups. To conclude, the thesis discussed the potential use of tactics employed in other crime investigations, including covert policing and the use of victim reception centres, when conducting future ICST operations.

Introduction: Photodynamic therapy (PDT) is a minimally invasive treatment that requires a light-activated drug, known as a photosensitiser (PS), light of a specific wavelength and oxygen. 5,10,15,20-meta-tetra(hydroxyphenyl)chlorin (m-THPC) is one of the most potent PS’s currently available for use in PDT, however, its undesirable accumulation in healthy tissues has prompted research to improve its uptake and selectivity into tumour tissue for the treatment of certain malignant diseases, whilst reducing adverse skin photosensitivity. In this investigation, a range of nanocarriers, eliciting a host of different properties, were developed to achieve more efficient delivery of m-THPC in vivo. These included liposomes, organically modified silica (ORMOSIL) nanoparticles (NPs) and polymeric NPs, which were additionally surface modified with a biocompatible polymer (PEG) coating to improve blood circulation times and the bioavailability of m-THPC. Further to this, studies investigating the conjugation of ligands, over-expressed on many cancers, to the NP surface aimed to increase active m-THPC-delivery via targeted nanocarriers. The overall objective of this study was to compare the pharmacokinetics of m-THPC delivery between the nanocarrier formulations and standard Foscan®, in normal rat and tumour-bearing animal models, to ultimately improve the efficacy of PDT. Materials & Methods: The biodistribution of m-THPC in its standard formulation (Foscan) compared to its incorporation in untargeted and targeted pegylated NPs was assessed through quantitative chemical extraction methods and pharmacokinetic analysis. A range of tissue samples were collected over different time periods following i.v. administration of NPs (m-THPC dose equivalent) in both healthy and tumour-bearing murine models. Confocal and fluorescence microscopy techniques were employed for in vitro uptake studies and to examine ex vivo tissue localisation of encapsulated m-THPC. Finally PDT and skin photosensitivity studies were carried out in vivo to assess the efficacy of treatment to tumours and skin through histological analysis. Results & Conclusion: Pharmacokinetic data typically indicated an increase in blood plasma t1/2 of pegylated NPs in comparison to non-pegylated NPs or Foscan alone, indicative of the stealth properties conferred by the PEG corona. Peak accumulation of m-THPC in tumour tissue occurred between 6-24 h via passive uptake with untargeted NPs, attributed to the enhanced permeability and retention (EPR) effect. A significant improvement in tumour uptake, by a factor of three, was observed using pegylated liposomes compared to Foscan alone. Active targeting of NPs demonstrated a positive uptake into cells, unfortunately this did not translate to an improvement in m-THPC biodistribution or PDT results using in vivo models. Encouragingly anti-tumour PDT effects were observed with all NPs compared to Foscan, but treatment was most effective with untargeted pegylated liposomes.