Regulation of Topoisomerase IIa expression in humans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

In mammalian cells, the loss or down-regulation of tumour-suppressor genes and/or the mutation or overexpression of proto-oncogenes, whose products promote unregulated proliferation in cells, characterise the process of malignant transformation. This generates mitogenic signals that promote abnormal cell growth resulting in tumour progression. Topoisomerase IIα (topo IIα) is an enzyme present in elevated concentrations in highly proliferating cells due to the requirement for untwisting and unknotting of the DNA which is essential for replication. Because of this requirement, a number of anti-cancer drugs have been designed with topo IIα as their primary target. The effectiveness of these drugs however is limited by the development of resistance. One factor linked to drug resistance is the down-regulation of topo IIα at the transcription level. Expression of topo IIα appears to be regulated through various transcription factors with members of the Spl family having a major contribution. Previous work has shown down regulation of topo IIα can occur at the level of transcription. Nucleotide sequencing of the topo IIα promoter in drug-resistant cell lines has not revealed any mutations thus far. Three known proteins and one uncharacterised protein are capable of interacting with the proximal topo IIα promoter region. The uncharacterised protein may act as a co-activator or a co-repressor depending on the complement of transcription factors associated with the DNA in this region. Because drug resistant cell lines showed modulated expression of these transcription factors, it is important to identify the unknown protein and characterise its role in regulating topo IIα expression. This research aimed to identify the minimal binding site and DNA elements required for the uncharacterised protein to bind, as well as introduce mutations into this proximal region and examine their functional significance. The results of this study could provide insights into the molecular mechanisms responsible for the development of drug resistance, contributing to more efficient and effective methods for the treatment of cancer

Accessing elite nurses for research: reflections on the theoretical and practical issues of telephone interviewing

Abstract Elite groups are interesting as they frequently are powerful (in terms of position, knowledge and influence) and enjoy considerable authority. It is important, therefore, to involve them in research concerned with understanding social contexts and processes. This is particularly pertinent in healthcare where considerable strategic development and change are features of everyday practice that may be guided, or perceived as being guided, by elites. This paper evolved from a study investigating the availability and role of nurses whose remit involved leading nursing research and development within acute NHS Trusts in two heath regions in Southern England. The study design included telephone interviews with Directors of Nursing Services during which time the researchers engaged in a reflective analysis of conducting research with an 'elite' group. Important issues identified were the role of the gatekeepers, engagement with elites and the use of the telephone interview method in this context. The paper examines these issues and makes a case for involving executive nurses in further research. The paper also offered strategies to help researchers design and implement telephone interview studies successfully to maximise access to the views and experiences of 'hard to reach groups', such as elites, whilst minimising the associated disruption.

On the Dialectics of Charisma in Marina Abramović’s The Artist is Present

While ‘charisma’ can be found in dramatic and theatrical parlance, the term enjoys only minimal critical attention in theatre and performance studies, with scholarly work on presence and actor training methods taking the lead in defining charisma’s supposed ‘undefinable’ quality. Within this context, the article examines the appearance of the term ‘charismatic space’ in relation to Marina Abramovic’s retrospective The Artist is Present at New York’s Museum of Modern Art in 2010. Here Abramovic uses this term to describe the shared space in which performer and spectator connect bodily, psychically, and spiritually through a shared sense of presence and energy in the moment of performance. Yet this is a space arguably constituted through a number of dialectical tensions and contradictions which, in dialogue with existing theatre scholarship on charisma, can be further understood by drawing on insights into charismatic leaders and charismatic authority in leadership studies. By examining the performance and its documentary traces in terms of dialectics we consider the political and ethical implications for how we think about power relations between artist/spectator in a neoliberal, market-driven art context. Here an alternative approach to conceiving of and facilitating a charismatic space is proposed which instead foregrounds what Bracha L. Ettinger calls a ‘matrixial encounter-event’: A relation of coexistence and compassion rather than dominance of self over other; performer over spectator; leader over follower. By illustrating the dialectical tensions in The Artist is Present, we consider the potential of the charismatic space not as generated through the seductive power or charm of an individual whose authority is tied to his/her ‘presence’, but as something co-produced within an ethical and relational space of trans-subjectivity

Efficiency and safety of varying the frequency of whole blood donation (INTERVAL): a randomised trial of 45 000 donors

Background: Limits on the frequency of whole blood donation exist primarily to safeguard donor health. However, there is substantial variation across blood services in the maximum frequency of donations allowed. We compared standard practice in the UK with shorter inter-donation intervals used in other countries. Methods: In this parallel group, pragmatic, randomised trial, we recruited whole blood donors aged 18 years or older from 25 centres across England, UK. By use of a computer-based algorithm, men were randomly assigned (1:1:1) to 12-week (standard) versus 10-week versus 8-week inter-donation intervals, and women were randomly assigned (1:1:1) to 16-week (standard) versus 14-week versus 12-week intervals. Participants were not masked to their allocated intervention group. The primary outcome was the number of donations over 2 years. Secondary outcomes related to safety were quality of life, symptoms potentially related to donation, physical activity, cognitive function, haemoglobin and ferritin concentrations, and deferrals because of low haemoglobin. This trial is registered with ISRCTN, number ISRCTN24760606, and is ongoing but no longer recruiting participants. Findings: 45 263 whole blood donors (22 466 men, 22 797 women) were recruited between June 11, 2012, and June 15, 2014. Data were analysed for 45 042 (99·5%) participants. Men were randomly assigned to the 12-week (n=7452) versus 10-week (n=7449) versus 8-week (n=7456) groups; and women to the 16-week (n=7550) versus 14-week (n=7567) versus 12-week (n=7568) groups. In men, compared with the 12-week group, the mean amount of blood collected per donor over 2 years increased by 1·69 units (95% CI 1·59–1·80; approximately 795 mL) in the 8-week group and by 0·79 units (0·69–0·88; approximately 370 mL) in the 10-week group (p<0·0001 for both). In women, compared with the 16-week group, it increased by 0·84 units (95% CI 0·76–0·91; approximately 395 mL) in the 12-week group and by 0·46 units (0·39–0·53; approximately 215 mL) in the 14-week group (p<0·0001 for both). No significant differences were observed in quality of life, physical activity, or cognitive function across randomised groups. However, more frequent donation resulted in more donation-related symptoms (eg, tiredness, breathlessness, feeling faint, dizziness, and restless legs, especially among men [for all listed symptoms]), lower mean haemoglobin and ferritin concentrations, and more deferrals for low haemoglobin (p<0·0001 for each) than those observed in the standard frequency groups. Interpretation: Over 2 years, more frequent donation than is standard practice in the UK collected substantially more blood without having a major effect on donors' quality of life, physical activity, or cognitive function, but resulted in more donation-related symptoms, deferrals, and iron deficiency. Funding: NHS Blood and Transplant, National Institute for Health Research, UK Medical Research Council, and British Heart Foundation

Digging deep: how organisational culture affects care home residents' experiences

Organisational culture of institutions providing care for older people is increasingly recognised as influential in the quality of care provided. There is little research, however, that specifically examines the processes of care home culture and how these may be associated with quality of care. In this paper we draw from an empirical study carried out in the United Kingdom (UK) investigating the relationship between care home culture and residents’ experience of care. Eleven UK care homes were included in an in-depth comparative case study design using extensive observation and interviews. Our analysis indicates how organisational cultures of care homes impact on the quality of care residents receive. Seven inter-related cultural elements were of key importance to quality of care. Applying Schein’s conceptualisation of organisational culture, we examine the dynamic relationship between these elements to show how organisational culture is locally produced and shifting. A particular organisational culture in a care home cannot be achieved simply by importing a set of organisational values or the ‘right’ leader or staff. Rather, it is necessary to find ways of resolving the everyday demands of practice in ways that are consistent with espoused values. It is through this everyday practice that assumptions continuously evolve, either consistent with or divergent from, espoused values. Implications for policy makers, providers and practitioners are discussed

Sympatric woodland Myotis bats form tight-knit social groups with exclusive roost home ranges

Background: The structuring of wild animal populations can influence population dynamics, disease spread, and information transfer. Social network analysis potentially offers insights into these processes but is rarely, if ever, used to investigate more than one species in a community. We therefore compared the social, temporal and spatial networks of sympatric Myotis bats (M. nattereri (Natterer's bats) and M. daubentonii (Daubenton's bats)), and asked: (1) are there long-lasting social associations within species? (2) do the ranges occupied by roosting social groups overlap within or between species? (3) are M. daubentonii bachelor colonies excluded from roosting in areas used by maternity groups? Results: Using data on 490 ringed M. nattereri and 978 M. daubentonii from 379 colonies, we found that both species formed stable social groups encompassing multiple colonies. M. nattereri formed 11 mixed-sex social groups with few (4.3%) inter-group associations. Approximately half of all M. nattereri were associated with the same individuals when recaptured, with many associations being long-term (>100 days). In contrast, M. daubentonii were sexually segregated; only a quarter of pairs were associated at recapture after a few days, and inter-sex associations were not long-lasting. Social groups of M. nattereri and female M. daubentonii had small roost home ranges (mean 0.2 km2 in each case). Intra-specific overlap was low, but inter-specific overlap was high, suggesting territoriality within but not between species. M. daubentonii bachelor colonies did not appear to be excluded from roosting areas used by females. Conclusions: Our data suggest marked species- and sex-specific patterns of disease and information transmission are likely between bats of the same genus despite sharing a common habitat. The clear partitioning of the woodland amongst social groups, and their apparent reliance on small patches of habitat for roosting, means that localised woodland management may be more important to bat conservation than previously recognised

Galleria mellonella Infection Model Identifies Both High and Low Lethality of Clostridium perfringens Toxigenic Strains and Their Response to Antimicrobials

Research progress into mechanisms of the anaerobe Clostridium perfringens and associated diseases has been frustrated by the lack of reliable infection models. Wax moth larvae (Galleria mellonella) have emerged as a viable alternative to other models of infection since they are economic, survive at 37�C and require no specialist equipment. This study aims to establish to what extent G. mellonella larvae can be used to study the virulence of C. perfringens strains and its suitability for studying novel treatment strategies by an improved time-lapse approach to data collection. Mortality and morbidity rates of larvae challenged with 105 CFU of C. perfringens isolates from various sources were observed over 72 h and dose response data obtained. Phenoloxidase enzyme activity was investigated as a marker for immune response and tissue burden assessed by histopathological techniques. Results demonstrate that C. perfringens is pathogenic toward G. mellonella although potency varies dramatically between C. perfringens isolates and the reference strain ATCC 13124 was shown to be avirulent. Infection with C. perfringens strains activated the melanisation pathway resulting in melanin deposition but no increase in enzyme activity was observed. Efficacy of antibiotic therapy (penicillin G, bacitracin, neomycin, and tetracycline) administered parenterally to some extent correlates with that of in vitro analysis. The findings suggest G. mellonella might be a useful in vivo model of infection and convenient as a prescreening assay for virulence of C. perfringens strains or as a simple, cheap and rapid in vivo assay in the first stage development of novel therapeutics against anaerobes

Concordance in diabetic foot ulceration : a cross-sectional study of agreement between wound swabbing and tissue sampling in infected ulcers

BACKGROUND: There is inadequate evidence to advise clinicians on the relative merits of swabbing versus tissue sampling of infected diabetic foot ulcers (DFUs). OBJECTIVES: To determine (1) concordance between culture results from wound swabs and tissue samples from the same ulcer; (2) whether or not differences in bacterial profiles from swabs and tissue samples are clinically relevant; (3) concordance between results from conventional culture versus polymerase chain reaction (PCR); and (4) prognosis for patients with an infected DFU at 12 months' follow-up. METHODS: This was a cross-sectional, multicentre study involving patients with diabetes and a foot ulcer that was deemed to be infected by their clinician. Microbiology specimens for culture were taken contemporaneously by swab and by tissue sampling from the same wound. In a substudy, specimens were also processed by PCR. A virtual 'blinded' clinical review compared the appropriateness of patients' initial antibiotic regimens based on the results of swab and tissue specimens. Patients' case notes were reviewed at 12 months to assess prognosis. RESULTS: The main study recruited 400 patients, with 247 patients in the clinical review. There were 12 patients in the PCR study and 299 patients in the prognosis study. Patients' median age was 63 years (range 26-99 years), their diabetes duration was 15 years (range 2 weeks-57 years), and their index ulcer duration was 1.8 months (range 3 days-12 years). Half of the ulcers were neuropathic and the remainder were ischaemic/neuroischaemic. Tissue results reported more than one pathogen in significantly more specimens than swabs {86.1% vs. 70.1% of patients, 15.9% difference [95% confidence interval (CI) 11.8% to 20.1%], McNemar's p-value < 0.0001}. The two sampling techniques reported a difference in the identity of pathogens for 58% of patients. The number of pathogens differed in 50.4% of patients. In the clinical review study, clinicians agreed on the need for a change in therapy for 73.3% of patients (considering swab and tissue results separately), but significantly more tissue than swab samples required a change in therapy. Compared with traditional culture, the PCR technique reported additional pathogens for both swab and tissue samples in six (50%) patients and reported the same pathogens in four (33.3%) patients and different pathogens in two (16.7%) patients. The estimated healing rate was 44.5% (95% CI 38.9% to 50.1%). At 12 months post sampling, 45 (15.1%) patients had died, 52 (17.4%) patients had a lower-extremity ipsilateral amputation and 18 (6.0%) patients had revascularisation surgery. LIMITATIONS: We did not investigate the potential impact of microbiological information on care. We cannot determine if the improved information yield from tissue sampling is attributable to sample collection, sample handling, processing or reporting. CONCLUSIONS: Tissue sampling reported both more pathogens and more organisms overall than swabbing. Both techniques missed some organisms, with tissue sampling missing fewer than swabbing. Results from tissue sampling more frequently led to a (virtual) recommended change in therapy. Long-term prognosis for patients with an infected foot ulcer was poor. FUTURE WORK: Research is needed to determine the effect of sampling/processing techniques on clinical outcomes and antibiotic stewardship. FUNDING: The National Institute for Health Research Health Technology Assessment programme

Mapping enzyme-substrate interactions: its potential to study the mechanism of enzymes

With the increase of the need to use more sustainable processes for the industry in our society, the modeling of enzymes has become crucial to fully comprehend their mechanism of action and use this knowledge to enhance and design their properties. A lot of methods to study enzymes computationally exist and they have been classified on sequence-based, structure-based, and the more new artificial intelligence-based ones. Albeit the abundance of methods to help predict the function of an enzyme, molecular modeling is crucial when trying to understand the enzyme mechanism, as they aim to correlate atomistic information with experimental data. Among them, methods that simulate the system dynamics at a molecular mechanics level of theory (classical force fields) have shown to offer a comprehensive study. In this book chapter, we will analyze these techniques, emphasizing the importance of precise modeling of enzyme-substrate interactions. In the end, a brief explanation of the transference of the information from research studies to the industry is given accompanied with two examples of family enzymes where their modeling has helped their exploitation.Peer ReviewedObjectius de Desenvolupament Sostenible::9 - Indústria, Innovació i InfraestructuraPostprint (author's final draft

An Overview of Three Promising Mechanical, Optical, and Biochemical Engineering Approaches to Improve Selective Photothermolysis of Refractory Port Wine Stains

During the last three decades, several laser systems, ancillary technologies, and treatment modalities have been developed for the treatment of port wine stains (PWSs). However, approximately half of the PWS patient population responds suboptimally to laser treatment. Consequently, novel treatment modalities and therapeutic techniques/strategies are required to improve PWS treatment efficacy. This overview therefore focuses on three distinct experimental approaches for the optimization of PWS laser treatment. The approaches are addressed from the perspective of mechanical engineering (the use of local hypobaric pressure to induce vasodilation in the laser-irradiated dermal microcirculation), optical engineering (laser-speckle imaging of post-treatment flow in laser-treated PWS skin), and biochemical engineering (light- and heat-activatable liposomal drug delivery systems to enhance the extent of post-irradiation vascular occlusion)