An investigation of the molecular interactions between statins and bacterial pathogens, and their combined impact on the human immune system

Statins are a class of drug that inhibits cholesterol biosynthesis, and are used to treat patients with high serum cholesterol levels. They exert this function by competitively binding to the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA reductase (HMGR), which catalyses the formation of mevalonate, a rate-limiting step in cholesterol biosynthesis. In addition, statins have what are called “pleiotropic effects”, which include the reduction of inflammation, immunomodulation, and antimicrobial effects. Statins can also improve survival of patients with sepsis and pneumonia. Cystic fibrosis (CF) is the most common recessive inherited disease in the Caucasian population, which is characterised by factors including, but not limited to, excessive lung inflammation and increased susceptibility to infection. Therefore, the overall objective of this study was to examine the effects of statins on CFassociated bacterial pathogens and the host response. In this work, the prevalence of HMGR was examined in respiratory pathogens, and several CF-associated pathogens were found to possess homologues of this enzyme. HMGR homology was analysed in Staphylococcus aureus, Burkholderia cenocepacia and Streptococcus pneumoniae, and the HMGR of B. cenocepacia was found to have significant conservation to that of Pseudomonas mevalonii, which is the most widely-characterised bacterial HMGR. However, in silico analysis revealed that, unlike S. aureus and S. pneumoniae, B. cenocepacia did not possess homologues of other mevalonate pathway proteins, and that the HMGR of B. cenocepacia appeared to be involved in an alternative metabolic pathway. The effect of simvastatin was subsequently tested on the growth and virulence of S. aureus, B. cenocepacia and S. pneumoniae. Simvastatin inhibited the growth of all 3 species in a dose-dependent manner. In addition, statin treatment also attenuated biofilm formation of all 3 species, and reduced in vitro motility of S. aureus. Interestingly, simvastatin also increased the potency of the aminoglycoside antibiotic gentamicin against B. cenocepacia. The impact of statins was subsequently tested on the predominant CF-associated pathogen Pseudomonas aeruginosa, which does not possess a HMGR homologue. Mevastatin, lovastatin and simvastatin did not influence the growth of this species. However, sub-inhibitory statin concentrations reduced the swarming motility and biofilm formation of P. aeruginosa. The influence of statins was also examined on Type 3 toxin secretion, quorum sensing and chemotaxis, and no statin effect was observed on any of these phenotypes. Statins did not appear to have a characteristic effect on the P. aeruginosa transcriptome. However, a mutant library screen revealed that the effect of statins on P. aeruginosa biofilm was mediated through the PvrR regulator and the Cup fimbrial biosynthesis genes. Furthermore, proteomic analysis demonstrated that 6 proteins were reproducibly induced by simvastatin in the P. aeruginosa swarming cells. The effect of statins on the regulation of the host-P. aeruginosa immune response was also investigated. Statin treatment increased expression of the pro-inflammatory cytokines IL-8 and CCL20 in lung epithelial cells, but did not attenuate P. aeruginosa-mediated inflammatory gene induction. In fact, simvastatin and P. aeruginosa caused a synergistic effect on CCL20 expression. The expression of the transcriptional regulators KLF2 and KLF6 was also increased by statins and P. aeruginosa, with the induction of KLF6 by simvastatin proving to be a novel effect. Interestingly, both statins and P. aeruginosa were capable of inducing alternative splicing of KLF6. P. aeruginosa was found to induce KLF6 alternative splicing by way of the type 3 secreted toxin ExoS. In addition, a mechanistic role was elucidated for KLF6 in the lung, as it was determined that statin-mediated induction of this protein was responsible for the induction of the host response genes CCL20 and iNOS. Moreover, statin treatment caused a slight increase in infection-related cytotoxicity, and increased bacterial adhesion to cells. Taken together, these data demonstrate that statins can reduce the virulence of CFassociated bacterial pathogens and alter host response effectors. Furthermore, novel statin effectors were identified in both bacterial and host cells.

Total synthesis of furospongolide and related furanolipid analogues as potential anti-tumour agents

This thesis details the design, development and execution of innovative methodology in the total synthesis of the terpene-derived marine natural product, furospongolide. It also outlines the synthetic routes used to prepare a novel range of furanolipids derivatives and subsequent evaluation of their potential as antitumour agents. The first chapter is a review of the literature describing efforts undertaken towards the synthesis of biologically active furanosesterterpenoid marine natural products. A brief discussion on the sources and biological activity exhibited by furan natural products is also provided. In addition, a concise account of the role of hypoxia in cancer, and the increasing interest in HIF-1 inhibition as a target for chemotherapeutics is examined. The second chapter discusses the concise synthesis of the marine HIF-1 inhibitor furospongolide, which was achieved in five linear steps from (E,E)-farnesyl acetate. The synthetic strategy features a selective oxidation reaction, a Schlosser sp3-sp3 cross-coupling, a Wittig cross-coupling and an elaborate one-pot selective reduction, lactonisation and isomerization reaction to install the butenolide ring. The structure-activity relationship of furospongolide was also investigated. This involved the design and synthesis of a library of structurally modified analogues sharing the same C1-C13 subunit. This was achieved by exploiting the brevity and high level of convergence of our synthetic route together with the readily amenable structure of our target molecule. Exploiting the Schlosser cross-coupling allowed for replacement of furan with other heterocycles in the preparation of various furanolipid and thiophenolipid derivatives. The employment of reductive amination and Wittig chemistry further added to our novel library of structural derivatives. The third chapter discusses the results obtained from the NCI from biological evaluation From a collection of 28 novel compounds evaluated against the NCI-60 cancer cell array, six drug candidates were successfully selected for further biological evaluation on the basis of antitumour activity. COMPARE analysis revealed a strong correlation between some of our design analogues and the blockbuster anticancer agent tamoxifen, further supporting the potential of furanolipids in the treatment of breast cancer. The fourth chapter, details the full experimental procedures, including spectroscopic and analytical data for all the compounds prepared during this research.

Towards a model for exploring the relationship between managerial decision problems and decision support opportunities

The organisational decision making environment is complex, and decision makers must deal with uncertainty and ambiguity on a continuous basis. Managing and handling decision problems and implementing a solution, requires an understanding of the complexity of the decision domain to the point where the problem and its complexity, as well as the requirements for supporting decision makers, can be described. Research in the Decision Support Systems domain has been extensive over the last thirty years with an emphasis on the development of further technology and better applications on the one hand, and on the other hand, a social approach focusing on understanding what decision making is about and how developers and users should interact. This research project considers a combined approach that endeavours to understand the thinking behind managers’ decision making, as well as their informational and decisional guidance and decision support requirements. This research utilises a cognitive framework, developed in 1985 by Humphreys and Berkeley that juxtaposes the mental processes and ideas of decision problem definition and problem solution that are developed in tandem through cognitive refinement of the problem, based on the analysis and judgement of the decision maker. The framework facilitates the separation of what is essentially a continuous process, into five distinct levels of abstraction of manager’s thinking, and suggests a structure for the underlying cognitive activities. Alter (2004) argues that decision support provides a richer basis than decision support systems, in both practice and research. The constituent literature on decision support, especially in regard to modern high profile systems, including Business Intelligence and Business analytics, can give the impression that all ‘smart’ organisations utilise decision support and data analytics capabilities for all of their key decision making activities. However this empirical investigation indicates a very different reality.

Investigation of factors driving the costs of operating the 2020 Irish power system with large-scale wind generation

The main goal of this work is to determine the true cost incurred by the Republic of Ireland and Northern Ireland in order to meet their EU renewable electricity targets. The primary all-island of Ireland policy goal is that 40% of electricity will come from renewable sources in 2020. From this it is expected that wind generation on the Irish electricity system will be in the region of 32-37% of total generation. This leads to issues resulting from wind energy being a non-synchronous, unpredictable and variable source of energy use on a scale never seen before for a single synchronous system. If changes are not made to traditional operational practices, the efficient running of the electricity system will be directly affected by these issues in the coming years. Using models of the electricity system for the all-island grid of Ireland, the effects of high wind energy penetration expected to be present in 2020 are examined. These models were developed using a unit commitment, economic dispatch tool called PLEXOS which allows for a detailed representation of the electricity system to be achieved down to individual generator level. These models replicate the true running of the electricity system through use of day-ahead scheduling and semi-relaxed use of these schedules that reflects the Transmission System Operator's of real time decision making on dispatch. In addition, it carefully considers other non-wind priority dispatch generation technologies that have an effect on the overall system. In the models developed, three main issues associated with wind energy integration were selected to be examined in detail to determine the sensitivity of assumptions presented in other studies. These three issues include wind energy's non-synchronous nature, its variability and spatial correlation, and its unpredictability. This leads to an examination of the effects in three areas: the need for system operation constraints required for system security; different onshore to offshore ratios of installed wind energy; and the degrees of accuracy in wind energy forecasting. Each of these areas directly impact the way in which the electricity system is run as they address each of the three issues associated with wind energy stated above, respectively. It is shown that assumptions in these three areas have a large effect on the results in terms of total generation costs, wind curtailment and generator technology type dispatch. In particular accounting for these issues has resulted in wind curtailment being predicted in much larger quantities than had been previously reported. This would have a large effect on wind energy companies because it is already a very low profit margin industry. Results from this work have shown that the relaxation of system operation constraints is crucial to the economic running of the electricity system with large improvements shown in the reduction of wind curtailment and system generation costs. There are clear benefits in having a proportion of the wind installed offshore in Ireland which would help to reduce variability of wind energy generation on the system and therefore reduce wind curtailment. With envisaged future improvements in day-ahead wind forecasting from 8% to 4% mean absolute error, there are potential reductions in wind curtailment system costs and open cycle gas turbine usage. This work illustrates the consequences of assumptions in the areas of system operation constraints, onshore/offshore installed wind capacities and accuracy in wind forecasting to better inform the true costs associated with running Ireland's changing electricity system as it continues to decarbonise into the near future. This work also proposes to illustrate, through the use of Ireland as a case study, the effects that will become ever more prevalent in other synchronous systems as they pursue a path of increasing renewable energy generation.

National, inter-institutional, graduate modules – blended learning approaches for PhD education in Ireland

This paper is a case study that describes the design and delivery of national PhD lectures with 40 PhD candidates in Digital Arts and Humanities in Ireland simultaneously to four remote locations, in Trinity College Dublin, in University College Cork, in NUI Maynooth and NUI Galway. Blended learning approaches were utilized to augment traditional teaching practices combining: face-to-face engagement, video-conferencing to multiple sites, social media lecture delivery support – a live blog and micro blogging, shared, open student web presence online. Techniques for creating an effective, active learning environment were discerned via a range of learning options offered to students through student surveys after semester one. Students rejected the traditional lecture format, even through the novel delivery method via video link to a number of national academic institutions was employed. Students also rejected the use of a moderated forum as a means of creating engagement across the various institutions involved. Students preferred a mix of approaches for this online national engagement. The paper discusses successful methods used to promote interactive teaching and learning. These included Peer to peer learning, Workshop style delivery, Social media. The lecture became a national, synchronous workshop. The paper describes how allowing students to have a voice in the virtual classroom they become animated and engaged in an open culture of shared experience and scholarship, create networks beyond their institutions, and across disciplinary boundaries. We offer an analysis of our experiences to assist other educators in their course design, with a particular emphasis on social media engagement.

The international diffusion of project management

This paper examines the international diffusion of one business practice, project management, through the prism of prior literature and data on the diffusion of ISO 9000. The study took an inductive approach, building theory through the iterative collection and analysis of quantitative and qualitative data. The findings problematise the central position accorded to the S-curve model and neo-institutional theory in explaining technology diffusion. The research posits three distinct processes driving the diffusion process: utility, institutional isomorphism, and competitive isomorphism, with the latter consisting of three primary mechanisms: competitive imitation, trendslators and fashion retailers. Contrary to prior literature, national, quasi-professional associations are found to be central to the diffusion process and play a key role in advocating and containing management technologies.

The effects of centrally acting ACE inhibitors on the rate of cognitive and functional decline in dementia: a KDD approach

Alzheimer’s Disease and other dementias are one of the most challenging illnesses confronting countries with ageing populations. Treatment options for dementia are limited, and the costs are significant. There is a growing need to develop new treatments for dementia, especially for the elderly. There is also growing evidence that centrally acting angiotensin converting enzyme (ACE) inhibitors, which cross the blood-brain barrier, are associated with a reduced rate of cognitive and functional decline in dementia, especially in Alzheimer’s disease (AD). The aim of this research is to investigate the effects of centrally acting ACE inhibitors (CACE-Is) on the rate of cognitive and functional decline in dementia, using a three phased KDD process. KDD, as a scientific way to process and analysis clinical data, is used to find useful insights from a variety of clinical databases. The data used are from three clinic databases: Geriatric Assessment Tool (GAT), the Doxycycline and Rifampin for Alzheimer’s Disease (DARAD), and the Qmci validation databases, which were derived from several different geriatric clinics in Canada. This research involves patients diagnosed with AD, vascular or mixed dementia only. Patients were included if baseline and end-point (at least six months apart) Standardised Mini-Mental State Examination (SMMSE), Quick Mild Cognitive Impairment (Qmci) or Activities Daily Living (ADL) scores were available. Basically, the rates of change are compared between patients taking CACE-Is, and those not currently treated with CACE-Is. The results suggest that there is a statistically significant difference in the rate of decline in cognitive and functional scores between CACE-I and NoCACE-I patients. This research also validates that the Qmci, a new short assessment test, has potential to replace the current popular screening tests for cognition in the clinic and clinical trials.

Parasuicide and general practice: a pilot study

General Practitioners from Cork City and its environs were sent a questionnaire regarding their experience of parasuicide in the previous twelve months. Replies were received from 133 of the 185 GPs. 189 individuals, accounting for 212 episodes of parasuicide, were seen by 78 doctors, indicating a lower level of repetition than that found in hospital-referred cases. Almost a third of doctors saw no cases, just over one fifth saw one episode and the same proportion dealt with two. A small number of general practitioners saw many cases. Regarding management, 128 (60%) were referred to Casualty, 31 of whom were also referred for psychiatric care. Thirty percent were referred directly for psychiatric care. While only fourteen were retained within general practice without referral, 40% of the GPs felt that, ideally, acts of parasuicide should be retained with more specialised advice being obtained. Furthermore, 88.1% believed that management of parasuicide should form part of an integral part of post-graduate or continued general practitioner medical training. Clearly, GPs are willing to play a more active role in the management of parasuicide.

Prevalence and control of Clostridium difficile in patients with cystic fibrosis

The aim of this thesis was to investigate the high prevalence of Clostridium difficile in patients with cystic fibrosis (CF), and to control its dissemination. To determine the carriage rate of C. difficile in CF patients, 60 patients were tested for C. difficile and its toxin. In total, 50% of patients were found to be asymptomatic carriers of C. difficile despite toxin being detected in 31.66% of patients. Ribotyping of the C. difficile isolates revealed 16 distinct ribotypes, including the hyper virulent RT078. All isolates were sensitive to both Vancomycin and Metronidazole. The effect of CF and its treatment on the gut microbiota of CF patients was assessed by 16s sequencing of the gut microbiota of 68 CF patients. When compared to a healthy control group, CF patient gut microbiota was found to be less diverse and had an increased Firmicutes to Bacteriodetes ratio. Interestingly, CF patients who were carriers of C. difficile had a less diverse gut microbiota than C. difficile negative CF patients. Multilocus sequence typing was found to be comparable to PCR-ribotyping for typing C. difficile isolates from high risk patient groups. The sequence type ST 26 is potentially associated with CF patients as all seven isolates were found in this group and this sequence type has been previously reported in CF patients in a geographically distinct study. The bacteriophage ФCD6356 was assessed as a targeted antimicrobial against C. difficile in an ex-vivo model of the human distal colon. Despite reducing viable C. difficile by 1.75 logs over 24 hours, this bacteriophage was not suitable due to its lysogenic nature. Following treatment, all surviving C. difficile were immune to reinfection due to prophage integration. However, the ФCD6356 encoded endolysin was capable of reducing viable C. difficile by 2.9 over 2 hours in vitro after being cloned and expressed in Escherichia coli.

End-of-life care: identifying an appropriate legal framework for specialist palliative care in Ireland

This thesis argues that the legal framework in Ireland for specialist palliative care is inadequate and consequently a more appropriate legal framework must be identified. This research is guided by three central research questions. The first central research question examines the legitimacy of the distinction between specialist palliative care and euthanasia. The second central research question asks what legal framework currently exists in Ireland for specialist palliative care. The third central research question examines an alternative legal framework for specialist palliative. This thesis is composed of seven chapters. The first Chapter is an introduction to the thesis and defines the terminology and the central research questions. Chapter Two explores the development and practice of palliative care in Ireland. Chapter Three examines the distinction in criminal law between specialist palliative care practices and euthanasia. Chapter Four examines the human rights framework for specialist palliative care. Chapter Five critiques the regulatory framework in Ireland for specialist palliative care. Having gained a thorough understanding of palliative care and the related legal framework, this thesis then engages in comparative analysis of the Netherlands which is used as a source of ideas for reform in Ireland. Chapter Seven is the concluding chapter and, in it, the main findings of this thesis are summarised. The main findings being that: the distinction between specialist palliative care and euthanasia is not sufficiently supported by justifications such as a double effect or the acts and omissions distinction, there is no clear decision-making framework in Ireland for specialist palliative care, and the current legal framework lacks clarity and does not promote consistency between providers of specialist palliative care. This Chapter also proposes that detailed professional standards and guidelines are likely to be the most appropriate way to effect individual and institutional change in the provision of specialist palliative care.