Characterising novel anti-biofilm targets for the treatment of chronic Pseudomonas aeruginosa infection in the cystic fibrosis lung

The global rise in antibiotic resistance is a significant problem facing healthcare professionals. In particular within the cystic fibrosis (CF) lung, bacteria can establish chronic infection and resistance to a wide array of antibiotic therapies. One of the principle pathogens associated with chronic infection in the CF lung is Pseudomonas aeruginosa. P. aeruginosa can establish chronic infection in the CF lung partly through the use of the biofilm mode of growth. This biofilm mode of growth offers a considerable degree of protection from a wide variety of challenges such as the host immune system or antibiotic therapy. The threat posed by the emergence of chronic pathogens is prompting the development of next generation antimicrobials. The biofilm mode of growth is often central to the establishment of chronic infection and the development of antibiotic resistance. Thus, targeting biofilm formation has emerged as one of the principle strategies for the development of next generation antimicrobials. In this thesis two separate approaches were used to identify potential anti - biofilm targets. The first strategy focused on the identification of novel genes with a role in a biofilm formation. High throughput screening identified almost 300 genes which had a role in biofilm formation. A number of these genes were characterised at a phenotypic and a molecular level. The second strategy focused on the identification of compounds capable of inhibiting biofilm formation. A collection of marine sponge isolated bacteria were screened for the ability to inhibit the central pathway regulating biofilm formation, quorum sensing. A number of distinct isolates were identified that had quorum sensing inhibition activity from which, a Pseudomonas isolate was selected for further characterisation. A specific compound capable of inhibiting quorum sensing was identified using chemical analytical technologies in the supernatant of this marine isolate.

The role of bacterial interspecies communication in polymicrobial infection of the cystic fibrosis lung

There is an increasing appreciation of the polymicrobial nature of bacterial infections associated with Cystic Fibrosis (CF) and of the important role for interactions in influencing bacterial virulence and response to therapy. Patients with CF are co-infected with Pseudomonas aeruginosa, Burkholderia cenocepacia and Stenotrophomonas maltophilia. These latter bacteria produce signal molecules of the diffusible signal factor (DSF) family, which are cis-2-unsaturated fatty acids. Previous studies showed that DSF from S. maltophilia leads to altered biofilm formation and increased tolerance to antibiotics in P. aeruginosa and that these responses require the P. aeruginosa sensor kinase PA1396. The work in this thesis aims of further elucidate the influence and mechanism of DSF signalling on P. aeruginosa and examine the role that such interspecies signalling play in infection of the CF airway. Next generation sequencing technologies targeting the 16S ribosomal RNA gene were applied to DNA and RNA isolated from sputum taken from cohorts of CF and non-CF subjects to characterise the bacterial community. In parallel, metabolomics analysis of sputum provided insight into the environment of the CF airway. This analysis revealed a number of observations including; that differences in metabolites occur in sputum taken from clinically stable CF patients and those with exacerbation and DNA- and RNA-based methods suggested that a strong relationship existed between the abundance of specific strict anaerobes and fluctuations in the level of metabolites during exacerbation. DSF family signals were also detected in the sputum and a correlation with the presence of DSFproducing organisms was observed. To examine the signal transduction mechanisms used by P. aeruginosa, bioinformatics with site directed mutagenesis were employed to identify signalling partners for PA1396. A pathway suggesting a role for a number of proteins in the regulation of several factors following DSF recognition by PA1396 were observed.

Skin and bone: the face in the archaeological imagination

Thirteen unique archaeological countenances from Ireland were produced through the Manchester method of facial reconstruction. Their gaze prompts a space for a broad discourse regarding the face found within human and artefactual remains of Ireland. These faces are reminders of the human element which is at the core of the discipline of archaeology. These re-constructions create a voyeuristic relationship with the past. At once sating a curiosity about the past, facial reconstructions also provide a catharsis to our presently situated selves. As powerful visual documents, archaeological facial reconstructions illustrate re-presentations of the past as well as how the present can be connected to the past. Through engagment with Emmanuel Levinas’s (1906- 1995) main philosophical themes, the presence of the face is examined in a diachronic structure. The ‘starting point’ is the Neolithic period which has been associated with the notion of visuality with a reconstruction from the early Neolithic site of Annagh, Co. Limerick. The following layer of analysis appears with attention to intersubjectivity in the early medieval period with facial reconstructions from Dooey, Co. Donegal and Owenbristy, Co. Galway. Building upon the past concepts, the late medieval period is associated with the notion of alterity and paired with faces from Ballinderry, Co. Kildare and a sample of males from Gallen Priory, Co. Offaly. The final layer of examination culminates with the application of response and respons-ibility to the post-medieval Irish landscape with facial reconstructions from the prison on Spike Island, Co. Cork. These layers of investigation are similar to the stratigraphical composition of both the archaeological landscape and the skeletal/soft tissue landscape of the face. The separation of the neglected phenomenon of the face from the overwhelming embrace of the field of craniometrics is necessary. Through this detachment a new manner in which to discuss the face and its place within the (bio)archaeological record is possible. Encountering the faces seen in mortuary contexts, material culture, and archaeological facial reconstructions, inform and shape the archaeological imagination.