Search

Cardiac arrests in the infant population result in undesirable rates of both mortality and morbidity. Cardiopulmonary resuscitation (CPR) is a potentially life-saving emergency procedure, performed during cardiac arrest, which aims to promote blood flow to vital organs through the provision of external chest compressions. To improve cardiac arrest outcomes, current international resuscitation guidelines emphasise the provision of high quality chest compressions during infant CPR. The principle goal of this research was to monitor, assess and engineer an improvement in the quality of chest compressions performed during simulated infant CPR. This was investigated in three experimental stages that evaluated: (i) the current quality of chest compressions performed on a commercially available manikin, (ii) the effects of a more ‘physiological’ infant CPR training manikin design on chest compression quality and thoracic over-compression and (iii) the effects of a real-time performance feedback program, developed to aid resuscitators in achieving high quality chest compressions. Performance was benchmarked against infant specific evidence based quality targets, with highly trained resuscitators achieving these targets in <9% of chest compressions during unassisted simulated infant CPR. The potential for thoracic over-compression in clinical practice was also highlighted when investigating the effects of the more ‘physiological’ infant CPR manikin design. The provision of real-time performance feedback considerably improved chest compression quality, with resuscitators achieving quality targets in >70% of all chest compressions. This research indicates that unassisted chest compressions rarely comply with evidence based quality targets during simulated infant CPR. Real-time performance feedback, in combination with a more ‘physiological’ infant CPR manikin, can considerably improve the quality of chest compressions performed by resuscitators. Importantly, these findings provide an excellent foundation for translation into clinical practice, particularly for assisting resuscitators in providing high quality chest compressions during infant CPR, which may contribute to improving the future outcomes of infant cardiac arrest.

Shigella flexneri is the causative agent of bacillary dysentery and utilises the Type III Secretion System (T3SS) for cellular invasion. The central component of the T3SS is the needle complex(NC), which resembles a molecular 'syringe' and is used by S. flexneri to secrete proteins directly into the host cell, thereby instigating the invasive process. After host cell sensing and activation via the tip complex (TC), which sits at the extremity of a 50 nm-Iong needle, a signal is thought to be transmitted from the needle to the base, resulting in activation of protein secretion. Precisely how host cell sensing occurs and how this leads to protein secretion in the base remains to be fully elucidated. Here, we used electron microscopy (EM) and mass spectrometry (MS) to investigate the structure of the NC with a view to decipher the structural basis of its function. Utilising cryoEM, we solved the structure of the needle in both wild type and activated states to sub-nanometer resolution, allowing us to visualise key inter-subunit interactions made by the needle subunit, MxiH. Whilst no structural differences were seen between wild type and activated needles, a previously unidentified hairpin motif was elucidated, which was shown to form part of a crucial inter-subunit junction required for needle polymerisation and possibly signal transduction. Using negative stain EM, we produced a moderate resolution density map of the wild type TC, revealing four copies of protein IpaD and one copy of IpaB. The reconstruction showed the subunits to be tilted, resulting in a proposed metastable TC. Reconstructions of TC's in partially activated and fully activated states revealed a correlation between subunit tilting/bending/rotating and activation, allow us to propose a mechanism for TC function. Furthermore, we show that the TC subunits probably interact with the needle via their C termini and with the crucial needle junction. In order to elucidate the architecture of the base, we used EM and Absolute Quantification (AQUA) peptides to show that the NC outer membrane ring(OMR) has C IS rotationally symmetry and that the inner membrane ring (IMR) has 24 subunits, possibly arranged in a C 12 configuration. We also show that each NC possibly has one copy of the export apparatus protein MxiA. We also report here the transmission of a 3.9MDa complex by native MS, the largest membrane protein analysed by this method and possibly representing the mass of the full intact Ne. Finally, we detail a new method for cryoEM grid preparation that overcomes a long standing problem in cryoEM of macromolecular complexes - sample adsorption to the grid surface - which has the potential to be applied to a diverse range of samples.

Investigating the mechanisms of regulation of drug disposition is important in understanding pharmacokinetic (PK) variability and rationalising drug-drug interactions. Ultimately, drug absorption in the intestine and drug elimination from the liver are the major determinants of PK. Key proteins involved in these processes are cytochrome P450 enzymes (CYPs) and ABC-transporters. These proteins are transcriptionally regulated by a complex network of nuclear receptor (NR) type transcription factors and many of these NRs are activated by endogenous or exogenous chemicals. The study of these mechanisms in rodent . models has been complicated by the reported species differences in activation of NRs. The aim of this thesis was to assess the spectrum of induction of CYPs, transporters and NRs in response to paradigm NR-activators in a battery of human and rat in-vitro systems. The aim of chapter two was to validate robust methodology for quantitatively assessing mRNA and protein expression of CYP2B6, CYP3A4, ABCB1, ABCC1, ABCC2, CAR, FXR, PXR and their rat homologues. In addition, since there are many conflicting reports in the literature, the impact of using dexamethasone (OEX) as a media supplement was assessed. Previous studies have illustrated species differences with pregnenalone-16a-carbonitrile (PCN) activating rodent but not human PXR, and rifampicin (RIF) activating human but not rodent PXR. The data presented in this chapter illustrate that OEX induces human PXR, CYP3A4, ABCB1 and rat orthologues. Furthermore, in the absence of OEX, RIF and PCN increased expression of human CYP3A4, ABCB1 and rat orthologues to a similar extent. These data suggest that there is a maximal induction and the addition of OEX effectively increases the baseline. However, since the maximum is fixed a reduction in the observed effect is evident. Therefore, although there are species differences in activation of PXR, the impact on target gene expression appears to be similar. For this reason, OEX was not incorporated into the culture media in SUbsequent chapters. The aim of chapter three was to investigate the impact of Phenobarbital (PB; CAR activator), PCN (rodent PXR activator), chenodeoxycholic acid (COCA; FXR activator), RIF (human PXR activator) and 9-cis-retinoic acid (9-cisRA; RXR activator) on expression of CYPs, transporters and NRs in human (Caco-2) and rat (iec-6) intestinal cell lines. Significant induction of transcripts in both human and rat intestinal cell lines were observed. However, some differences were seen in the induction profiles in Caco-2 versus lec-6 cells. In general, logarithmic correlations between the changes in mRNA and protein were also observed. Lower changes in mRNA elicited linear increases in protein but there appeared to be a threshold beyond which no additional increase in protein was observed. In chapter four, these experiments were repeated in the human (HepG2) and rat (H411e) hepatic cell lines. Again, induction of transcripts in both human and rat hepatic cell lines was observed. In hepatic and intestinal cell lines concentration-dependent responses were observed in most cases. However, i~ some cases, bell shaped concentration-response profiles were observed. In most cases these were related to toxicity but in some cases sub-maximal induction was observed at concentrations that were non-toxic. These data indicate that using a single concentration may result in gross underestimation of induction and may also explain some of the discrepancies in the literature. Logarithmic correlations between the changes in mRNA and protein in HepG2 and H411 e cells were also observed. Similar to intestinal cell lines, lower changes in mRNA elicited linear increases in protein. This is most likely due to the time which analysis was conducted; mRNA induction being more rapid than that of protein. The aim of chapter five was to investigate these observations in human (cryopreserved from two donors) and rat (fresh) hepatocYtes. In these studies, a sub·toxic concentration (1IJM) causing maximal induction of most transcripts in cell lines was chosen and a time course was conducted to circumvent problems associated with a decrease in viability over time. Protein was not investigated because insufficient material was observed after incubation with typical NR activators. Generally, maximal induction was observed at 6 hours in human and at 4 hours in rat hepatocytes, and in most instances time-dependent induction was observed. Interestingly, there was variation in the observed induction between the human donors. These data were in broad agreement with data generated in cell lines, and where overlap existed were also in agreement with previous studies in the literature. The aim of chapter six was to investigate activation of a range of human NRsby the activators utilised in previous chapters. This was particularly important given that similar induction of target genes was observed, even where species differences have been documented. In order to probe the mechanisms that underpin these observations, mammalian 1 hybrid ligand binding assays were employed for PXR, VOR, PPARy, LXRa, FXRa and RXRa in Caco-2 and CHO-K1 cells. All NRs showed some degree of promiscuity, with LXRa appearing the most promiscuous. PPARy was activated by PCN, which is particularly interesting in light of previously reported PPAR response elements within the PXR proximal promoter. COCA significantly activated FXRa and LXRa in CHO-K1 cells. In addition, LXRa, FXRa and VOR were all activated by COCA in Caco-2 cells. In summary, variation in induction was observed between model systems for both tissue and species. However, in the absence of OEX, both rat and human PXR and PXR target genes were activated by PCN and RIF to a similar extent. In addition, a number of novel observations were made. For example upregulation of NRs by their purported activators was observed, which raises the possibility of self regulation. Further studies, which utilise reporter constructs from the target gene promoters coupled with NR expression vectors, are now required to investigate which NRs are involved. Work has now also begun to utilise targeted knockdown of NRs (with SiRNA), in order to determine the relative contribution of individual NRs in the reported induction.