Epithelial branching morphogenesis is a critical step in the development of many epithelial organs including the lungs and the salivary glands. Many of the signalling pathways that orchestrate this process show a resurgence in the diseased state. We sought to determine the role of a common disease pathway, the phosphoinositide 3-kinase (PI3K) pathway, in the branching program of the lung. We utilised ex-vivo cultures of embryonic murine lungs that best recapitulates the developmental process. Inhibition of PI3Kα, or its downstream signalling components Akt and mammalian target of rapamycin, was able to greatly enhance the branching potential of embryonic lungs, implying a negative role for PI3K in the lung branching morphogenesis. Moreover inhibition of PI3K was sufficient to alter the morphogenic properties of fibroblast growth factor 7 on isolated murine lung epithelium from a cystic to a branching response. We also investigated protein mediated drug delivery tools to provide a means of enhancing the delivery of PI3K based therapeutics. Cell-penetrating peptides (CPPs) are small proteins that are able to transport a drug cargo across the membrane of a cell. However, given that many CPPs are derived from viral proteins and venoms we sought to determine if common CPPs could induce an immune response. CPPs we capable of delivering a protein cargo into the interior of epithelial cells without inducing an immune response as measured by a lack of NFκB activation and no observable increase in interleukin-6 or -8 secretion. We finally explored the potential of repurposing bacterial toxins for therapeutic applications. The toxins Pseudomonas aeruginosa exotoxin A (PEx) and Cholix (Chx) are natively capable of entering cells and subsequently being transported to either the cytoplasm of non-polarised cells, or undergoing transcytosis across polarised cells. We were able to identify that the C16:1 ceramide chain variant of the ganglioside GM1 is capable of enhancing the intracellular transport of PEx to the Golgi network. Moreover, we demonstrated that PEx is able to transport an siRNA cargo across polarised monolayers of Caco-2 cells and deliver said cargo into cells present in the basolateral compartment.

Glucocorticoids have diverse physiological functions: they affect central nervous system function, intermediary metabolism and restore homeostasis after stress. Secretion of glucocorticoids is regulated by the hypothalamic-pituitary-adrenal (HPA) axis; negative feedback at the hypothalamus and pituitary suppresses glucocorticoid secretion while the hippocampus exerts additional control over HPA axis activity. Glucocorticoids exert most of their actions, including negative feedback, via the glucocorticoid receptor (GR). The glucocorticoid sensitivity of a given cell/tissue is dependent on the level of GR expression. The regulation of the GR gene is complex; GR levels in adult animals are subject to glucocorticoid regulation and can be permanently "programmed" by early life events, with hippocampal GR permanently increased by neonatal handling (via alterations in serotonin turnover) and decreased by prenatal dexamethasone exposure. Evidence suggests that these effects may be mediated through differential regulation of variant exon-1 containing GR mRNAs; in rats the GR gene contains 8 protein-coding exons (exons 2-9) and at least 11 alternate untranslated exons 1 (exons li-ln) which may reflect transcription regulated by alternate promoters. The aim of this thesis was to further investigate the distribution of these variant GR transcripts and examine whether glucocorticoids themselves differentially regulate GR mRNA and its alternate exons 1 in a tissue and region-specific manner. Tissue and region-specific differences in the expression of variant GR mRNA transcripts were found in rat and mouse. Most GR mRNA variants were ubiquitously expressed, but those containing exon 11 were restricted to rat thymus, liver and hippocampus and mouse spleen, while those containing exon 14 were absent from rat cerebral cortex and mouse lung, heart and abdominal fat. In situ mRNA hybridisation on rat brain showed that all the exons 1 studied showed differences in their regional expression when compared to distribution of the total population of GR mRNAs. In contrast, in rat liver and thymus GR mRNA variants showed the same regional distribution as total GR mRNA with highest expression in periportal region of the liver and the thymic cortex. To investigate whether glucocorticoids differentially regulate variant GR mRNAs, in situ mRNA hybridisation was used to investigate the expression variant exons 1 in the hippocampus of adult rats subjected to 72h (ST) or 3-week (LT) adrenalectomy with glucocorticoid replacement. Variant GR mRNAs containing exons 1₅, 1₇, 1₁₀ and 1₁₁ were not affected by adrenalectomy or supraphysiological glucocorticoid replacement in ST or LT animals. However, both adrenalectomy and supraphysiological glucocorticoid replacement significantly upregulated total GR mRNA in the hippocampus of ST animals while adrenalectomy significantly upregulated total GR mRNA in the hippocampus of LT animals. In situ mRNA hybridisation and RNase protection analysis were used to investigate expression of variant GR mRNAs in the liver. Glucocorticoid manipulations did not significantly affect expression of variant GR mRNAs containing exons 1₅, 1₆, 1₁₀ and 1₁₁. However, in ST adrenalectomised animals glucocorticoid replacement significantly downregulated GR mRNA levels compared to adrenalectomised animals given vehicle alone. Adrenalectomy had no effect on total GR mRNA expression in the LT animals, although in these animals the periportal :perivenous ratio of GR expression was significantly increased by adrenalectomy compared to sham. Preliminary data from DNase I hypersensitive site mapping in control animals showed an area of DNase I sensitive chromatin around the position of exon 1₁₀, present in the majority of GR mRNA in the liver. In the thymus, although adrenalectomy with either high and low dose glucocorticoid replacement in ST animals caused a significant downregulation of GR mRNA in the cortex and medulla compared to sham, the expression of exons 1₁, 1₆ and 1₁₀ of the GR gene was not significantly affected by glucocorticoid manipulations. There was no effect of glucocorticoid manipulation on GR or its variants in the LT animals. These results demonstrate tissue-specific differences in the distribution of GR mRNA variants, suggesting that variations in promoter usage may have a role in determining the "set-point" of GR expression in different tissues. The observed tissue-specific effects of glucocorticoids on GR mRNA expression could not be accounted for by changes in expression of any of the variant GR mRNA transcripts studied. This suggests that either the expression of another variant mRNA (known or novel) is regulated by glucocorticoids or that expression of all or a subset of the variant GR mRNAs changed with a magnitude too small to be detected in this study.

Drugs of abuse alter the neurochemistry of the human brain. This is axiomatic, for if they did not, they would be inactive and hence not abused. My interest in the study of the neurobiology of dependence has centred on the use of the techniques of functional neuroimaging to examine the responses and changes of the living human brain to the processes of substance misuse. As one would expect, this thesis therefore follows that path. After a brief description of the social history of substance misuse, it is intended to begin with an explanation of the variety of techniques available today. This will not be an exhaustive, nor complete, list but will restrict itself to those techniques that feature in the rest of the thesis. A complete list and descriptions of the relative merits of them all would be sufficient to fill an entire thesis alone. Following on from the description of neuroimaging techniques, this thesis will discuss the previous use of these to the field of opioid addiction and dependence. As the final chapter will be largely discussing the common findings and changes in neurobiology found across the spectrum of drugs of abuse, this review will not be confined solely to the study of opioid dependence; however, all the research work carried out for this thesis focused entirely on this disorder. The middle section of this thesis will focus on the empirical research carried out using Positron Emission Tomography (PET) techniques to examine the neurobiology of opioid dependence. There are two separate studies contained within this section; each beginning with a detailed discussion of the scanning and analysis techniques developed and used, and each using a different sub-type of PET imaging to examine different components of the disorder. The final chapter will attempt to draw together the results from the review of the published literature with the experimental results. The aim is to show the increasing parallels being found from different methodologies examining different drugs of abuse and hence provide evidence for core underlying neurobiological changes that are implicated in the central concept of addiction.

In the biopharmaceutical industry being first to market is often key to product success. However process development still needs to deliver an economic, regulatory compliant and effective process. Scale-down studies are commonly used for this purpose since they are quicker and cheaper than pilot plant work. However it is necessary that scale-down methods be created which not only examine the conditions of process stages but also allow production of realistic output streams. These output streams can then be used in the development of subsequent purification operations and facilitate rapid and efficient process development while minimising early investment and risk. Scale-down techniques were used to predict the effects of process changes in a plasma fractionation operation. These predictions were compared to pilot plant data and the full- scale process to assess their accuracy. The scale-down techniques used did not predict the performance of full-scale operations reliably despite being in close agreement with pilot plant results. This is due to the limitations of current scale-down techniques, which focus on a few core parameters and fail to examine engineering and operational details of larger processes. Traditionally, predicting filtration operations is via a bench-top pressure filter, using constant pressure tests to examine the effect of pressure on filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (?) and compressibility (n) is used to predict the pressure profile required to maintain a constant, predetermined flux rate. This thesis reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to those produced at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down method gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional laboratory batch filter. Very small chromatography columns can be useful tools for narrowing down the myriad of process options involved with this operation, but operational practicalities mean that traditional scale-down rules must be broken. Additionally a number of factors make their operation and subsequent interpretation of results problematic. By identifying and then quantifying the effects of these phenomena it is possible to compensate for their impact. By making several calculated adjustments to the outputs from a small column it was possible to predict the performance of much larger laboratory columns.

No description available.

Biopharmaceutical development is often delayed in part by the extensive stability analysis and formulation refinement entailed in the early stages. Determination of a suitable therapeutic protein or peptide and the ideal formulation requires consideration of t he delivery route, dose and the stability of both the active ingredient and the formulation as a whole. The selection of appropriate buffering agents and other excipients is of utmost importance in this process, but can be time consuming due to their huge variety and the necessity for thorough testing of each component. Currently this testing is performed by a number of different techniques eit her to observe changes in protein structure or by detecting aggregation once it has occurred. Considering this, we identi› fied a need for an approach which, using a small amount of material, had the capability to detect the propensity for a given biopharmaceutical to be unstable and/or aggregate in a part icular set of formulation conditions. Such a technique could allow additional screening to be performed earlier in the drug development process, enabling the early identification and further refinement of drug candid ates and formulations. An atomic force microscopy force measurement based technique was proposed to meet t his need. This thesis details the first steps in developing an assay fit for this purpose.

Background: Sepsis is a common reason for admission to adult, paediatric or neonatal intensive care and mortality remains high. Evidence of increased pharmacokinetic (PK) variability in critically ill adults compared to non-critically ill has raised concerns about antimicrobial target attainment and dosing strategies. Paediatric data is lacking. Aims: 1. Summarise published data on the PK of commonly used antibiotics across all age groups, in healthy and critically ill populations. 2. Describe the PK of commonly used antibiotics in patients receiving treatment in adult, paediatric and neonatal intensive care and describe the effect of age on antibiotic PK. 3. Describe attainment of pharmacokinetic/pharmacodynamic (PK/PD) targets for antibiotics in critically ill patients of all ages and outline associations between PK/PD target attainment and clinical outcome. Methods: 1. Systematic review of PK data in adult, paediatric and neonatal populations. 2. Prospective, observational study of the PK of 10 commonly used antimicrobials in critically ill adults, children and neonates. 3. Population-PK techniques (NONMEM) to model antibiotic PK and PK/PD target attainment in critical illness, using data from all age groups. Inclusion of a maturation function to model the effect of age on PK parameters Results: 1. Parameters from 143 studies were pooled to estimate PK maturation- decline through life. There was marked uncertainty in parameter estimates. 2. PK study of 230 critically ill participants (300 antibiotic prescriptions) and 1283 PK samples. Age range 24-weeks post-menstrual age to 90-years. 3. Population-PK models of 10 antibiotics using all age groups. Maturation-decline function describes changes in PK through life well. Only 74% of antibiotic courses achieved minimum PK/PD targets. Paediatric participants had lower target attainment than adults. Conclusion: Age need not be a barrierto inclusion in a PK trial. A new standard method for modelling PK maturation throughout life has been proposed that could be used in future age-inclusive PK studies. The first age-inclusive PK models of beta-lactams have been presented with the PK of intravenous benzylpenicillin and clarithromycin described for the first time in critically ill populations. Standard doses fail to achieve PK/PD targets in critically ill adults, children and neonates.

No description available.

This thesis describes an investigation of the permeability of three antiretroviral (ARV) drugs (that are under development as vaginal microbicides) using the human endometrial epithelial cell line, HEC-1A, as an in vitro model. Barrier properties of HEC-1A cells in transwell cultures were determined by measurement of transepithelial electrical resistance (TEER), immunofluorescent staining of tight junctions and determination of the bi-directional permeability of, the paracellular marker, mannitol. Expression of specific uptake and efflux transporters were demonstrated by western blotting and compared with human tissue. Findings indicate that HEC-1A cells provide a physiologically relevant model to investigate permeability of candidate vaginal microbicides. In contrast, the commercially available EpiVagina™ model showed sub-optimal barrier properties. Permeabilities of three ARV drugs Tenofovir (TFV), Darunavir (DRV) and Dapivirine (DPV) were investigated in the HEC-1A model. Efflux ratios of all drugs were approximately 1 indicating transporter-independent permeability across the epithelium. Apparent permeability (Papp) values for TFV were consistent with paracellular diffusion while those for DRV and DPV were indicative of transcellular diffusion. No drug-drug interactions were observed when drugs were coadministered in double combinations. To model the effects of inflammation on drug uptake, HEC-1A cultures were stimulated with a variety of toll-like receptor (TLR) ligands. The TLR-3 ligand Poly I:C stimulated pro-inflammatory cytokine production by HEC-1A cells but did not affect permeability of the drugs and no effect on TEER values was evident. Permeability of DRV was also assessed using ex vivo rat and macaque cervicovaginal epithelial tissue. Consistent with findings from the HEC-1A model, DRV transfer across rat epithelium was transporter-independent although the Papp values were significantly lower. Preliminary data from macaque tissue indicate DRV may be effluxed. Development of a robust and physiologically-relevant in vitro model will contribute to development of vaginal microbicides by allowing rapid measurement of drug transporter dependency, drug-drug interactions and testing of formulations aimed at optimising tissue distribution. In this study, all of the ARVs showed transporterindependent transfer across the epithelium with no drug-drug interactions.

This thesis examines the effects of chronic use of ketamine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, on subjective experience and cognition. It is important to explore the chronic effects of ketamine as the number of individuals using the drug recreationally is increasing both in the UK and worldwide. There is a paucity of research exploring the chronic effects of ketamine. Many studies have shown that acutely ketamine induces psychotic-like symptomatology and specific cognitive dysfunction in healthy, drug-naive volunteers. For this reason, a ketamine model of the psychoses has been proposed. However the few studies of the effects of chronic ketamine have provided mixed findings. Part 1 of the thesis comprises a literature review, which investigates the psychotomimetic effects of ketamine, through the synthesis of current research findings, to determine whether ketamine is a useful model of the symptomatology characteristic of the psychoses. It presents an overview of ketamine and its association with the psychoses, before providing a detailed account of the functional psychoses and drug models of the psychoses (namely the dopamine hypothesis, the serotonin hypothesis and the glutamate hypothesis). The review then synthesises the acute and chronic ketamine studies to date, highlighting which states appear to be best modelled (i.e. the pre-psychotic, acute or chronic state experienced by individuals with idiopathic psychoses). Finally, the review briefly considers the treatment implications of the ketamine model of psychoses, and the risk chronic ketamine use poses to users in terms of developing fully-manifest psychotic symptomatology. In Part 2, an investigation of the chronic effects of ketamine on subjective experiences and cognitive functioning is reported, in order to determine whether chronic ketamine models symptomatology associated with the pre-psychotic state of idiopathic psychoses (where the term idiopathic refers to psychotic symptomatology of unknown aetiology, i.e. that which occurs in the majority of the general population and is not drug-induced). This investigation was part of a joint project conducted with 2 other trainees to investigate the chronic effects of ketamine, cannabis and cocaine on subjective experiences and cognitive functioning (See Appendix 1 for details of the contribution made by each trainee). The empirical paper reports a between subjects study which compared 21 frequent ketamine users (who used ketamine daily), 20 infrequent ketamine users (who used ketamine a maximum of once or twice a week) and 20 controls (who reported no illicit drug use). On a clinical index of symptomatology (SPI-A), a 'frequency' effect was observed: frequent ketamine users were found to be higher in psychotic-like symptomatology (i.e. basic symptoms) than infrequent users, who in turn were found to be higher in symptomatology than controls. Both groups of ketamine users were also found to be higher in psychosis proneness on a general population index of psychotic-like markers (OLIFE) compared with controls. Furthermore, both groups of ketamine users demonstrated impaired episodic memory and working memory compared to controls. Group differences were found in executive functioning. Part 3 comprises a critical appraisal of the research. It includes reflections on my experience of the research process and conducting research with the ketamine using population, as well as reflections on clinically relevant observations.