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The rapid rise in the prevalence of pathogenic bacteria that are resistant to front-line antibiotic drugs has necessitated a simultaneous upsurge in the use of "last-resort" antimicrobial agents. Colistin is one such antibiotic of last-resort that is increasingly used in the clinic as a salvage therapy to treat infections caused by multi-drug resistant Gram-negative microorganisms, including Pseudomonas aeruginosa and Escherichia coli. Unfortunately, despite its growing importance, colistin treatment is toxic, frequently fails, and resistance to the antibiotic is an intensifying threat. There is, therefore, a crucial requirement to augment the efficacy of colistin therapy, but efforts to do so are hampered by a lack of understanding about the drug's mechanism of action. The work in this thesis initially uncovered a novel process by which colistin-susceptible P. aeruginosa cells survive exposure to the antibiotic, through the extracellular release of lipopolysaccharide (LPS) molecules that inactivate colistin in the external environment. In attempting to overcome this mode of drug tolerance by inhibiting LPS biosynthesis, critical insight into colistin's bactericidal mechanism was obtained - namely, that the antibiotic kills Gram-negative bacteria by targeting LPS in the cytoplasmic membrane, not by interacting with membrane phospholipids, as previously thought. This finding in turn led to investigations about the site where colistin resistance, mediated by cationic chemical modifications to LPS, was conferred. It was shown that resistance to colistin in P. aeruginosa and E. coli, acquired through the harbouring of diverse plasmid-borne mobile colistin resistance (mcr) genes or chromosomal mutations, was in fact conferred at the cytoplasmic membrane, as opposed to the outer membrane of bacterial cells. Subsequent experiments revealed that intrinsic colistin resistance in Burkholderia cenocepacia was mediated at the outer membrane, and that strains of Enterobacter cloacae possess a unique inducible form of hetero-resistance to colistin. After characterising the mode of action of colistin and a number of potential causes of colistin treatment failure, new combination treatment strategies were designed to improve the effectiveness of colistin therapy. Using murepavadin, an inhibitor of the LPS transport system in pre-clinical development, to accumulate LPS in the cytoplasmic membrane proved to be particularly potent at amplifying the bactericidal activity of colistin in vitro against clinical isolates, in an in vivo murine lung infection model, and for overcoming colistin resistance. Furthermore, the capacity for colistin to permeabilise the outer membrane of colistin-resistant bacteria was exploited to re-sensitise Gram-negative pathogenic organisms to rifampicin, an antibiotic which normally cannot traverse the cell envelope. In summary, this work has identified how colistin works and why it often fails clinically, as well as providing urgently-needed novel solutions to enhance colistin efficacy and patient outcomes.

Rectangular channel geometries are widely encountered in supersonic flows, such as in wind tunnels and in aircraft inlets. Shock-boundary-layer interactions in these flows are known to exhibit significant three-dimensionality, due to the presence of sidewalls and associated corner boundary layers. The main effect is on the local separation of these corner regions, which then affects the wider flow field. Successful prediction of the overall flow therefore relies on the corner separation to be determined accurately. This, in turn, requires knowledge of the flow momentum distribution within the corner boundary layers. However, numerical methods struggle to reliably compute these flows and there is not much experimental data on supersonic corner boundary layers for comparison. This thesis addresses the outstanding gap in knowledge by performing validation-quality experiments on the corner regions of a Mach 2.5 channel flow, with a unit Reynolds number of approximately 40 million per metre. The experiments are conducted in the rectangular test section of a supersonic wind tunnel at the University of Cambridge. An analysis of the wind tunnel experiments, alongside computational data provided by the US Air Force Research Laboratory, reveals that the corner boundary layers are strongly influenced by the geometry of the two-dimensional nozzles used to produce the supersonic flow. The dominant effect is related to bulk vertical velocities within the sidewall boundary layers, induced by vertical pressure gradients in the nozzle. For some very particular geometries, a second influence may be associated with a region of separated flow immediately ahead of the nozzle, which generates vortices within the sidewall boundary layer. Through these mechanisms, the nozzle geometry is seen to strongly influence both the thickness and the structure of the corner boundary layers. High-quality experimental data in the corner regions are used to validate relevant numerical methods. Simple linear eddy-viscosity type turbulence models are found to compute these flows particularly poorly, with a 7% discrepancy in streamwise velocity. This is largely due to the fact that they do not capture known, stress-induced, corner vortices. However, the quadratic constitutive relation improves prediction of the corner boundary-layer structure, reducing experimental-computational differences by as much as half. This improvement is associated with vorticity generation in these corner regions, albeit with slightly different properties to the physical vortices. This production of vorticity depends only on the presence of a quadratic term in the eddy-viscosity model and not on which particular quadratic term is used. A more general form of the quadratic constitutive relation with one additional term is proposed, which appears to exhibit substantial improvements in the prediction of turbulent stress anisotropies. The nozzle geometry effects are exploited to produce two otherwise-identical experimental setups with distinctly different momentum distributions in the corner boundary layers. A full-span wedge introduces an oblique shock with flow deflection angle, 8 degrees, which impinges on the floor boundary layer. The two setups exhibit quite dissimilar separation behaviour, not only in the corner regions but also on the tunnel's centre span, with a difference in central separation length of as much as 35%. The observed behaviour is consistent with expectations based on local flow momentum affecting corner separation size, and on the displacement effect of this corner separation influencing the wider flow.

The feasibility of hot melt extrusion (HME) was explored for development of amorphous solid dispersion systems. Controlled release formulations were developed using a cellulose based derivative, AffinisolTMHPMC 100cP and 4M grades. BCS class II drugs ibuprofen and posaconazole were selected due to their difference in glass transition temperature and lipophilicity. This study focused on investigation of the impact the material attributes and process parameters on the critical quality attributes in preparation of amorphous solid dispersions using hot melt extrusion. The critical quality attributes were sub divided into three main attributes of material, process and product. Rheology of ibuprofen-Affinisol 100cP from melt phase to extrudate phase was tracked. A partial factorial design was carried out to investigate the critical parameters affecting HME. For optimisation of 40%IBU-Affinisol 100cP blends, a feed rate of 0.6kg/hr, screw speed of 500rpm and screw configuration with two mixing elements were found to be optimum for single phase extrudates. ATR-FTIR spectroscopy was found to be an indirect technique of choice in predicting the maximum ibuprofen drug load within extrudates. Prediction was based on the prepared extrudates without charging them to stability conditions. An alternative strategy of incorporation of di-carboxylic acids to increase the dissolution of posaconazole-Affinisol 4M blends was investigated. Succinic acid and L- malic acid incorporation was found to increase the dissolution of posaconazole. Although, the extrudates crystallised out quicker than the naïve posaconazole-Affinisol 4M, but free posaconazole formed eutectic and co-crystal with succinic and L-malic acid within extrudates. This lead to an increase in dissolution of the extrudates compared to day 0.

Background: Paediatric ependymomas are the second most common malignant brain tumours in children. Tumour recurrence, chemoresistance and invasion of surrounding critical structures are the hallmarks of ependymomas. These features are consistent with the cancer stem cell (CSC) hypothesis which states that tumours harbour a sub-population of stem-like cells which underlie therapeutic resistance. This study investigates the role of the radial glial stem cell marker BLBP, the multidrug transporter ABCB1, and the DNA repair enzyme MGMT in therapy failure in ependymomas with particular emphasis on the role of CSCs. Material and Methods: Database analyses were performed to assess the expression of the aforementioned markers in patients from 3 publicly available gene expression datasets. Furthermore, samples from 2 European paediatric ependymoma trial cohorts were screened for ABCB1, BLBP and MGMT expression by immunohistochemistry to elucidate their prognostic value. The expression of these markers was also determined in a panel of 5 ependymoma derived cell lines by QRT-PCR or western blotting analysis. Roles in chemoresistance (clonogenic & cytotoxicity assays) and tumour invasion (wound healing & 3D invasion assay) were then investigated. Results: Poor survival in the chemotherapy-led paediatric ependymoma CNS9204 trial was significantly associated with ABCB1 (P=0.007) and BLBP (P=0.03) expression whilst MGMT (P<0.001) and BLBP (P=0.002) expression predicted poor survival in the radiotherapy-led CNS9904 trial cohort. ABCB1 and BLBP expression was consistent with the CSC hypothesis whilst MGMT was expressed in both CSCs as well as the tumour bulk. Inhibition of ABCB1 and BLBP, with the phosphodiesterase-5 inhibitor vardenafil and PPAR-ϒ antagonist GW9662 respectively, potentiated response to chemotherapy and also inhibited the ability of ependymoma cell lines to migrate and invade. Finally, whilst each of the tested cell lines were resistant to the alkylating agent temozolomide, they were sensitive to the novel N3-propargyl analogue of temozolomide. Conclusion: ABCB1, BLBP and MGMT were not only markers of robust prognostic value but they also contributed functionally to the aggressive behaviour of ependymoma. Inhibition of ABCB1 and BLBP by vardenafil and GW9662 may represent effective approaches to overcome chemoresistance and invasion in ependymoma patients. The N3-propargyl analogue of temozolomide could also represent a novel treatment option for MGMT expressing ependymoma patients.

The technique of ATR-FTIR has been used to study the rates of diffusion of water and two different protein solutions into hydrogels, in the form of thin films, synthesised "in-situ" onto the surface of an ATR crystal. The work involved making copolymers and crosslinked gels of glycerol monomethacrylate (GMMA), lauryl methacrylate (LMA) and ethylene glycol dimethacrylate (EGDMA).Bands characteristic of the individual monomers could not be identified once a stock solution was made up. The rates of reaction were determined from the integrated area of the broad band at 815 cm[-1], which was designated as a monomer band. This band decreased in intensity as the polymerisation progressed. Similarly, a band at 750 cm[-1], designated as a polymer band, made an appearance and increased in intensity throughout the course of the polymerisation. Attempts were made to fit the reaction kinetics to first and second order reactions, using the linear part of the plot of the integrated area of the 815 cm[-1] band. However no particular trend could be found with increase in amounts of crosslinker for each formulation. The reaction possibly followed complex kinetics and this simplistic approach was not really applicable. Diffusion of deionised water into the synthesised gels was studied for three differentformulations. It was found that the diffusion coefficient was of the order of 10[-5] cm[2] s[-1] for the diffusion of water into the gels and decreased with increasing crosslinker concentration, except in the case of 100% GMMA with 0% crosslinker. The dissolution of the GMMA segments in the GMMA-EGDMA copolymer, was further confirmed by using ESEM. The gels with all other formulations were found to be morphologically featureless when looked at in the ESEM chamber. Four water bands were fitted to the &ugr;(OH) stretching region of each spectrum during diffusion and diffusion coefficients of each types of water were calculated. Diffusion of a 30% saline solution of bovine serum albumin was studied for the synthesised gels. It was found in each case, that only the water from the saline solution permeated through the polymer, leaving the protein in the form of a deposit on the top of the gel. The molecular weight of the protein (65kDa) was responsible for the latter not diffusing through the gel. The pores on the gel surface could not swell to an extent large enough to allow the passage of this bulky protein. Hence the gel acted as a selectively permeable membrane allowing passage of molecules based on their molecular weights. The order of magnitude of the diffusion coefficient for the saline water was the same as that of pure water. Diffusion of a solution of lysosyme containing 1% by volume of lysosyme, 49% by volume of water and 50% by volume of glycerol was characterised by a typical two step diffusion process. The first step was rather rapid and could possibly be due to water uptake from the solution. The second step showed evidence of glycerol permeating through the gel. It was impossible to distinguish the protein from the water and glycerol due to its concentration. Diffusion experiments were also carried out with a 10% solution of lysosyme in D[2]O. It was found that a single stage diffusion profile resulted in this case for the D2O . The protein definitely permeated into the gel in this case and it was possible to distinguish the amide I band. All the D[2]O first permeated into the gel and increased the pore size by swelling of the gel. The protein was then seen to permeate into the polymer.

The uptake of the auxins indole-3-acetic acid (IAA) and 1-naphthylacetic acid (NAA) was investigated using fluorimetric methods of assay. Uptake by the following plant material was examined - segments of Avena coleoptile, maize mesocotyl and the hypocotyl of Phaseolus radiatus. The growth regulating substances 2,4-dichlorophenoxyacetic acid, triiodobenzoic acid (TIBA), N-l-naphthyl-phthalamic acid, (1-naphthyl-methyl-sulphide) - propionic acid, and 3:5-diiodo- and 3:5-dibromo-4-hydroxybenzonitrile were examined for their effects on the entry and accumulation of IAA and NAA. Experiments were conducted to distinguish between the initial physical phase of uptake and subsequent metabolic uptake by pretreatment of the tissue with metabolic inhibitors and poisons. As fluorescence assay can be employed, under the experimental conditions, only for measuring loss of the auxins from the external medium, C14-labelled IAA and I131-labelled TIBA were used to assay actual uptake by the plant material. As it was suspected that the effects of TIBA on the uptake of IAA were exerted through metabolic action on a sulfhydryl-enzyme system, various SH-enzyme protectors (2,3-dimercaptopropanol, cysteine) were tested in attempts towards tracing the mechanism of TIBA action. Chromatographic and fluorimetric analysis of variously treated material has been undertaken. Autoradiographic analysis to check the nature and degree of IAA metabolism and the effects of TIBA on it was conducted, and measurements made of respiratory decarboxylation of IAA - C14 under the experimental conditions. The results have been critically discussed with reference to the literature dealing with synergism and antagonism between growth regulators, uptake and transport of auxins, mechanism of auxin action in growth, general aspects of membrane permeability and solute transport, and auxin kinetics.

Despite the availability of current anti-tuberculosis treatment regiments, high incidence and mortality rates of tuberculosis (TB) infections has necessitated the development of new treatment options for this deadly disease. There is a renewed interest in the third line anti-TB drug clofazimine due to its potent activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB strains. Poor oral pharmacokinetics and adverse effect profile of clofazimine limits the use of this agent making it a last resort drug for TB treatment. In this master’s project, targeted delivery of clofazimine (CLF) in the form of dry powder inhaler formulation with dipalmitoylphosphatidylcholine (DPPC) was explored to provide improved treatment option for tuberculosis. Four formulations of clofazimine viz., “CLF : 100” (containing 100% clofazimine), two clofazimine /DPPC mixtures “CLF : DPPC_80 : 20 or 50 : 50” (containing 20% and 50% of DPPC respectively) and “CLF : LEU_80 : 20” (containing 80% clofazimine and 20% leucine) were prepared by spray drying and evaluated for their aerodynamic, physicochemical and anti-tubercular properties. “CLF : DPPC_80 : 20” formulation showed the best aerodynamic performance with mass median aerodynamic diameter of 3.2 µm and highest fine particle fraction of 47% which was attributed to spherical particle morphology and low aggregation tendency observed in this formulation. Minimum inhibitory concentration (MIC) of this formulation was comparable to that of unformulated clofazimine indicating, no loss of the anti-TB activity of clofazimine after spray drying with DPPC. Clofazimine was present in crystalline form in all the formulations and was chemically compatible with DPPC. Salt forms of clofazimine were also prepared with the aim to identify high solubility clofazimine salt as a candidate for TB therapy. Crystalline salts of clofazimine with sulfonic acid (methanesulfonic, benzenesulfonic and toluenesulfonic acid) and hydroxyl acids (citric, lactic and tartaric acid) were formed and preliminary physicochemical characterization was performed. All the salts showed higher solubility than clofazimine and Methanesulfonic acid salt of clofazimine was found to be the highest solubility salt among the clofazimine salts tested. MIC values obtained for the sulfonic acid salts were qualitatively comparable to MIC of raw clofazimine.

Heart disease, cancer, diabetes and other complex diseases account for more than half of human mortality in the United States. Other diseases such as AIDS, asthma, Parkinson’s disease, Alzheimer’s disease and cerebrovascular ailments such as stroke not only augment this mortality but also severely deteriorate the quality of human life experience. In spite of enormous financial support and global scientific effort over an extended period of time to combat the challenges posed by these ailments, we find ourselves short of sighting a cure or vaccine. It is widely believed that a major reason for this failure is the traditional reductionist approach adopted by the scientific community in the past. In recent times, however, the systems biology based research paradigm has gained significant favor in the research community especially in the field of complex diseases. One of the critical components of such a paradigm is computational systems biology which is largely driven by mathematical modeling and simulation of biochemical systems. The most common methods for simulating a biochemical system are either: a) continuous deterministic methods or b) discrete event stochastic methods. Although highly popular, none of them are suitable for simulating multi-scale models of biological systems that are ubiquitous in systems biology based research. In this work a novel method for simulating biochemical systems based on a deterministic solution is presented with a modification that also permits the incorporation of stochastic effects. This new method, through extensive validation, has been proven to possess the efficiency of a deterministic framework combined with the accuracy of a stochastic method. The new crossover method can not only handle the concentration and spatial gradients of multi-scale modeling but it does so in a computationally efficient manner. The development of such a method will undoubtedly aid the systems biology researchers by providing them with a tool to simulate multi-scale models of complex diseases.

The notion of dependent types has been around for more than three decades, but a major complication resulting from introducing such a type discipline is that pure type inference for the enriched system is no longer possible. However, in the late nineties, a restricted form of dependent types was introduced in practical programming to capture more program properties through types and thereby detect more program errors at compile-time than effected by the strong type discipline in Standard ML or Java. Xanadu, a dependently typed imperative programming language, was the result of enriching the type system of imperative programming paradigm with a restricted form of dependent types. For the effective compilation of Xanadu program, it is crucial to successfully solve the linear constraints generated during its type-checking. This study is focussed on solving and verifying the linear integer constraints generated during the type-checking of Xanadu program. We present a two-tier constraint solver, based on the well-known linear programming algorithm, the Simplex method. The first tier uses the two-phase Simplex method to find a rational solution for a given set of constraints. If no rational solution exists, then no integer solution could be found. However, if a rational solution exists, the second tier uses branch and bound method, an integer programming algorithm, to seek an integer solution. As a significant application, we also study the use of our solver in eliminating the need for array bounds checking at runtime. The solver also generates a data structure, which we designate as history matrix, that records the operations of the Simplex algorithm. We claim that verifying the constraints using this data structure is faster than verifying them through the solver. Several constraints, gathered from the type-checking of Xanadu programs are solved and verified using the history matrix. The results presented, validate our claim. Moreover, we regard this matrix as a representation of a proof, which along with the compiled code would form an effort aimed towards producing a certifying code or a proof carrying code for Xanadu.