Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells

The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 μM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 μM 1α,25-dihydroxyvitamin D3 to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213±54.35%) compared to that of parent cells (100±6.05%). Furthermore, they retained significantly less [3H]vincristine sulphate (p<0.05), a P-gp substrate, after efflux (272.89±11.86 fmol/mg protein) than the parent cells (381.39±61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1α,25-dihydroxyvitamin D3 treatment was quantified to be 76.2±7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0±20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0±20.0 to 1057.0±46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association.

Systems biology approach to study permeability of paracetamol and its solid dispersion

Physiological changes that take place at cellular level are usually reflective of their level of gene expression. Different formulation excipients have an impact on physiological behavior of the exposed cells and in turn affect transporter genes, enterocyte-mediated metabolism and toxicity biomarkers. The aim of this study was to prepare solid dispersion of paracetamol and evaluate genetic changes that occur in Caco-2 cell lines during the permeability of paracetamol alone and paracetamol solid dispersion formulations. Paracetamol-PEG 8000 solid dispersion was prepared by melt fusion method and the formulation was characterised using differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Formulation of solid dispersion resulted in the conversion of crystalline drug into an amorphous form. Permeability studies showed that paracetamol absorption was higher from the solid dispersion formulation. DNA microarrays analysis was carried out in order to investigate the involvement of any efflux/uptake transporters in paracetamol or its solid dispersion permeability. Neither transporter carriers nor efflux proteins were found to be involved in the absorption of paracetamol or its PEG solid dispersion. Gene expression analysis established that paracetamol toxicity was potentially reduced upon formulation into solid dispersion when ATP binding cassette (ABC) and solute carrier transporter (SLC) genes were analyzed.

The properties of advanced alumimium alloy systems

2XXX and 7XXX series aluminium alloys have been the accepted materials for airframe construction for many decades. However, only minor improvements in properties have been possible by the development of these alloys since the early 1970's. The constant need to reduce weight in aircraft has therefore led to a resurgence in the research for higher performance aluminium alloys. The reason for this investigation was to evaluate possible alternatives for the existing conventional aluminium alloy 2014 for aircraft wheel applications. Three new technologies in alloy development were considered: a metal matrix composite, an aluminium-lithium alloy and a powder metallurgical alloy. The basic mechanical properties of these advanced materials have already been established to an extent, but their fatigue behaviour has yet to be fully understood. The purpose of this work was to investigate the fatigue properties of the materials concerned, in both air and an aerated 3.5% NaCl solution, and compare these properties to 2014-T6. As well as the basic mechanical properties, fatigue crack propagation data is presented for all of the materials concerned. Additionally, fatigue crack initiation data is presented for the aluminium-lithium alloy and 2014. The D.C. electrical potential method was used to monitor crack growth. Of the materials investigated, the most promising was the aluminium-lithium alloy. However, short transverse properties need to be increased and the commercial cost of the material needs to be decreased before it can be considered as a direct replacement for 2014 for aircraft structural applications. It was considered that the cost of the powder metallurgical alloy would limit its further use. The metal matrix composite material proved to be unsuitable for most ambient temperature applications

Distributed control of computer communication systems

Flow control in Computer Communication systems is generally a multi-layered structure, consisting of several mechanisms operating independently at different levels. Evaluation of the performance of networks in which different flow control mechanisms act simultaneously is an important area of research, and is examined in depth in this thesis. This thesis presents the modelling of a finite resource computer communication network equipped with three levels of flow control, based on closed queueing network theory. The flow control mechanisms considered are: end-to-end control of virtual circuits, network access control of external messages at the entry nodes and the hop level control between nodes. The model is solved by a heuristic technique, based on an equivalent reduced network and the heuristic extensions to the mean value analysis algorithm. The method has significant computational advantages, and overcomes the limitations of the exact methods. It can be used to solve large network models with finite buffers and many virtual circuits. The model and its heuristic solution are validated by simulation. The interaction between the three levels of flow control are investigated. A queueing model is developed for the admission delay on virtual circuits with end-to-end control, in which messages arrive from independent Poisson sources. The selection of optimum window limit is considered. Several advanced network access schemes are postulated to improve the network performance as well as that of selected traffic streams, and numerical results are presented. A model for the dynamic control of input traffic is developed. Based on Markov decision theory, an optimal control policy is formulated. Numerical results are given and throughput-delay performance is shown to be better with dynamic control than with static control.

Ultra-long haul optical fibre transmission systems

This thesis examines experimentally options for optical fibre transmission over oceanic distances. Its format follows the chronological evolution of ultra-long haul optical systems, commencing with opto-electronic regenerators as repeaters, progressing to optically amplified NRZ systems and finally solitonic propagation. In each case recirculating loop techniques are deployed to simplify the transmission experiments. Advances in high speed electronics have allowed regenerators operating at 10 Gbit/s to become a practical reality. By augmenting such devices with optical amplifiers it is possible to greatly enhance the repeater spacing. Work detailed in this thesis has culminated in the propagation of 10 Gbit/s data over 400,000 km with a repeater spacing of 160 km. System reliability and robustness are enhanced by the use of a directly modulated DFB laser transmitter and total insensitivity of the system to the signal state of polarisation. Optically amplified ultra-long haul NRZ systems have taken on particular importance with the impending deployment of TAT 12/13 and TPC 5. The performance of these systems is demonstrated to be primarily limited by analogue impairments such as the accumulation of amplifier noise, polarisation effects and optical non-linearities. These degradations may be reduced by the use of appropriate dispersion maps and by scrambling the transmitted state of signal polarisation. A novel high speed optically passive polarisation scrambler is detailed for the first time. At bit rates in excess of 10 Gbit/s it is shown that these systems are severely limited and do not offer the advantages that might be expected over regenerated links. Propagation using solitons as the data bits appears particularly attractive since the dispersive and non-linear effects of the fibre allow distortion free transmission. However, the generation of pure solitons is difficult but must be achieved if the uncontrolled transmission distance is to be maximised. This thesis presents a new technique for the stabilisation of an erbium fibre ring laser that has aUowed propagation of 2.5 Gbit/s solitons to the theoretical limit of ~ 18,000 km. At higher bit rates temporal jitter becomes a significant impairment and to aUow an increase in the aggregate line rate multiplexing in both time and polarisation domains has been proposed. These techniques are shown to be of only limited benefit in practical systems and ultimately some form of soliton transmission control is required. The thesis demonstrates synchronous retiming by amplitude modulation that has allowed 20 Gbit/s data to propagate 125,000 km error free with an amplifier spacing approaching the soliton period. Ultimately the speed of operation of such systems is limited by the electronics used and, thus, a new form of soliton control is demonstrated using all optical techniques to achieve synchronous phase modulation.

Liposomes:Formulation and characterisation as contrast agents and as vaccine delivery systems

Liposome systems are well reported for their activity as vaccine adjuvants; however novel lipid-based microbubbles have also been reported to enhance the targeting of antigens into dendritic cells (DCs) in cancer immunotherapy (Suzuki et al 2009). This research initially focused on the formulation of gas-filled lipid coated microbubbles and their potential activation of macrophages using in vitro models. Further studies in the thesis concentrated on aqueous-filled liposomes as vaccine delivery systems. Initial work involved formulating and characterising four different methods of producing lipid-coated microbubbles (sometimes referred to as gas-filled liposomes), by homogenisation, sonication, a gas-releasing chemical reaction and agitation/pressurisation in terms of stability and physico-chemical characteristics. Two of the preparations were tested as pressure probes in MRI studies. The first preparation composed of a standard phospholipid (DSPC) filled with air or nitrogen (N2), whilst in the second method the microbubbles were composed of a fluorinated phospholipid (F-GPC) filled with a fluorocarbon saturated gas. The studies showed that whilst maintaining high sensitivity, a novel contrast agent which allows stable MRI measurements of fluid pressure over time, could be produced using lipid-coated microbubbles. The F-GPC microbubbles were found to withstand pressures up to 2.6 bar with minimal damage as opposed to the DSPC microbubbles, which were damaged at above 1.3 bar. However, it was also found that DSPC-filled with N2 microbubbles were also extremely robust to pressure and their performance was similar to that of F-GPC based microbubbles. Following on from the MRI studies, the DSPC-air and N2 filled lipid-based microbubbles were assessed for their potential activation of macrophages using in vitro models and compared to equivalent aqueous-filled liposomes. The microbubble formulations did not stimulate macrophage uptake, so studies thereafter focused on aqueous-filled liposomes. Further studies concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyldioctadecylammonium (DDA) and immunomodulatory trehalose dibehenate (TDB) with the addition of polyethylene glycol (PEG). One of the proposed hypotheses for the mechanism behind the immunostimulatory effect obtained with DDA:TDB is the ‘depot effect’ in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. The depot effect has been suggested to be primarily due to their cationic nature. Results reported within this thesis demonstrate that higher levels of PEG i.e. 25 % were able to significantly inhibit the formation of a liposome depot at the injection site and also severely limit the retention of antigen at the site. This therefore resulted in a faster drainage of the liposomes from the site of injection. The versatility of cationic liposomes based on DDA:TDB in combination with different immunostimulatory ligands including, polyinosinic-polycytidylic acid (poly (I:C), TLR 3 ligand), and CpG (TLR 9 ligand) either entrapped within the vesicles or adsorbed onto the liposome surface was investigated for immunogenic capacity as vaccine adjuvants. Small unilamellar (SUV) DDA:TDB vesicles (20-100 nm native size) with protein antigen adsorbed to the vesicle surface were the most potent in inducing both T cell (7-fold increase) and antibody (up to 2 log increase) antigen specific responses. The addition of TLR agonists poly(I:C) and CpG to SUV liposomes had small or no effect on their adjuvanticity. Finally, threitol ceramide (ThrCer), a new mmunostimulatory agent, was incorporated into the bilayers of liposomes composed of DDA or DSPC to investigate the uptake of ThrCer, by dendritic cells (DCs), and presentation on CD1d molecules to invariant natural killer T cells. These systems were prepared both as multilamellar vesicles (MLV) and Small unilamellar (SUV). It was demonstrated that the IFN-g secretion was higher for DDA SUV liposome formulation (p<0.05), suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

Computational mechanics reveals nanosecond time correlations in molecular dynamics of liquid systems

Statistical complexity, a measure introduced in computational mechanics has been applied to MD simulated liquid water and other molecular systems. It has been found that statistical complexity does not converge in these systems but grows logarithmically without a limit. The coefficient of the growth has been introduced as a new molecular parameter which is invariant for a given liquid system. Using this new parameter extremely long time correlations in the system undetectable by traditional methods are elucidated. The existence of hundreds of picosecond and even nanosecond long correlations in bulk water has been demonstrated. © 2008 Elsevier B.V. All rights reserved.

The molecular design of a new solvent for the absorption of carbon dioxide

Gas absorption, the removal of one or more constitutents from a gas mixture, is widely used in chemical processes. In many gas absorption processes, the gas mixture is already at high pressure and in recent years organic solvents have been developed for the process of physical absorption at high pressure followed by low pressure regeneration of the solvent and recovery of the absorbed gases. Until now the discovery of new solvents has usually been by expensive and time consuming trial and error laboratory tests. This work describes a new approach, whereby a solvent is selected from considerations of its molecular structure by applying recently published methods of predicting gas solubility from the molecular groups which make up the solvent molecule. The removal of the acid gases of carbon dioxide and hydrogen sulfide from methane or hydrogen was used as a commercially important example. After a preliminary assessment to identify promising moecular groups, more than eighty new solvent molecules were designed and evaluated by predicting gas solubility. The other important physical properties were also predicted by appropriate theoretical procedures, and a commercially promising new solvent was chosen to have a high solubility for acid gases, a low solubility for methane and hydrogen, a low vapour pressure, and a low viscosity. The solvent chosen, of molecular structure Ch3-COCH2-CH2-CO-CH3, was tested in the laboratory and shown to have physical properties, except for vapour pressures, close to those predicted. That is gas solubilities were within 10% but lower than predicted. Viscosity within 10% but higher than predicted and a vapour pressure significantly lower than predicted. A computer program was written to predict gas solubility in the new solvent at the high pressures (25 bar) used in practice. This is based on the group contribution method of Skold Jorgensen (1984). Before using this with the new solvent, Acetonyl acetone, the method was show to be sufficiently accurate by comparing predicted values of gas solubility with experimental solubilities from the literature for 14 systems up to 50 bar. A test of the commercial potential of the new solvent was made by means of two design studies which compared the size of plant and approximate relative costs of absorbing acid gases by means of the new solvent with other commonly used solvents. These were refrigerated methanol(Rectisol process) and Dimethyl Ether or Polyethylene Glycol(Selexol process). Both studies showed in terms of capital and operating cost some significant advantage for plant designed for the new solvent process.

Chemical and physical methods of enhancing the percutaneous absorption of antimicrobial agents

Contrary to previously held beliefs, it is now known that bacteria exist not only on the surface of the skin but they are also distributed at varying depths beneath the skin surface. Hence, in order to sterilise the skin, antimicrobial agents are required to penetrate across the skin and eliminate the bacteria residing at all depths. Chlorhexidine is an antimicrobial agent with the widest use for skin sterilisation. However, due to its poor permeation rate across the skin, sterilisation of the skin cannot be achieved and, therefore, the remaining bacteria can act as a source of infection during an operation or insertion of catheters. The underlying theme of this study is to enhance the permeation of this antimicrobial agent in the skin by employing chemical (enhancers and supersaturated systems) or physical (iontophoresis) techniques. The hydrochloride salt of chlorhexidine (CHX), a poorly soluble salt, was used throughout this study. The effect of ionisation on in vitro permeation rate across the excised human epidennis was investigated using Franz-type diffusion cells. Saturated solutions of CHX were used as donor and the variable studied was vehicle pH. Permeation rate was increased with increasing vehicle pH. The pH effect was not related to the level of ionisation of the drug. The effect of donor vehicle was also studied using saturated solutions of CHX in 10% and 20% ethanol as the donor solutions. Permeation of CHX was enhanced by increasing the concentration of ethanol which could be due to the higher concentration of CHX in the donor phase and the effect of ethanol itself on the membrane. The interplay between drug diffusion and enhancer pretreatment of the epidennis was studied. Pretreatment of the membrane with 10% Azone/PG demonstrated the highest diffusion rate followed by 10% olcic acid/PG pretreatment compared to other pretreatment regimens (ethanol, dimethyl sulfoxide (DMSO), propylene glycol (PG), sodium dodecyl sulphate (SDS) and dodecyl trimethyl ammonium bromide (DT AB). Differential Scanning Calorimetry (DSC) was also employed to study the mode of action of these enhancers. The potential of supersaturated solutions in enhancing percutaneous absorption of CHX was investigated. Various anti-nucleating polymers were screened in order to establish the most effective agent. Polyvinylpyrrolidone (PVP, K30) was found to be a better candidate than its lower molecular weight counterpart (K25) and hydroxypropyl methyleellulose (HPMC). The permeation studies showed an increase in diffusion rate by increasing the degree of saturation. Iontophoresis is a physical means of transdemal drug delivery enhancement that causes an increased penetration of molecules into or through the skin by the application of an electric field. This technique was employed in conjunction with chemical enhancers to assess the effect on CHX permeation across the human epidermis. An improved transport of CHX, which was pH dependant was observed upon application of the current. Combined use of iontophoresis and chemical enhancers further increased the CHX transport indicating a synergistic effect. Pretreatment of the membrane with 10% Azone/PG demonstrated the greatest effect.

Typical case behaviour of spin systems in random graph and composite ensembles

This thesis includes analysis of disordered spin ensembles corresponding to Exact Cover, a multi-access channel problem, and composite models combining sparse and dense interactions. The satisfiability problem in Exact Cover is addressed using a statistical analysis of a simple branch and bound algorithm. The algorithm can be formulated in the large system limit as a branching process, for which critical properties can be analysed. Far from the critical point a set of differential equations may be used to model the process, and these are solved by numerical integration and exact bounding methods. The multi-access channel problem is formulated as an equilibrium statistical physics problem for the case of bit transmission on a channel with power control and synchronisation. A sparse code division multiple access method is considered and the optimal detection properties are examined in typical case by use of the replica method, and compared to detection performance achieved by interactive decoding methods. These codes are found to have phenomena closely resembling the well-understood dense codes. The composite model is introduced as an abstraction of canonical sparse and dense disordered spin models. The model includes couplings due to both dense and sparse topologies simultaneously. The new type of codes are shown to outperform sparse and dense codes in some regimes both in optimal performance, and in performance achieved by iterative detection methods in finite systems.

Structural characterization of titanium-doped Bioglass using isotopic substitution neutron diffraction

Melt quenched silicate glasses containing calcium, phosphorus and alkali metals have the ability to promote bone regeneration and to fuse to living bone. Of these glasses 45S5 Bioglass® is the most widely used being sold in over 35 countries as a bone graft product for medical and dental applications; particulate 45S5 is also incorporated into toothpastes to help remineralize the surface of teeth. Recently it has been suggested that adding titanium dioxide can increase the bioactivity of these materials. This work investigates the structural consequences of incorporating 4 mol% TiO2 into Bioglass® using isotopic substitution (of the Ti) applied to neutron diffraction and X-ray Absorption Near Edge Structure (XANES). We present the first isotopic substitution data applied to melt quench derived Bioglass or its derivatives. Results show that titanium is on average surrounded by 5.2(1) nearest neighbor oxygen atoms. This implies an upper limit of 40% four-fold coordinated titanium and shows that the network connectivity is reduced from 2.11 to 1.97 for small quantities of titanium. Titanium XANES micro-fluorescence confirms the titanium environment is homogenous on the micron length scale within these glasses. Solid state magic angle spinning (MAS) NMR confirms the network connectivity model proposed. Furthermore, the results show the intermediate range order containing Na-O, Ca-O, O-P-O and O-Si-O correlations are unaffected by the addition of small quantities of TiO2 into these systems.

Regeneration limit of classical Shannon capacity

Since Shannon derived the seminal formula for the capacity of the additive linear white Gaussian noise channel, it has commonly been interpreted as the ultimate limit of error-free information transmission rate. However, the capacity above the corresponding linear channel limit can be achieved when noise is suppressed using nonlinear elements; that is, the regenerative function not available in linear systems. Regeneration is a fundamental concept that extends from biology to optical communications. All-optical regeneration of coherent signal has attracted particular attention. Surprisingly, the quantitative impact of regeneration on the Shannon capacity has remained unstudied. Here we propose a new method of designing regenerative transmission systems with capacity that is higher than the corresponding linear channel, and illustrate it by proposing application of the Fourier transform for efficient regeneration of multilevel multidimensional signals. The regenerative Shannon limit -the upper bound of regeneration efficiency -is derived. © 2014 Macmillan Publishers Limited. All rights reserved.

The evolution of learning systems:to Bayes or not to be

Bayesian algorithms pose a limit to the performance learning algorithms can achieve. Natural selection should guide the evolution of information processing systems towards those limits. What can we learn from this evolution and what properties do the intermediate stages have? While this question is too general to permit any answer, progress can be made by restricting the class of information processing systems under study. We present analytical and numerical results for the evolution of on-line algorithms for learning from examples for neural network classifiers, which might include or not a hidden layer. The analytical results are obtained by solving a variational problem to determine the learning algorithm that leads to maximum generalization ability. Simulations using evolutionary programming, for programs that implement learning algorithms, confirm and expand the results. The principal result is not just that the evolution is towards a Bayesian limit. Indeed it is essentially reached. In addition we find that evolution is driven by the discovery of useful structures or combinations of variables and operators. In different runs the temporal order of the discovery of such combinations is unique. The main result is that combinations that signal the surprise brought by an example arise always before combinations that serve to gauge the performance of the learning algorithm. This latter structures can be used to implement annealing schedules. The temporal ordering can be understood analytically as well by doing the functional optimization in restricted functional spaces. We also show that there is data suggesting that the appearance of these traits also follows the same temporal ordering in biological systems. © 2006 American Institute of Physics.

Comments on multiple oscillatory solutions in systems with time-delay feedback

A complex Ginzburg-Landau equation subjected to local and global time-delay feedback terms is considered. In particular, multiple oscillatory solutions and their properties are studied. We present novel results regarding the disappearance of limit cycle solutions, derive analytical criteria for frequency degeneration, amplitude degeneration, frequency extrema. Furthermore, we discuss the influence of the phase shift parameter and show analytically that the stabilization of the steady state and the decay of all oscillations (amplitude death) cannot happen for global feedback only. Finally, we explain the onset of traveling wave patterns close to the regime of amplitude death.

Sonogashira cross-coupling and homocoupling on a silver surface:chlorobenzene and phenylacetylene on Ag(100)

Scanning tunneling microscopy, temperature-programmed reaction, near-edge X-ray absorption fine structure spectroscopy, and density functional theory calculations were used to study the adsorption and reactions of phenylacetylene and chlorobenzene on Ag(100). In the absence of solvent molecules and additives, these molecules underwent homocoupling and Sonogashira cross-coupling in an unambiguously heterogeneous mode. Of particular interest is the use of silver, previously unexplored, and chlorobenzene—normally regarded as relatively inert in such reactions. Both molecules adopt an essentially flat-lying conformation for which the observed and calculated adsorption energies are in reasonable agreement. Their magnitudes indicate that in both cases adsorption is predominantly due to dispersion forces for which interaction nevertheless leads to chemical activation and reaction. Both adsorbates exhibited pronounced island formation, thought to limit chemical activity under the conditions used and posited to occur at island boundaries, as was indeed observed in the case of phenylacetylene. The implications of these findings for the development of practical catalytic systems are considered.