All-fiber polarization interference filters based on 45°-tilted fiber gratings

We report all-fiber polarization interference filters, known as Lyot and Lyot-Ohman filters, based on alternative concatenation of UV-inscribed fiber gratings with structure tilted at 45° and polarization maintaining (PM) fiber cavities. Such filters generate comb-like transmission of linear polarization output. The free spectral range (FSR) of a single-stage (Lyot) filter is PM fiber cavity length dependent, as a 20 cm long cavity showed a 26.6 nm FSR while the 40 cm one exhibited a 14.8 nm FSR. Furthermore, we have theoretically and experimentally demonstrated all-fiber 2-stage and 3-stage Lyot-Ohman filters, giving more freedom in tailoring the transmission characteristics.

Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

Mathematical modelling and control of agitated extraction columns

Liquid-liquid extraction has long been known as a unit operation that plays an important role in industry. This process is well known for its complexity and sensitivity to operation conditions. This thesis presents an attempt to explore the dynamics and control of this process using a systematic approach and state of the art control system design techniques. The process was studied first experimentally under carefully selected. operation conditions, which resembles the ranges employed practically under stable and efficient conditions. Data were collected at steady state conditions using adequate sampling techniques for the dispersed and continuous phases as well as during the transients of the column with the aid of a computer-based online data logging system and online concentration analysis. A stagewise single stage backflow model was improved to mimic the dynamic operation of the column. The developed model accounts for the variation in hydrodynamics, mass transfer, and physical properties throughout the length of the column. End effects were treated by addition of stages at the column entrances. Two parameters were incorporated in the model namely; mass transfer weight factor to correct for the assumption of no mass transfer in the. settling zones at each stage and the backmixing coefficients to handle the axial dispersion phenomena encountered in the course of column operation. The parameters were estimated by minimizing the differences between the experimental and the model predicted concentration profiles at steady state conditions using non-linear optimisation technique. The estimated values were then correlated as functions of operating parameters and were incorporated in·the model equations. The model equations comprise a stiff differential~algebraic system. This system was solved using the GEAR ODE solver. The calculated concentration profiles were compared to those experimentally measured. A very good agreement of the two profiles was achieved within a percent relative error of ±2.S%. The developed rigorous dynamic model of the extraction column was used to derive linear time-invariant reduced-order models that relate the input variables (agitator speed, solvent feed flowrate and concentration, feed concentration and flowrate) to the output variables (raffinate concentration and extract concentration) using the asymptotic method of system identification. The reduced-order models were shown to be accurate in capturing the dynamic behaviour of the process with a maximum modelling prediction error of I %. The simplicity and accuracy of the derived reduced-order models allow for control system design and analysis of such complicated processes. The extraction column is a typical multivariable process with agitator speed and solvent feed flowrate considered as manipulative variables; raffinate concentration and extract concentration as controlled variables and the feeds concentration and feed flowrate as disturbance variables. The control system design of the extraction process was tackled as multi-loop decentralised SISO (Single Input Single Output) as well as centralised MIMO (Multi-Input Multi-Output) system using both conventional and model-based control techniques such as IMC (Internal Model Control) and MPC (Model Predictive Control). Control performance of each control scheme was. studied in terms of stability, speed of response, sensitivity to modelling errors (robustness), setpoint tracking capabilities and load rejection. For decentralised control, multiple loops were assigned to pair.each manipulated variable with each controlled variable according to the interaction analysis and other pairing criteria such as relative gain array (RGA), singular value analysis (SVD). Loops namely Rotor speed-Raffinate concentration and Solvent flowrate Extract concentration showed weak interaction. Multivariable MPC has shown more effective performance compared to other conventional techniques since it accounts for loops interaction, time delays, and input-output variables constraints.

The development of a model system and high throughput assay for the study of interactions between zinc finger proteins and DNA

It has been recognised for some time that a full code of amino acid-based recognition of DNA sequences would be useful. Several approaches, which utilise small DNA binding motifs called zinc fingers, are presently employed. None of the current approaches successfully combine a combinatorial approach to the elucidation of a code with a single stage high throughput screening assay. The work outlined here describes the development of a model system for the study of DNA protein interactions and the development of a high throughput assay for detection of such interactions. A zinc finger protein was designed which will bind with high affinity and specificity to a known DNA sequence. For future work it is possible to mutate the region of the zinc finger responsible for the specificity of binding, in order to observe the effect on the DNA / protein interactions. The zinc finger protein was initially synthesised as a His tagged product. It was not possible however to develop a high throughput assay using the His tagged zinc finger protein. The gene encoding the zinc finger protein was altered and the protein synthesised as a Glutathione S-Transferase (GST) fusion product. A successful assay was developed using the GST protein and Scintillation Proximity Assay technology (Amersham Pharmacia Biotech). The scintillation proximity assay is a dynamic assay that allows the DNA protein interactions to be studied in "real time". This assay not only provides a high throughput method of screening zinc finger proteins for potential ligands but also allows the effect of addition of reagents or competitor ligands to be monitored.

The effect of thermochemical treatment on the fatigue properties of alloy steels

The effect of thermochemical treatment namely carburising on the fatigue behaviour of one carbon and two alloy steels has been studied in rotating and unidirectional bending. The effect of carbon profile on the unidirect¬ional bending fatigue strength of 63SA14 was assessed, and it was found that single stage carburising with a surface carbon content of 0.8% has resulted in a higher fatigue strength than other types of carbon profiles. Residual stresses and other metallurgical variables arising from different carbon profiles, were also considered. The highest compressive stresses h~e resulted from boost-diffuse-carburising. On the other hand surface decarburisation was associated with tensile residual stresses and a reduced fatigue strength. Retained austenite was found to be detrimental in unidirectional bending fatigue; however its presence in carburised 83SAIS did not seem to influence the rotating bending fatigue strength. Carbide particles in globular and/or intergranular form were detrimental to compressive residual stresses; the unidirectional bending fatigue strength is markedly lowered. The highest fatigue strength was accomplished by vacuum carburising. The absence of internal oxidation was the key factor in the increased fatigue strength; the presence of uniformly distributed fine carbide particles did not upset the superior fatigue strength of vacuum carburised pieces. The effect of mean stress on the fatigue strength of carburised 63SA14 was studied. Increasing the mean stress as would be expected resulted in a decreased fatigue strength. Carburisation showed its advantages at low mean stress, but at high mean stress it offers little advantage over the uncarburised hardened conditions. Notch effect was also studied in unidirectional bending of carburised 080MlS. The general trend showed that the fatigue strength decreases with increasing the stress concentration factor. But different carburising conditions have different effect on notch sensitivity.

Characterisation and performance of a Terfenol-D coated femtosecond laser inscribed optical fibre Bragg sensor with a laser ablated microslot for the detection of static magnetic fields

We present a novel device for the characterisation of static magnetic fields through monitoring wavelength shifts of femtosecond inscribed fibre Bragg grating and micromachined slot, coated with Terfenol-D. The device was sensitive to static magnetic fields and can be used as a vectoral sensor for the detection of magnetic fields as low as 0.046 mT with a resolution of ± 0.3mT in transmission and ± 0.7mT in reflection. The use of a femtosecond laser to both inscribe the FBGs and micromachine the slot in a single stage prior to coating the device significantly simplifies the fabrication.

Fabrication of high aspect ratio microfluidic devices using direct FS ablation

We present a single stage direct fs ablation results which show that it is possible to make high quality and high aspect ratio devices in a single stage process using a CAD optimised approach.

Application of space-charge electrostatic precipitator for collection of oil mist from pyrolysis gases

A novel electrostatic precipitator CAROLA® is developed for collection of fine oil mists. It operates on the principle of unipolar particle charging in the corona discharge and particle precipitation under the field of space charge. The pilot precipitator was tested at different gas temperatures. It is shown that the increase of gas temperature changes the characteristics of the corona discharge and particle size distribution, especially for droplets sub-micron droplets. The CAROLA® precipitator was used for collection of oil mist from pyrolysis gases at the HALOCLEAN® plant. The flow rate of biomass in the HALOCLEAN® plant was 15-30 kg/h. The particle mass concentration in the raw gas was over 100 g/Nm. The operation voltage of the precipitator was 10-12 kV and corona current up to 0,1 mA. Single stage electrostatic precipitator ensured mass collection efficiency 97-99,5% for pyrolysis oil mist.

The antioxidant enzyme peroxiredoxin-2 is depleted in lymphocytes seven days after ultra-endurance exercise

Purpose: Peroxiredoxin-2 (PRDX-2) is an antioxidant and chaperone-like protein critical for cell function. This study examined whether the levels of lymphocyte PRDX-2 are altered over one month following ultra-endurance exercise. Methods: Nine middle-aged men undertook a single-stage, multi-day 233 km (145 mile) ultra-endurance running race. Blood was collected immediately before (PRE), upon completion/retirement (POST), and following the race at DAY 1, DAY 7 and DAY 28. Lymphocyte lysates were examined for PRDX-2 by reducing SDS-PAGE and western blotting. In a sub-group of men who completed the race (n = 4) PRDX-2 oligomeric state (indicative of redox status) was investigated. Results: Ultra-endurance exercise caused significant changes in lymphocyte PRDX-2 (F (4,32) 3.409, p=0.020, ?(2) =0.299): seven-days after the race, PRDX-2 levels in lymphocytes had fallen to 30% of pre-race values (p=0.013) and returned to near-normal levels at DAY 28. Non-reducing gels demonstrated that dimeric PRDX-2 (intracellular reduced PRDX-2 monomers) was increased in 3 of 4 race completers immediately post-race, indicative of an "antioxidant response". Moreover, monomeric PRDX-2 was also increased immediately post-race in 2 of 4 race-completing subjects, indicative of oxidative damage, which was not detectable by DAY 7. Conclusions: Lymphocyte PRDX-2 was decreased below normal levels 7 days after ultra-endurance exercise. Excessive accumulation of reactive oxygen species induced by ultra-endurance exercise may underlie depletion of lymphocyte PRDX-2 by triggering its turnover after oxidation. Low levels of lymphocyte PRDX-2 could influence cell function and might, in part, explain reports of dysregulated immunity following ultra-endurance exercise.

Phase discrimination and simultaneous frequency conversion of the orthogonal components of an optical signal by four-wave mixing in an SOA

Simultaneous conversion of the two orthogonal phase components of an optical input to different output frequencies has been demonstrated by simulation and experiment. A single stage of four-wave mixing between the input signal and four pumps derived from a frequency comb was employed. The nonlinear device was a semiconductor optical amplifier, which provided overall signal gain and sufficient contrast for phase sensitive signal processing. The decomposition of a quadrature phase-shift keyed signal into a pair of binary phase-shift keyed outputs at different frequencies was also demonstrated by simulation.

Femtosecond laser inscribed Bragg sensor in Terfenol-D coated optical fibre with ablated microslot for the detection of static magnetic fields

A novel device for the detection and characterisation of static magnetic fields is presented. It consists of a femtosecond laser inscribed fibre Bragg grating (FBG) that is incorporated into an optical fibre with a femtosecond laser micromachined slot. The symmetry of the fibre is broken by the micro-slot, producing non-uniform strain across the fibre cross section. The sensing region is coated with Terfenol-D making the device sensitive to static magnetic fields, whereas the symmetry breaking results in a vectorial sensor for the detection of magnetic fields as low as 0.046 mT with a resolution of ±0.3mT in transmission and ±0.7mT in reflection. The sensor output is directly wavelength encoded from the FBG filtering, leading to simple demodulation through the monitoring of wavelength shifts that result as the fibre structure changes shape in response to the external magnetic field. The use of a femtosecond laser to both inscribe the FBG and micro-machine the slot in a single stage, prior to coating the device, significantly simplifies the sensor fabrication.

Femtosecond laser inscribed Bragg sensor in Terfenol-D coated optical fibre with ablated microslot for the detection of static magnetic fields

A novel device for the detection and characterisation of static magnetic fields is presented. It consists of a femtosecond laser inscribed fibre Bragg grating (FBG) that is incorporated into an optical fibre with a femtosecond laser micromachined slot. The symmetry of the fibre is broken by the micro-slot, producing non-uniform strain across the fibre cross section. The sensing region is coated with Terfenol-D making the device sensitive to static magnetic fields, whereas the symmetry breaking results in a vectorial sensor for the detection of magnetic fields as low as 0.046 mT with a resolution of ±0.3mT in transmission and ±0.7mT in reflection. The sensor output is directly wavelength encoded from the FBG filtering, leading to simple demodulation through the monitoring of wavelength shifts that result as the fibre structure changes shape in response to the external magnetic field. The use of a femtosecond laser to both inscribe the FBG and micro-machine the slot in a single stage, prior to coating the device, significantly simplifies the sensor fabrication.

Fabrication of high aspect ratio microfluidic devices using direct FS ablation

We present a single stage direct fs ablation results which show that it is possible to make high quality and high aspect ratio devices in a single stage process using a CAD optimised approach.

Waveguide fabrication in lithium-niobo-phosphate glasses by high repetition rate femtosecond laser:route to non-equilibrium material’s states

We study waveguide fabrication in lithium-niobo-phosphate glass, aiming at a practical method of single-stage fabrication of nonlinear integrated-optics devices. We observed chemical transformations or material redistribution during the course of high repetition rate femtosecond laser inscription. We believe that the laser-induced ultrafast heating and cooling followed by elements diffusion on a microscopic scale opens the way toward the engineering non-equilibrium sates of matter and thus can further enhance Refractive Index (RI) contrasts by virtue of changing glass composition in and around the fs tracks. © 2014 Optical Society of America.

Serial OTDM for 100 Gbit-Ethernet applications

A novel transmitter for 100 Gbit-Ethernet applications is proposed, based on the serial cascade of two 50 Gbit/s inverse-return-to-zero (also known as dark soliton) transmitters based on Mach-Zehnder modulators. The proposed transmitter and demultiplexer system uses commercially available components optimised for 40 Gbit/s applications. A 2.9 dB penalty at 100 Gbit/s is obtained using a single-stage OTDM demultiplexer and a preamplified receiver.