Protein mass spectrometry for bioprocess development & monitoring

Bioprocesses for therapeutic protein production typically require significant resources to be invested in their development. This development could be improved with technologies that can elucidate the physicochemical properties of process stream components with small sample volumes in a rapid and readily performed manner. This is especially true in early phase development when material and established analytical methods are limiting. This thesis has investigated various process materials but was focussed mainly on those existing in an ApolipoproteinA-IM (ApoA-IM) process, produced using an Eschericia coli (E. coli) host. Using a mass spectrometric technique this project began by monitoring the product and contaminant during the ApoA-IM process and how this analytical approach compares to traditional analytical methods such as high performance liquid chromatography (HPLC). Results showed that, unlike many other analytical methods, surface enhanced laser desorption ionisation mass spectrometry (SELDI-MS) can handle early process samples that contain complex mixtures of biological molecules with limited sample pre-treatment and thereby provide meaningful process-relevant information. The change in material during the flocculation/centrifugation stage of the process was then examined. When only a change in cellular debris was observed an existing methodology developed at University College London (UCL) was implemented to maximise cellular debris removal. The predictive scale down methodology enabled rapid optimization of the operating conditions for a flocculation followed with a centrifugation step using only small volumes (20mL) of a high solids (~20% w/w) E. coli heat extract. These experiments suggested that adding a higher level of a cationic polymer could substantially increase the strength of the flocculated particles produced, thereby enhancing overall clarification performance in a large scale centrifuge. This was subsequently validated at pilot scale.The proteins remaining from this flocculation/centrifugation stage were then compared using the mass spectrometric technique to calculate the difficulty of removing each protein contaminant from the ApoA-IM product and suggested conditions for future sorbent scouting runs

Saturated 13.2 nm high-repetition-rate laser in nickel-like cadmium

Includes bibliographical references (page 2583).We report gain-saturated operation of a 13.2 nm tabletop laser in Ni-like Cd at a 5 Hz repetition rate. A gain-length product G×L=17.6 was obtained by heating a precreated plasma with 8 ps duration Ti:sapphirelaser pulses with an energy of only 1 J impinging at a grazing angle of 23°. With an average power of ~1 mW, this laser is an attractive coherent source for at-wavelength metrology of extreme UV lithography optics and other applications

Pulse duration measurements of grazing-incidence-pumped high repetition rate Ni-like Ag and Cd transient soft x-ray lasers

Includes bibliographical references (page 3045).We have measured the pulse duration of gain-saturated 13.9 nm Ni-like Ag and 13.2 nm Ni-like Cd transient collisional lasers excited by grazing-incidence-pumping for several pumping conditions. High-resolution streak-camera measurements yielded FWHM pulse durations close to 5 ps for both lasers under optimum pumping conditions. The very high brightness and short pulse duration of these new high repetition tabletop soft x-ray lasers make them an attractive source for dynamic applications

A decrease in photoperiod shortly after first feeding influences the development of Atlantic salmon (Salmo salar)

Four groups of Atlantic salmon fry (n=2000) were exposed to continuous light (LD24:0) from first feeding on 18th April 2001, after which they were exposed to either an 8 or 12 week period of short days (LD10:14) starting on either the 21st May or the 18th June. Each group was then returned to LD24:0 until the conclusion of the experiment the following March. In August 200 fish per treatment were individually PIT tagged. All groups were maintained under an ambient temperature regime. The highest levels of sexual maturation in 0+ male parr were recorded in the 12 week/May group (>11% of the entire male and female population), with the lowest levels (6%) in the 8 week/May and 8 week/June groups (

Saturated high-repetition-rate 18.9-nm tabletop laser in nickel-like molybdenum

Includes bibliographical references (page 167).We report saturated operation of an 18.9-nm laser at 5-Hz repetition rate. An amplification with a gain-length product GL of 15.5 is obtained in the 4d 1S0―4p 1P1 laser line of Ni-like Mo in plasmas heated at grazing incidence with ~1-J pulses of 8.1-ps duration from a tabletop laser system. Lasing is obtained over a broad range of time delays and pumping conditions. We also measure a GL of 13.5 in the 22.6-nm transition of the same ion. The results are of interest for numerous applications requiring high-repetition-rate lasers at wavelengths below 20 nm

Integrated Radiation Transport and Nuclear Fuel Performance for Assembly-Level Simulations

The Advanced Multi-Physics (AMP) Nuclear Fuel Performance code (AMPFuel) is focused on predicting the temperature and strain within a nuclear fuel assembly to evaluate the performance and safety of existing and advanced nuclear fuel bundles within existing and advanced nuclear reactors. AMPFuel was extended to include an integrated nuclear fuel assembly capability for (one-way) coupled radiation transport and nuclear fuel assembly thermo-mechanics. This capability is the initial step toward incorporating an improved predictive nuclear fuel assembly modeling capability to accurately account for source-terms and boundary conditions of traditional (single-pin) nuclear fuel performance simulation, such as the neutron flux distribution, coolant conditions, and assembly mechanical stresses. A novel scheme is introduced for transferring the power distribution from the Scale/Denovo (Denovo) radiation transport code (structured, Cartesian mesh with smeared materials within each cell) to AMPFuel (unstructured, hexagonal mesh with a single material within each cell), allowing the use of a relatively coarse spatial mesh (10 million elements) for the radiation transport and a fine spatial mesh (3.3 billion elements) for thermo-mechanics with very little loss of accuracy. In addition, a new nuclear fuel-specific preconditioner was developed to account for the high aspect ratio of each fuel pin (12 feet axially, but 1 4 inches in diameter) with many individual fuel regions (pellets). With this novel capability, AMPFuel was used to model an entire 17 17 pressurized water reactor fuel assembly with many of the features resolved in three dimensions (for thermo-mechanics and/or neutronics), including the fuel, gap, and cladding of each of the 264 fuel pins; the 25 guide tubes; the top and bottom structural regions; and the upper and lower (neutron) reflector regions. The final, full assembly calculation was executed on Jaguar using 40,000 cores in under 10 hours to model over 162 billion degrees of freedom for 10 loading steps. The single radiation transport calculation required about 50% of the time required to solve the thermo-mechanics with a single loading step, which demonstrates that it is feasible to incorporate, in a single code, a high-fidelity radiation transport capability with a high-fidelity nuclear fuel thermo-mechanics capability and anticipate acceptable computational requirements. The results of the full assembly simulation clearly show the axial, radial, and azimuthal variation of the neutron flux, power, temperature, and deformation of the assembly, highlighting behavior that is neglected in traditional axisymmetric fuel performance codes that do not account for assembly features, such as guide tubes and control rods

High repetition rate collisional soft x-ray lasers based on grazing incidence pumping

We discuss the demonstration of gain-saturated high repetition rate table-top soft x-ray lasers producing microwatt average powers at wavelengths ranging from 13.9 to 33 nm. The results were obtained heating a pre-created plasma with a picosecond optical laser pulse impinging at grazing incidence onto a pre-created plasma. This pumping geometry increases the energy deposition efficiency of the pump beam into the gain region, making it possible to saturate soft x-ray lasers in this wavelength range with a short pulse pump energy of only 1 J at 800 nm wavelength. Results corresponding to 5 Hz repetition rate operation of gain-saturated 14.7 nm Ni-like Pd and 32.6 nm line Ne-like Ti lasers pumped by a table-top Ti:sapphire laser are reported. We also discuss results obtained using a 1 {omega} 1054 nm pre-pulse and 2{omega} 527 nm short pulse from a Nd:glass pump laser. This work demonstrates the feasibility of producing compact high average power soft x-ray lasers for applications

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies