Pryolytic and kinetic study of Chlorella Vulgaris under isothermal and non-isothermal conditions

Algae are a new potential biomass for energy production but there is limited information on their pyrolysis and kinetics. The main aim of this thesis is to investigate the pyrolytic behaviour and kinetics of Chlorella vulgaris, a green microalga. Under pyrolysis conditions, these microalgae show their comparable capabilities to terrestrial biomass for energy and chemicals production. Also, the evidence from a preliminary pyrolysis by the intermediate pilot-scale reactor supports the applicability of these microalgae in the existing pyrolysis reactor. Thermal decomposition of Chlorella vulgaris occurs in a wide range of temperature (200-550°C) with multi-step reactions. To evaluate the kinetic parameters of their pyrolysis process, two approaches which are isothermal and non-isothermal experiments are applied in this work. New developed Pyrolysis-Mass Spectrometry (Py-MS) technique has the potential for isothermal measurements with a short run time and small sample size requirement. The equipment and procedure are assessed by the kinetic evaluation of thermal decomposition of polyethylene and lignocellulosic derived materials (cellulose, hemicellulose, and lignin). In the case of non-isothermal experiment, Thermogravimetry- Mass Spectrometry (TG-MS) technique is used in this work. Evolved gas analysis provides the information on the evolution of volatiles and these data lead to a multi-component model. Triplet kinetic values (apparent activation energy, pre-exponential factor, and apparent reaction order) from isothermal experiment are 57 (kJ/mol), 5.32 (logA, min-1), 1.21-1.45; 9 (kJ/mol), 1.75 (logA, min-1), 1.45 and 40 (kJ/mol), 3.88 (logA, min-1), 1.45- 1.15 for low, middle and high temperature region, respectively. The kinetic parameters from non-isothermal experiment are varied depending on the different fractions in algal biomass when the range of apparent activation energies are 73-207 (kJ/mol); pre-exponential factor are 5-16 (logA, min-1); and apparent reaction orders are 1.32–2.00. The kinetic procedures reported in this thesis are able to be applied to other kinds of biomass and algae for future works.

Non-English-speaking defendants in the magistrates court:a comparative study of face-to-face and prison video link interpreter-mediated hearings in England

This study compares interpreter-mediated face-to-face Magistrates Court hearings with those conducted through prison video link in which interpreters are located in court and non- English-speaking defendants in prison. It seeks to examine the impact that the presence of video link has on court actors in terms of interaction and behaviour. The data comprises 11 audio-recordings of face-to-face hearings, 10 recordings of prison video link hearings, semistructured interviews with 27 court actors, and ethnographic observation of hearings as viewed by defendants in Wormwood Scrubs prison in London. The over-arching theme is the pervasive influence of the ecology of the courtroom upon all court actors in interpretermediated hearings and thus on the communication process. Close analysis of the court transcripts shows that their relative proximity to one another can be a determinant of status, interpreting role, mode and volume. The very few legal protocols which apply to interpretermediated cases (acknowledging and ratifying the interpreter, for example), are often forgotten or dispensed with. Court interpreters lack proper training in the specific challenges of court interpreting, whether they are co-present with the defendant or not. Other court actors often misunderstand the interpreter’s role. This has probably come about because courts have adjusted their perceptions of what they think interpreters are supposed to do based on their own experiences of working with them, and have gradually come to accept poor practice (the inability to perform simultaneous interpreting, for example) as the norm. In video link courts, mismatches of sound and image due to court clerks’ failure to adequately track current speakers, poor image and sound quality and the fact that non-English-speaking defendants in pre-and post-court consultations can see and hear interpreters but not their defence advocates are just some of the additional layers of disadvantage and confusion already suffered by non- English-speaking defendants. These factors make it less likely that justice will be done.

The N-terminus of CD14 acts to bind apoptotic cells and confers rapid-tethering capabilities on non-myeloid cells:CD14 and rapid tethering of apoptotic cells

Cell death and removal of cell corpses in a timely manner is a key event in both physiological and pathological situations including tissue homeostasis and the resolution of inflammation. Phagocytic clearance of cells dying by apoptosis is a complex sequential process comprising attraction, recognition, tethering, signalling and ultimately phagocytosis and degradation of cell corpses. A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within this process. The role of myeloid cell CD14 in mediating apoptotic cell interactions with macrophages has long been known though key molecules and residues involved have not been defined. Here we sought to further dissect the function of CD14 in apoptotic cell clearance. A novel panel of THP-1 cell-derived phagocytes was employed to demonstrate that CD14 mediates effective apoptotic cell interactions with macrophages in the absence of detectable TLR4 whilst binding and responsiveness to LPS requires TLR4. Using a targeted series of CD14 point mutants expressed in non-myeloid cells we reveal CD14 residue 11 as key in the binding of apoptotic cells whilst other residues are reported as key for LPS binding. Importantly we note that expression of CD14 in non-myeloid cells confers the ability to bind rapidly to apoptotic cells. Analysis of a panel of epithelial cells reveals that a number naturally express CD14 and that this is competent to mediate apoptotic cell clearance. Taken together these data suggest that CD14 relies on residue 11 for apoptotic cell tethering and it may be an important tethering molecule on so called 'non-professional' phagocytes thus contributing to apoptotic cell clearance in a non-myeloid setting. Furthermore these data establish CD14 as a rapid-acting tethering molecule, expressed in monocytes, which may thus confer responsiveness of circulating monocytes to apoptotic cell derived material. © 2013 Thomas et al.

ProxiMAX randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering.

ProxiMAX Randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

ProxiMAX randomisation achieves saturation mutagenesis of contiguous codons without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, it uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents and as such, requires no specialised chemistry, reagents nor equipment. When particular residues are known to affect protein activity/specificity, their combinatorial replacement with all 20 amino acids, or a subset thereof, can provide a rapid route to generating proteins with desirable characteristics. Conventionally, saturation mutagenesis replaced key codons with degenerate ones. Although simple to perform, that procedure resulted in unnecessarily large libraries, termination codons and inherent uneven amino acid representation. ProxiMAX randomisation is an enzyme-based technique that can encode unbiased representation of all or selected amino acids or else can provide required codons in pre-defined ratios. Each saturated position can be defined independently of the others. ProxiMAX randomisation is achieved via saturation cycling: an iterative process comprising blunt end ligation, amplification and digestion with a Type IIS restriction enzyme. We demonstrate both unbiased saturation of a short 6-mer peptide and saturation of a hypervariable region of a scfv antibody fragment, where 11 contiguous codons are saturated with selected codons, in pre-defined ratios. As such, ProxiMAX randomisation is particularly relevant to antibody engineering. The development of ProxiMAX randomisation from concept to reality is described.

Investigation of two-wave mixing in arbitrary oriented sillenite crystals

A generalized systematic description of the Two-Wave Mixing (TWM) process in sillenite crystals allowing for arbitrary orientation of the grating vector is presented. An analytical expression for the TWM gain is obtained for the special case of plane waves in a thin crystal (|g|d«1) with large optical activity (|g|/?«1, g is the coupling constant, ? the rotatory power, d the crystal thickness). Using a two-dimensional formulation the scope of the nonlinear equations describing TWM can be extended to finite beams in arbitrary geometries and to any crystal parameters. Two promising applications of this formulation are proposed. The polarization dependence of the TWM gain is used for the flattening of Gaussian beam profiles without expanding them. The dependence of the TWM gain on the interaction length is used for the determination of the crystal orientation. Experiments carried out on Bi12GeO20 crystals of a non-standard cut are in good agreement with the results of modelling.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.

Process re-engineering for effective implementation of projects

Time, cost and quality are the prime objectives of any project. Unfortunately, today’s project management does not always ensure the realisation of these objectives. The main reasons of project non-achievement are changes in scope and design, changes in Government policies and regulations, unforeseen inflation, under-estimation and mis-estimation. An overall organisational approach with the application of appropriate management philosophies, tools and techniques can only solve the problem. The present study establishes a methodology for achieving success in implementing projects using a business process re-engineering (BPR) framework. Internal performance characteristics are introspected through condition diagnosis that identifies and prioritises areas of concern requiring attention. Process re-engineering emerges as a most critical area for immediate attention. Project process re-engineering is carried out by eliminating non-value added activities, taking up activities concurrently by applying information systems rigorously and applying risk management techniques throughout the project life cycle. The overall methodology is demonstrated through applications to cross country petroleum pipeline project organisation in an Indian scenario.

ProxiMAX randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.

Knowledge transfer in website design:exploring the processes and benefits of design collaboration for non-creative Micros

This thesis explores the interaction between Micros (<10 employees) from non-creative sectors and website designers ("Creatives") that occurred when creating a website of a higher order than a basic template site. The research used Straussian Grounded Theory Method with a longitudinal design, in order to identify what knowledge transferred to the Micros during the collaboration, how it transferred, what factors affected the transfer and outcomes of the transfer including behavioural additionality. To identify whether the research could be extended beyond this, five other design areas were also examined, as well as five Small to Medium Enterprises (SMEs) engaged in website and branding projects. The findings were that, at the start of the design process, many Micros could not articulate their customer knowledge, and had poor marketing and visual language skills, knowledge core to web design, enabling targeted communication to customers through images. Despite these gaps, most Micros still tried to lead the process. To overcome this disjoint, the majority of the designers used a knowledge transfer strategy termed in this thesis as ‘Bi-Modal Knowledge Transfer’, where the Creative was aware of the transfer but the Micro was unaware, both for drawing out customer knowledge from the Micro and for transferring visual language skills to the Micro. Two models were developed to represent this process. Two models were also created to map changes in the knowledge landscapes of customer knowledge and visual language – the Knowledge Placement Model and the Visual Language Scale. The Knowledge Placement model was used to map the placement of customer knowledge within the consciousness, extending the known Automatic-Unconscious -Conscious model, adding two more locations – Peripheral Consciousness and Occasional Consciousness. Peripheral Consciousness is where potential knowledge is held, but not used. Occasional Consciousness is where potential knowledge is held but used only for specific tasks. The Visual Language Scale was created to measure visual language ability from visually responsive, where the participant only responds personally to visual symbols, to visually multi-lingual, where the participant can use visual symbols to communicate with multiple thought-worlds. With successful Bi-Modal Knowledge Transfer, the outcome included not only an effective website but also changes in the knowledge landscape for the Micros and ongoing behavioural changes, especially in marketing. These effects were not seen in the other design projects, and only in two of the SME projects. The key factors for this difference between SMEs and Micros appeared to be an expectation of knowledge by the Creatives and failure by the SMEs to transfer knowledge within the company.

Pharmaceutical process optimisation of bulk lyophilisates:implications of powder handling

Lyophilisation or freeze drying is the preferred dehydrating method for pharmaceuticals liable to thermal degradation. Most biologics are unstable in aqueous solution and may use freeze drying to prolong their shelf life. Lyophilisation is however expensive and has seen lots of work aimed at reducing cost. This thesis is motivated by the potential cost savings foreseen with the adoption of a cost efficient bulk drying approach for large and small molecules. Initial studies identified ideal formulations that adapted well to bulk drying and further powder handling requirements downstream in production. Low cost techniques were used to disrupt large dried cakes into powder while the effects of carrier agent concentration were investigated for powder flowability using standard pharmacopoeia methods. This revealed superiority of crystalline mannitol over amorphous sucrose matrices and established that the cohesive and very poor flow nature of freeze dried powders were potential barriers to success. Studies from powder characterisation showed increased powder densification was mainly responsible for significant improvements in flow behaviour and an initial bulking agent concentration of 10-15 %w/v was recommended. Further optimisation studies evaluated the effects of freezing rates and thermal treatment on powder flow behaviour. Slow cooling (0.2 °C/min) with a -25°C annealing hold (2hrs) provided adequate mechanical strength and densification at 0.5-1 M mannitol concentrations. Stable bulk powders require powder transfer into either final vials or intermediate storage closures. The targeted dosing of powder formulations using volumetric and gravimetric powder dispensing systems where evaluated using Immunoglobulin G (IgG), Lactate Dehydrogenase (LDH) and Beta Galactosidase models. Final protein content uniformity in dosed vials was assessed using activity and protein recovery assays to draw conclusions from deviations and pharmacopeia acceptance values. A correlation between very poor flowability (p<0.05), solute concentration, dosing time and accuracy was revealed. LDH and IgG lyophilised in 0.5 M and 1 M mannitol passed Pharmacopeia acceptance values criteria with 0.1-4 while formulations with micro collapse showed the best dose accuracy (0.32-0.4% deviation). Bulk mannitol content above 0.5 M provided no additional benefits to dosing accuracy or content uniformity of dosed units. This study identified considerations which included the type of protein, annealing, cake disruption process, physical form of the phases present, humidity control and recommended gravimetric transfer as optimal for dispensing powder. Dosing lyophilised powders from bulk was demonstrated as practical, time efficient, economical and met regulatory requirements in cases. Finally the use of a new non-destructive technique, X-ray microcomputer tomography (MCT), was explored for cake and particle characterisation. Studies demonstrated good correlation with traditional gas porosimetry (R2 = 0.93) and morphology studies using microscopy. Flow characterisation from sample sizes of less than 1 mL was demonstrated using three dimensional X-ray quantitative image analyses. A platinum-mannitol dispersion model used revealed a relationship between freezing rate, ice nucleation sites and variations in homogeneity within the top to bottom segments of a formulation.

Freezing process optimisation of pharmaceutical formulations for freeze drying

The body of work presented in this thesis are in three main parts: [1] the effect of ultrasound on freezing events of ionic systems, [2] the importance of formulation osmolality in freeze drying, and [3] a novel system for increasing primary freeze drying rate. Chapter 4 briefly presents the work on method optimisation, which is still very much in its infancy. Aspects of freezing such as nucleation and ice crystal growth are strongly related with ice crystal morphology; however, the ice nucleation process typically occurs in a random, non-deterministic and spontaneous manner. In view of this, ultrasound, an emerging application in pharmaceutical sciences, has been applied to aid in the acceleration of nucleation and shorten the freezing process. The research presented in this thesis aimed to study the effect of sonication on nucleation events in ionic solutions, and more importantly how sonication impacts on the freezing process. This work confirmed that nucleation does occur in a random manner. It also showed that ultrasonication aids acceleration of the ice nucleation process and increases the freezing rate of a solution. Cryopreservation of animal sperm is an important aspect of breeding in animal science especially for endangered species. In order for sperm cryopreservation to be successful, cryoprotectants as well as semen extenders are used. One of the factors allowing semen preservation media to be optimum is the osmolality of the semen extenders used. Although preservation of animal sperm has no relation with freeze drying of pharmaceuticals, it was used in this thesis to make a case for considering the osmolality of a formulation (prepared for freeze drying) as a factor for conferring protein protection against the stresses of freeze drying. The osmolalities of some common solutes (mostly sugars) used in freeze drying were determined (molal concentration from 0.1m to 1.2m). Preliminary investigation on the osmolality and osmotic coefficients of common solutes were carried out. It was observed that the osmotic coefficient trend for the sugars analysed could be grouped based on the types of sugar they are. The trends observed show the need for further studies to be carried out with osmolality and to determine how it may be of importance to protein or API protection during freeze drying processes. Primary drying is usually the longest part of the freeze drying process, and primary drying times lasting days or even weeks are not uncommon; however, longer primary drying times lead to longer freeze drying cycles, and consequently increased production costs. Much work has been done previously by others using different processes (such as annealing) in order to improve primary drying times; however, these do not come without drawbacks. A novel system involving the formation of a frozen vial system which results in the creation of a void between the formulation and the inside wall of a vial has been devised to increase the primary freeze drying rate of formulations without product damage. Although the work is not nearly complete, it has been shown that it is possible to improve and increase the primary drying rate of formulations without making any modifications to existing formulations, changing storage vials, or increasing the surface area of freeze dryer shelves.

Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100mL stirred spinner flasks at a density of 3×10⁵cells.mL^-1 in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63±0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8±1.4% with a similar 3h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation.

One's own country and familiar places in the mind's eye:different topological representations for navigational and non-navigational contents

Visual mental imagery is a process that draws on different cognitive abilities and is affected by the contents of mental images. Several studies have demonstrated that different brain areas subtend the mental imagery of navigational and non-navigational contents. Here, we set out to determine whether there are distinct representations for navigational and geographical images. Specifically, we used a Spatial Compatibility Task (SCT) to assess the mental representation of a familiar navigational space (the campus), a familiar geographical space (the map of Italy) and familiar objects (the clock). Twenty-one participants judged whether the vertical or the horizontal arrangement of items was correct. We found that distinct representational strategies were preferred to solve different categories on the SCT, namely, the horizontal perspective for the campus and the vertical perspective for the clock and the map of Italy. Furthermore, we found significant effects due to individual differences in the vividness of mental images and in preferences for verbal versus visual strategies, which selectively affect the contents of mental images. Our results suggest that imagining a familiar navigational space is somewhat different from imagining a familiar geographical space. © 2014 Elsevier Ireland Ltd.