The engineering of biological systems with predictable behavior is a challenging problem. One reason for this difficulty is that engineered biological systems are embedded within complex and variable host cells. To help enable the future engineering of biological systems, we are studying and optimizing the interface between an engineered biological system and its host cell or ``chassis''. Other engineering disciplines use modularity to make interacting systems interchangeable and to insulate one system from another. Engineered biological systems are more likely to work as predicted if system function is decoupled from the state of the host cell. Also, specifying and standardizing the interfaces between a system and the chassis will allow systems to be engineered independent of chassis and allow systems to be interchanged between different chassis. To this end, we have assembled orthogonal transcription and translation systems employing dedicated machinery, independent from the equivalent host cell machinery. In parallel, we are developing test systems and metrics to measure the interactions between an engineered system and its chassis. Lastly, we are exploring methods to``port'' a simple engineered system from a prokaryotic to a eukaryotic organism so that the system can function in both organisms. / Poster presented at the 2005 ICSB meeting, held at Harvard Medical School in Boston, MA.

The purpose of this study was to evaluate the performance of scanning mobility particle sizers in the characterization of nanoaerosols. A sampling chamber was constructed from aluminum and tempered glass, had a volume of 4.6 cubic feet, and was designed for the introduction of aerosols and dilution air, maintenance of aerosol concentration, and continuous exhaust of chamber air. Penetration and aerosol distribution tests were conducted within the chamber. An aerosol generation and measurement system comprised of nitrogen gas, BGI 3 jet Collison Nebulizer, diffusion dryer, aerosol charge neutralizer, mixing chamber, critical orifice, hygrometer, condensation particle counter, scanning mobility particle sizer, air sampling pump, air sampling cassettes, and a vacuum pump was assembled. A BGI 3 jet Collison Nebulizer was used to generate the nanoparticle aerosols. The two types of nanoparticle aerosols utilized in the experiment were salt (NaCl) and polystyrene latex (PSL) spheres. Relative humidity and temperature measurements were obtained within the chamber. Real-time, direct-reading particle measurement instruments including a condensation particle counter (CPC) (TSI, Model 3007), and three scanning mobility particle sizer (SMPS) instruments (Particle Measuring Systems, Nano-ID NPS500; TSI, NanoScan SMPS Nanoparticle Sizer Model 3910) were used for particle measurements. For each test run, two air samples were collected on membrane filters for electron microscopy (EM) analysis. Eight trials were conducted using NaCl nanoaerosols, and twelve trials were conducted using PSL spheres. The selected particle sizes for the experiments were 57 nm, 92 nm, 147 nm, and 220 nm. For the NaCl nanoaerosol suspensions, the SMPS lines of fit were log-normally distributed and predominantly parallel. The geometric standard deviation (GSD) of these distributions was approximately 1.7, which confirms that the distributions were approximately the same. In these experiments, instrument 3 identified a higher percentage of NaCl particles within the size range intervals of the selected NaCl size parameter, and the count median diameters (CMDs) for the instrument 3 measurements were closer to the selected NaCl size parameter more often than the other instruments. This suggests that instrument 3 was more responsive than the other instruments to the selected size range and the selected NaCl size parameters. The electron microscopy (EM) lines of fit for the NaCl experiments were predominantly parallel with the SMPS lines of fit, suggesting that the log-normally distributions are similar. The GSD of EM distributions was approximately 1.8, which confirms that the distributions were approximately the same as the SMPS distributions. Results from the regression plots demonstrated that the main effects and interaction were statistically significant with a p<0.0001. The coefficient of determination, R2, for the regression lines was 0.87. The post-hoc Tukey HSD results identified a significant difference between the instrument 3 dataset, and the datasets for instruments 1 and 2. For the PSL nanoaerosol suspensions, the SMPS lines of fit were log-normally distributed and predominantly parallel. The GSD of these distributions was approximately 1.3, which confirms that the distributions were approximately the same. In these experiments, instrument 2 identified a higher percentage of PSL particles within the size range intervals of the selected PSL size parameter, and instrument 2 CMDs were closer to the selected PSL size parameter more often than the other instruments. This suggests that instrument 2 was more responsive than the other instruments to the selected size range and the selected PSL size parameters. Results from the regression plots demonstrated that the main effects and interaction were statistically significant with a p<0.01. The coefficient of determination, R2, for the regression lines was 0.44. The post-hoc Tukey HSD test identified a significant difference between the instrument 3 dataset and the instrument 1 dataset. Potential sources of variability include solution water background contamination, surfactants in the PSL solution, and agglomeration. The performance of all the scanning mobility particle sizers compared in these experiments was acceptable for research and field applications, but caution should be taken when comparing the measurements of SMPS, especially SMPS from different manufacturers.

Engineered nanomaterials (ENMs) have been increasingly used in various applications. Often, the ENMs are functionalised with a surface coating to enhance their properties. Decades of research has provided information on mostly pristine and unmodified ENMs, while ecotoxicity of coated ENMs and how their hazard changes with age in soils is still uncertain. The thesis aimed to determine the toxic effects and bioaccumulation potential of CuO ENMs and CdTe quantum dots (QDs) with different chemical coatings (carboxylate, COOH; polyethylene glycol, PEG; ammonium, NH4+) on the earthworm (Eisenia fetida), and compare the effects to their metal salt (CuSO4) or micron-sized counterpart. Then, to determine if any observed toxicity was altered after ageing the soils for up to one year. Incidental plant growth was studied in the exposure soils to maximise the scientific value of the earthworm tests. Toxic effects of CuO ENMs were also assessed in Caenorhabditis elegans exposed in liquid and soil media to understand effects of the media and method of dosing on ENM toxicity. CuO ENMs were equally toxic to earthworms, or less toxic to plants than the dissolved Cu; whereas CdTe QD ENMs were more toxic than the micron-sized CdTe QDs. There was a coating effect in both, CuO and CdTe QD ENM experiments, the -COOH coated ENMs were most toxic in the fresh soil study, while -NH4+ coated ENMs were most toxic in the aged soil study. Despite the similarities in the toxicity ranking, the biological effects exerted were different between CuO and CdTe QD ENMs. In C. elegans exposures, the ENMs were more hazardous than dissolved Cu, but ranking of ENMs depended on the media and method of dosing. The results suggest the coating effect is determined by the reactivity of the coating in a given media, and it also depends on the core of the ENMs. As such, coating and ageing effects should be considered in the risk assessment of ENMs.

The rapid advancement of drug delivery systems (DDS) has raised the possibility of using functional engineered nano/micro-particles as drug carriers for the administration of active pharmaceutical ingredients (APIs) to the affected area. The major goals in designing these functional particles are to control the particle size, the surface properties and the pharmacologically active agents release in order to achieve the site-specification of the drug at the therapeutically optimal rate and dose regimen. Two different equipment (i.e. glass capillary microfluidic device and micro-engineered membrane dispersion cell) were utilised in this study for the formation of functional nano/micro-particles by antisolvent precipitation method. This method is based on micromixing/direct precipitation of two miscible liquids, which appear as a straightforward method, rapid and easy to perform, does not require high stirring rates, sonication, elevated temperatures, surfactants and Class 1 solvents can be avoided. Theoretical selection of a good solvent and physicochemical interaction between solvent-water-polymer with the aid of Bagley s two-dimensional graph were successfully elucidated the nature of anti-solvent precipitation method for the formation of desired properties of functional pharmaceutical nano/micro-engineered particles. For the glass capillary microfluidic experiment, the organic phase (a mixture of polymer and tetrahydrofuran/acetone) was injected through the inner glass capillary with a tapered cross section culminated in a narrow orifice. The size of nanoparticles was precisely controlled by controlling phase flow rates, orifice size and flow configuration (two- phase co-flow or counter-current flow focusing). The locations at which the nanoparticles would form were determined by using the solubility criteria of the polymer and the concentration profiles found by numerical modelling. This valuable results appeared as the first computational and experimental study dealing with the formation of polylactide (PLA) and poly(ε-caprolactone) (PCL) nanoparticles by nanoprecipitation in a co-flow glass capillary device. The optimum formulations and parameters interactions involved in the preparation of paracetamol encapsulated nanoparticles (PCM-PCL NPs) using a co-flow microfluidic device was successfully simulated using a 25-full factorial design for five different parameters (i.e. PCL concentration, orifice size, flow rate ratios, surfactant concentration and paracetamol amount) with encapsulation efficiency and drug loading percentage as the responses. PCM-loaded composite NPs composed of a biodegradable poly(D,L-lactide) (PLA) polymer matrix filled with organically modified montmorillonite (MMT) nanoparticles were also successfully formulated by antisolvent nanoprecipitation in a microfluidic co-flow glass capillary device. The incorporation of MMT in the polymer matrix improved the drug encapsulation efficiency and drug loading, and extended the rate of drug release in simulated intestinal fluid (pH 7.4). The encapsulation of MMT and PCM in the NPs were well verified using transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), x-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). PCL drug-carrier nanoparticles were also produced by rapid membrane micromixing combined with nanoprecipitation in a stirred cell employing novel membrane dispersion. The size of the NPs was precisely controlled by changing the aqueous-to-organic volumetric ratio, stirring rate, transmembrane flux, the polymer content in the organic phase, membrane type and pore morphologies. The particle size decreased by increasing the stirring rate and the aqueous-to-organic volumetric ratio, and by decreasing the polymer concentration in the aqueous phase and the transmembrane flux. The existence of the shear stress peak within a transitional radius and a rapid decline of the shear stress away from the membrane surface were revealed by numerical modelling. Further investigation on the PCL nanoparticles loaded immunosuppressive rapamycin (RAPA) drug were successfully synthesised by anti-solvent nanoprecipitation method using stainless steel (SS) ringed micro-engineered membrane. Less than 10 μm size of monohydrate piroxicam (PRX) micro-crystals also was successfully formed with the application of anti-solvent precipitation method combined with membrane dispersion cell that has been utilised in the formation of functional engineered nanoparticles. This study is believed to be a new insight into the development of integrated membrane crystallisation system.

Multiple sclerosis is a neurodegenerative disorder affecting the central nervous system (CNS). Currently, the disease is incurable and immunomodulating drugs are the only option to control the disease. CD49d is an adhesion receptor expressed on most immune cells. Antibodies that bind to CD49d and block immune cells from trafficking toward the CNS are being pursued as one class of therapeutics. In this work, by combining recombinant antibody and phage display technologies we isolated 10 anti-CD49d single domain antibodies from a synthetic antibody light chain variable domain (VL) phage display library. Isolated VLs (~ 12 kDa) were expressed in Escherichia coli, purified and analysed for biophysical characteristics. The majority were expressed in good yields and were non-aggregating. All 10 VLs bound recombinant CD49d by ELISA, and 7 bound to CD49d-expressing cells in flow cytometry experiments. To empower the VLs for better therapeutic efficacy (thru increasing avidity and half-life), three of the lead VLs were re-engineered as fusions to fragment crystallisable (Fc) of human immunoglobulin gamma (IgG). The engineered hFc-VL fragments (~ 70 – 90 kDa) retained their specificity for CD49d by flow cytometry. With (i) being less immunogenic due to their human nature, (ii) their efficient access to cryptic epitopes (iii) having half-lives comparable to IgGs’ and (iv) being more cost effective compared to IgGs, these novel antibody fragments (monovalent VLs and bivalent hFc-VLs) provide a promising therapeutic platform against multiple sclerosis.

Introduction: Venous disease impairs the quality of life, necessitates time off work and causes venous ulcers. The focus of this thesis is to explore strategies to prevent chronic venous insufficiency (CVI) and venous ulceration. Aims 1. To identify a population at risk of developing venous ulcers 2. To study the pressure profile required by elastic stockings to halve transit venous time 3. To explore the role of compression following ankle fracture Methods: Data was collected from 231 patients with venous ulcers and age and sex matched 210 controls to identify risk factors for venous ulceration. Univariate and multivariate analysis of potential risk factors was undertaken to identify those that independently predict this risk. After identifying the population at risk, prophylactic strategies were developed. The effect of Engineered Compression Stockings (ECS) delivering 15mmHg, 25mmHg and 35mmHg pressure at the ankle on the calf venous transit time and volume was measured to determine the ideal pressure profile required to halve transit venous time, which should be appropriate for DVT prophylaxis. A dorsal foot vein was cannulated in 15 healthy volunteers with no venous disease. The transit time (secs) for ultrasound contrast from a foot vein to the popliteal vein was measured using duplex ultrasound. Calf volumes were recorded by water displacement. ECS delivering 25mmHg of pressure around the ankle were compared with no compression in a randomized controlled trial (RCT) in 90 patients within 72 hours of ankle fracture. Patients were randomised to either i) ECS and air-cast boot or ii) a liner and air-cast boot and were followed at 2, 4, 8, 12 weeks and 6 months. The primary outcome was functional recovery measured using the Olerud Molander Ankle Score (OMAS). Secondary outcomes were i) The American Orthopaedic Foot and Ankle Score (AOFAS), ii) SF12v2 Quality of Life score (QoL), iii) pain, and iv) frequency of DVT. Results The risk factors significantly associated with venous ulceration on multivariate analysis included a history of Deep Vein Thrombosis (DVT), phlebitis, hip replacement, poor mobility, weight/kg>100Kg, varicose veins (VV), family history of VV and weight (kg) between 75-100kg. A simple diagnostic scoring system was derived from this regression analysis with scores of . 3 predicting a 6.7% annual risk and of < 1 a 0.6% risk. Mean transit time without compression was 35, 32 and 33 secs while standing, sitting and lying. Transit time was consistently halved by ECS delivering 25mmHg to 14, 13 and 14 secs respectively (p<0.001). Mean leg volume whilst standing was reduced significantly from 3447ml with no ECS to 3259ml, 3161ml and 3067ml with ECS applying 15, 25 and 35mmHg respectively (p<0.001). ECS in ankle fracture patients reduced ankle swelling at all time points and significantly improved mean OMAS score at six months to 98 compared with 67 for the liner (p<0.001). AOFAS and SF12v2 scores were also significantly improved (p<0.001, p= 0.016). Of 86 patients with duplex imaging at four weeks, only five (12%) of the 43 ECS patients had a DVT compared with 10 (23%) of the 43 controls (p= 0.26). Conclusions: The risk score for venous ulcers will allow us to undertake RCTs on the prevention of leg ulceration. The pressure profile required to halve transit venous time is 25mmHg. The frequency of asymptomatic DVT following ankle fracture is sufficient to justify prophylaxis. Compression has a potential role in the management of ankle fractures by improving functional outcome and QoL. These studies facilitate research into the prevention of venous disease.

No description available.

Yes / Diagnostics and prognostics have significant roles in the reliability enhancement of systems and are focused topics of active research. Engineered systems are becoming more complex and are subjected to miscellaneous failure modes that impact adversely their performability. This everincreasing complexity makes fault diagnostics and prognostics challenging for the system-level functions. A significant number of successes have been achieved and acknowledged in some review papers; however, these reviews rarely focused on the application of complex engineered systems nor provided a systematic review of diverse techniques and approaches to address the related challenges. To bridge the gap, this paper firstly presents a review to systematically cover the general concepts and recent development of various diagnostics and prognostics approaches, along with their strengths and shortcomings for the application of diverse engineered systems. Afterward, given the characteristics of complex systems, the applicability of different techniques and methods that are capable to address the features of complex systems are reviewed and discussed, and some of the recent achievements in the literature are introduced. Finally, the unaddressed challenges are discussed by taking into account the characteristics of automotive systems as an example of complex systems. In addition, future development and potential research trends are offered to address those challenges. Consequently, this review provides a systematic view of the state of the art and case studies with a reference value for scholars and practitioners.

Yes / The use of biological macromolecules like quince seed mucilage (QSM), as the common curative practice has a long history in traditional folk medicine to cure wounds and burns. However, this gel cannot be applied on exudative wounds because of the high water content and non-absorption of infection of open wounds. It also limits cell-to-cell interactions and leads to the slow wound healing process. In this study to overcome these problems, a novel QSM-based hybrid scaffold modified by PCL/PEG copolymer was designed and characterized. The properties of this scaffold (PCL/QSM/PEG) were also compared with four scaffolds of PCL/PEG, PCL/Chitosan/PEG, chitosan, and QSM, to assess the role of QSM and the combined effect of polymers in improving the function of skin tissue-engineered scaffolds. It was found, the physicochemical properties play a crucial role in regulating cell behaviors so that, PCL/QSM/PEG as a smart/stimuli-responsive bio-matrix promotes not only human-adipose stem cells (h-ASCs) adhesion but also supports fibroblasts growth, via providing a porous-network. PCL/QSM/PEG could also induce keratinocytes at a desirable level for wound healing, by increasing the mechanobiological signals. Immunocytochemistry analysis confirmed keratinocytes differentiation pattern and their normal phenotype on PCL/QSM/PEG. Our study demonstrates, QSM as a differentiation/growth-promoting biological factor can be a proper candidate for design of wound dressings and skin tissue-engineered substrates containing cell.

Phosphorus (P) in stormwater runoff has detrimental effects on water quality and ecosystem health when it reaches surface waters and promotes algal blooms. Constructed wetlands (CWs) have been utilized to combat this problem by containing stormwater and removing excess nutrients. Including filter materials in the design of CWs has shown promise for increasing their capacity for nutrient removal. This mesocosm scale study was conducted outdoors over a 12-month period to evaluate the effectiveness of three filter materials in their ability to adsorb phosphorus, retain water, and support plant life. The filter materials examined were electric arc furnace (EAF) slag, engineered biochar, and sand. All treatments demonstrated positive plant response and the ability to retain water. The EAF slag and biochar removed significant amounts of P from effluent and appear to be suitable materials for integrating into CW design. Sand was found to be ineffective as a P filter.