Biocatalysts offer advantages over their chemical counterparts in terms of their high enantioselectivity and the opportunity to develop more environmentally friendly processes. However, the widespread adoption of biocatalytic processes is hampered by the long development times for enzymes with novel and sufficient activity and adequate stability under operating conditions. Protein engineering, while extremely useful for modifying the properties of protein catalysts in select cases, still cannot be performed rapidly enough for many applications. In order for biocatalysts to become a competitive alternative to chemical catalysts, new tools to make the tailoring of biocatalysts by protein engineering methods speedier and more efficient are necessary. The aim of this work was to develop methods to aid in the faster production of novel biocatalysts. Protein engineering involves two steps: the generation of diversity and the screening or selection of variants with the desired properties. Both of these must be targeted to create a faster protein engineering process. In the case of the former, this work sought to clone and overexpress some template enzymes which would create smaller, more manageable libraries of mutants with a higher likelihood of function by the manipulation of a few focused amino acid residues. For the latter, this work developed and validated a Monte-Carlo simulation model of pooling to increase screening throughput and created a set of vectors to aid in high-throughput screening by eliminating unwanted mutants from the assay procedure entirely.

Segmented polyurethaneureas (SPUU) are thermoplastic elastomers with excellent elastic properties, high abrasion resistance and tear strength, making them very useful in numerous industrial applications ranging from microelectronics (slurry pad) to biomedical (artificial heart vessels) applications. The elastic and mechanical properties of these materials are strongly influenced by their two phase morphology. The factors that influence phase separation include difference in polarity between the hard and soft phases, composition and temperature. In general good phase separation results in materials with superior mechanical and elastic properties. Due to the immense potential applications of SPUU elastomers, there is a need for materials with higher strength. However, higher strength is not desired at the detriment of elasticity. If fact, stronger materials with enhanced elasticity are desired. In this thesis, high-strength SPUU elastomers were synthesized by incorporating reactive Laponite particles with surface-active free amine. The synthesis of pure SPUU is very complex, and addition of a reactive silicate further increases the complexity. To remedy this challenge, combinatorial methods and high-throughput screening techniques were used to optimize the diamine concentration and cure temperature. It was determined that pure SPUU elastomers prepared at a diamine stoichiometry of 85 100 mole %, and cured at 90 95 oC produced materials with higher strength and elongation at break. SPUU nanocomposites were prepared by maintaining the overall diamine stoichiometry at 95 mole %, and cured at 90 oC. Uniaxial tensile strength was optimized at a particle weight fraction of 1 wt. %, with a nearly 200 % increase in tensile strength and a 40 % increase in elongation at break, compared to pristine SPUU.

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Propylene oxide (PO) is an important raw material for the chemical industry, which is produced commercially by the chlorohydrin process and hydroperoxide process. However the deficiencies in these processes have given rise to considerable interest in the development of a direct route to PO that does not produce by-products or coproducts. The development of novel, active and selective catalysts for gas phase oxidation of propylene using molecular oxygen were studied via testing a large number of catalysts by high-throughput screening method over combinatorially prepared different catalytic system in this study. v The promoted and un-promoted silver (Ag), copper (Cu), manganese (Mn) mono and bimetallic catalytic system over high and low surface area silica, alumina, titanium oxide and titanium-silicate supports were prepared by single step sol-gel method and by incipient wetness method. The study to determine the most effective catalyst and promoter in the epoxidation reaction with different reaction conditions, showed that potassium (K)- promoted Cu metal supported over high surface area silica favored the PO production at a high reaction temperature (350 &deg / C) and oxygen rich atmosphere (C3H6/O2=1.0). The catalyst showed high and low propylene oxide productivity was investigated by some of the characterization techniques. The highlydispersed copper particle over silica support was determined by XRD, TEM and XPS techniques. The only change between promoted and un-promoted catalyst was found out in the temperature dependence of propylene consumption and PO production rate. It was inferred that potassium (K) only neutralizes the acid sites of silica.

The objective of this research is to develop automated and integrated microsystems for high-resolution imaging and high-throughput phenotyping / laser ablation of C. elegans. These microsystems take advantage of microfluidic technology for precisely handling animals and computer-aid automation for high-throughput processing. We demonstrated automated and high-throughput imaging / sorting and laser ablation of C. elegans. This thesis work is divided into four parts: development of a microsystem for imaging and sorting, development of a microsystem for laser cell ablation, development of a novel temperature measurement method, and development of pressure measurement method in microchannels. First, a microsystem was developed for high-throughput microscopy at high resolution and sorting. The microfluidic chip integrates novel microfluidic components to trap, position, immobilize, and sort/release animals. To characterize device operation and aid design of the device numerical models were developed. The experimental results demonstrate that the device operates robustly in a completely automatable manner. Additionally, a sophisticated control algorithm developed by Matthew Crane (Dr. Hang Lu¡¯s lab) automates the entire process of image acquisition, analysis, and sorting, which allows the system to operate without human intervention. This microsystem sorted worms based on their fluorescent expression pattern with over 95% accuracy per round at a rate of several hundred worms per hour. Secondly, the technologies developed for the imaging/sorting system were adapted and further improved to develop a microsystem for high-throughput cell laser ablation of C. elegans. The multiplex ablation module combined with the embryo trap module enables robust manipulation of embryos/L1-stage C. elegans. In addition, software for image processing and automation was developed to allow high-throughput cell ablations. This system performed ablation of a large number of animals and demonstrated accurate ablation by showing behavioral defects of the ablated worms in a chemotaxis avoidance assay. Thirdly, to aid future development of the microdevices, a novel in situ method for three-dimensionally resolved temperature measurement in microchannels was developed. This method uses video-microscopy in combination with image analysis software (developed by Jaekyu Cho in Dr. Victor Breedveld¡¯s group) to measure Brownian diffusion of nanoparticles that is correlated to temperature. This method offers superior reproducibility and reduced systematic errors. In addition, we demonstrated that this method can be used to measure spatial temperature variations in three dimensions in situ. Lastly, a method for pressure measurement in microdevices was also developed through collaboration with Hyewon Lee (Dr. Hang Lu¡¯s lab) to aid further device optimization. These micro pressure-sensors are composed of two flow layers with a polydimethylsiloxane (PDMS) membrane in between. The membrane deforms as a function of pressure and its deformation is quantified by a simple image-based method. These sensors offer high-precision pressure measurement in broad sensing ranges. In addition, a pressure transduction scheme combined with imaging-based method enables multiplex pressure measurement for simultaneously detecting pressures in multiple locations in a microsystem. Overall, the technologies developed in this thesis will establish a solid basis for continuous improvement of the microsystems for multi-cellular model organisms. This high-throughput technology will facilitate a broad range of biological and medical research.

<p>Robust and efficient cleaning procedures for protein A chromatography resins used for production of monoclonal antibody based biopharmaceuticals are crucial for safe and cost efficient processes. In this master thesis the effect of different cleaning regimes with respect to ligand stability of two protein A derived media, MabSelectTM and MabSelect SuReTM, has been investigated. A 96-well format has been used for preliminary screening of different cleaning agents, contact times and temperatures. NaCl as a ligand stabilizer during cleaning-in-place (CIP) was also included as a parameter. For optimal throughput and efficiency of screening, Rectangular Experimental Design for Multi-Unit Platforms; RED-MUP, and TECAN robotic platform have been utilized. For verification of screening, selected conditions were run in column format using the parallel chromatography system ÄKTAxpressTM. In the efficiency study, where a manual preparation of CIP solutions was compared with an automated mode performed in TECAN, the total process time ended up at eight hours versus three days respectively. However, the time measured included the learning process for the TECAN platform and for further preparations the automated mode is the superior choice. The study confirmed the higher alkaline stability of MabSelect SuRe compared to MabSelect. After exposure to 0.55 M NaOH during 24h MabSelect SuRe still retained 90% of the initial capacity. In contrast MabSelect had 60% of the initial binding capacity. When CIP with 10 mM NaOH was performed at 40 °C MabSelect reduced its capacity by half while MabSelect SuRe still had a binding capacity of 80%. The 96-well screening showed that an addition of NaCl during CIP had a significant positive effect on the stability of MabSelect, but needs to be verified on column format. The correlation between results from screening in 96-well filter plate and column format was good.</p>

In diesem Vortrag wird Condor als ein Job-Managementsystem für Rechen-Cluster vorgestellt. Dabei wird Funktionsweise an einem Beispiel demonstriert und erläutert. Besonders untersucht wird das Checkpointing und Migrieren von Prozessen zwischen verschiedenen Knoten.

Thesis (M.S.)--Ball State University, 2009. / Title from PDF t.p. (viewed on Nov. 30, 2009). Includes bibliographical references (p. 74-80).

Surgery of axons in C. elegans using ultrafast laser pulses, and observing their subsequent regrowth opens a new frontier in neuroscience, since such research holds a great potential for the development of novel therapies and cures to neurodegenerative diseases. In order to make the required large-scale genetic screenings in C. elegans possible and thus obtain statistically significant biological data, an automated laser axotomy system needs to be developed. Microfluidic devices hold the promise of improved throughput by integrating different functional modules into a single chip. The first step to developing a microfluidic device for laser axotomy is to devise an on-chip worm trapping method, which maintains a high degree of immobilization to sever axons without using anesthetics. In this thesis, we present a novel method that uses a thin, deflectable PDMS membrane that individually traps worms in a microfluidic device. Axons can successfully be severed with the same accuracy as those using conventional paralyzing techniques. This device also incorporates recovery chambers for housing worms after surgery and for time-lapse imaging of axonal regrowth without the repeated use of anesthetics. Towards accomplishing an automated, high-throughput laser axotomy system, we developed an improved microfluidic design based on the same mechanical immobilization technique. This second generation device allows for serially processing of a large quantity of worms rapidly using a semi-automated system. Integrated to the opto-mechanical platform, a software program utilizing image processing techniques is developed. This semi-automated program can automatically identify the location of worms, their neuronal cell bodies, focus on the axons of interest, and align the laser beam with the axon via a PID based viso-servo feedback algorithm. Statistic data demonstrate that there is no significant difference in axonal reconnection rates between surgeries performed on-chip and using anesthetics. To improve flow control, a three-dimensional novel microfluidic valve structure is designed and fabricated. This novel valve structure allows for a complete sealing of the flow channel, without degrading optical conditions for imaging and laser ablation in the trapping area. Finally, we developed a prototypical microfluidic assembly that will eventually be able to interface a well-plate to automatically deliver population of worms from individual wells to the automated chip for axotomy. This interface consists of a microfluidic multiplexer to significantly reduce the number of solenoid valves needed to individually address each well. / text

Ricin and Shiga toxin type 1 are potent cytotoxins known as ribosome inhibition proteins, abbreviated RIPs. Proteins of this family shut down protein synthesis by removing a critical adenine in the conserved stem-loop structure of 28S rRNA. Due to its exquisite cytotoxicity, the plant toxin ricin has been used as a biological warfare agent. Although great achievement has been made on ricin research, including catalytic mechanism and structure analysis, there is still no specific treatment available for ricin exposure. In addition, ricin A chain inhibitors may also be useful against the homologous bacterial proteins shiga toxins, which are responsible for dysentery, and diseases related to food poisoning, including hemolytic uremic syndrome. Previous study on RTA inhibitor search has provided a number of substrate analog inhibitors, all of which, however, are weaker inhibitors. Therefore, the goal of this work is to improve the binding affinity of known inhibitors and to discovery new scaffolds for inhibitor discovery and development. In this work, multiple approaches were employed for this purpose, including optimizing known inhibitors and searching new inhibitors by Virtual Drug Screening (VDS) and High Throughput Screening (HTS). A number of new RTA inhibitors were discovered by these strategies, which provide a variety of pharmacophores for RTA inhibitor design, and also added a new line of evidence for VDS as an advanced technology for drug discovery and development. / text