Two polysaccharide-degrading enzymes (endo-1,4-D-glucanase and β-mannanase) from blue mussel, Mytilus edulis, have been purified to homogeneity using a combination of several chromatographic steps. Each enzyme has been characterized with regard to its molecular weight, isoelectric point, pH and temperature stability, pH and temperature optimum and substrate specificity. The amino acid sequence of the endoglucanase has been determined at the protein level. The two enzymes are true blue mussel proteins as confirmed at the DNA level. The nucleotide sequences of synthesized cDNA from digestive gland and of genomic DNA from gill tissue were compared. Both genes contain introns, a property typical of eucaryotic organisms. Amino acid sequence based classification has revealed that the endoglucanase belongs to the glycoside hydrolase family 45, subfamily 2 while β-mannanase is a member of family 5. Both enzymes form insoluble inclusion bodies when expressed in Escherichia coli. Refolding attempts were unsuccessful. However, the β-mannanase was successfully expressed in the methylotropic yeast Pichia pastoris with an expression level above 100 mg/l in shaking culture. Crystals of the endoglucanase were made from the native protein and a dataset was collected to 1.85 Å resolution using an in-house rotating anode x-ray source. Crystals were also produced using recombinant β-mannanase and a dataset was collected to 1.4 Å resolution at the ESRF synchrotron beamline ID14-EH1. The three dimensional structure of the endoglucanase was solved by X-ray crystallography.

The objective of this study was to investigate the mechanisms and factors controlling growth and proliferation in serum-free Spodoptera frugiperda Sf9 cultures as well as the implications of these factors for protein production in the baculovirus expression system. The physiology of recently thawed, low passage (Lp) Sf9 cultures, were compared to high passage (Hp) cultures at p>100. Lp cells passed a switch in proliferation kinetics after 30-40 passages, characterized by a shorter lag-phase and an increased maximum specific proliferation rate, µN,max, from 0.03/h to 0.04/h. Conditioned medium (CM), 10 kDa CM filtrate and 10 kDa CM concentrate promoted proliferation of Lp Sf9 cells, but had no effect after the switch. Sf9 cell cycle dynamics were characterized by an initial G2/M arrest, which synchronized the cells and this feature was more pronounced for Hp than for Lp cells. CM addition decreased the initial arrest for Lp cultures, but did not affect Hp cells. Late in the culture, a final G2/M accumulation occurred. An octaploid population emerged during G2/M arrests. Further, a 49 kDa proform and a 39 kDa active form of Sf9 cathepsin L was identified from gelatine zymography of Sf9 CM, on basis of inhibitor profile and substrate range. Removal of procathepsin L during the course of an Sf9 culture had a negative effect on Sf9 proliferation. Procathepsin L was also identified in Trichoplusia ni High five CM. High five CM promoted growth of Sf9 cells, but when procathepsin L was removed no effect was observed. It is suggested that these observations are due to an autocrine system controlling proliferation. One Sf9 mitogen might be a <10 kDa peptide, while the effect of 10 kDa CM concentrate may originate from procathepsin L. A hypothesis is therefore that procathepsin L acts as a mitogen in Sf9 cultures, perhaps in concert with the <10 kDa peptide. The volumetric product yield (P) in baculovirus infected Sf9 cells increased linearly up to 68-75 h of culture. Beyond this point almost no product was detected. Medium renewal at infection prolonged the productivity phase until 117 h, but generated only a 10% increase in P. The specific product formation rate (YP/N) was highest at µN,max. YP/N of Lp cells decreased by 30-50% when 20% CM or 10 kDa CM filtrate was added, whereas addition of CM to cells having passed the switch on growth kinetics did not affect productivity. Further, Hp cells exhibited a two-fold higher YP/N than Lp cells, when infected during the initial 48 h of culture. This coincided with a high degree of synchronization. Yeastolate limitation was used to achieve artificial synchronization of an Lp culture, and YP/N could thereby be maintained high during a prolonged time, resulting in a 69% increased P. This suggests that a decreasing degree of synchronization during the course of a culture partly explains the cell-density dependent drop in productivity in Sf9 cells. Finally, ~10 kDa gel filtration fractions from Sf9 and High five CM were found to be bactericidal. Exposure of a Bacillus megaterium culture for eight min to an Sf9 CM fraction killed 99% of the population, and 60 min exposure killed 35% of an Escherichia coli population. In both cases cell lysis was observed. B. megaterium incubated in an High five CM fraction lost 97% viability in 40 min. The effect of the High five CM fraction most probably originated from a lysozyme precursor protein, whereas the Sf9 executor remains unknown. / QC 20100908

Clinical and engineering studies of carotid artery disease typically assume that the common carotid artery (CCA), proximal to the bifurcation, is relatively straight enough to assume fully-developed flow. However, a recent study from our group (Ford et al) showed the surprising presence, in vivo, of strongly skewed velocity profiles in mildly curved CCAs. In this thesis we aim to understand how CCA geometry affects velocity profile skewing. The left and right normal CCAs of 32 participants (62±13 yrs), randomly chosen from NIH’s VALIDATE study (N~450) were digitally segmented from aortic root to bifurcation. It was shown that each segmented CCA could be divided into nominal cervical and thoracic region and that each region could be approximated by planar circular arches. Subsequent CFD simulations of CCA parametric models suggested strong velocity profile skewing both at the inlet and outlet of cervical segment and the effect of various geometric parameters were investigated.

Clinical and engineering studies of carotid artery disease typically assume that the common carotid artery (CCA), proximal to the bifurcation, is relatively straight enough to assume fully-developed flow. However, a recent study from our group (Ford et al) showed the surprising presence, in vivo, of strongly skewed velocity profiles in mildly curved CCAs. In this thesis we aim to understand how CCA geometry affects velocity profile skewing. The left and right normal CCAs of 32 participants (62±13 yrs), randomly chosen from NIH’s VALIDATE study (N~450) were digitally segmented from aortic root to bifurcation. It was shown that each segmented CCA could be divided into nominal cervical and thoracic region and that each region could be approximated by planar circular arches. Subsequent CFD simulations of CCA parametric models suggested strong velocity profile skewing both at the inlet and outlet of cervical segment and the effect of various geometric parameters were investigated.

<p>A generalized quantum chemical approach for electron transport in molecular devices is developed. It allows to treat the devices where the metal electrodes and the molecule are either chemically or physically bonded on equal footing. Effects of molecular length and hydrogen bonding on the current-voltage (I-V) characteristics of molecular devices are discussed. An extension to include the vibration motions of the molecule has been derived and implemented. It provides the inelastic electron tunneling spectroscopy (IETS) of molecular devices with unprecedented accuracy, and reveals important information about the molecular structures that are not accessible in the experiment. The IETS is shown to be a powerful characterization tool for molecular devices.</p><p>An effective elongation method has been developed to study the electron transport in nanoand bio-electronic devices at hybrid density functional theory level. It enables to study electronic structures and transportation properties of a 40 nm long self-assembled conjugated polymer junction, a 21 nm long single-walled carbon nanotubes (SWCNT), and a 60 basepairs DNA molecule. It is the first time that systems consisting of more than 10,000 electrons have been described at such a sophisticated level. The calculations have shown that the electron transport in sub-22 nm long SWCNT and short DNA molecules is dominated by the coherent scattering through the delocalized unoccupied states. The derived length dependence of coherent electron transport in these nanostructured systems will be useful for the future experiments. Moreover, some unexpected behaviors of these devices have been discovered.</p>

The alignment of the femoral and tibial components of the Total Knee Arthoplasty (TKA) is one of the most important factors to implant survivorship. Hence, numerous ligament balancing techniques and devices have been developed in order to accurately balance the knee intra-operatively. Spacer block, tensioner and tram adapter are instruments that allow surgeons to qualitatively balance the flexion and extension gaps during TKA. However, even with these instruments, the surgical procedure still relies on the skill and experience of the surgeon. The objective of this thesis is to develop a computerized surgical instrument that can acquire intra-operative data telemetrically for surgeons and engineers. Microcantilever is chosen to be used as the strain sensing elements. Even though many high end off-the-shelf data acquisition components and integrated circuit (IC) chips exist on the market, yet multiple components are required to process the entire array of microcantilevers and achieve the desired functions. Due to the size limitation of the off-chip components, an Application Specific Integrated Circuit (ASIC) chip is designed and fabricated. Using a spacer block as a base, sensors, a data acquisition system as well as the transmitter and antenna are embedded into it. The electronics are sealed with medical grade epoxy.

Low back pain is a common occupational effect of the long term driving of vehicles. The sonoSens® Monitor ultrasonic device was utilized to model lumbar spinal motions while driving a tractor trailer. Further data was collected to determine if acceleration from a seat pad and EMG data correlated with the driver’s movements.Three testing bouts were completed and analyzed for this study. For each test the driver drove a 1998 Peterbuilt 379 fitted with a National Seating 2000 seat. One driver was used for all three tests.Data analysis was accomplished with the sonoSens® Analyzer and Microsoft® Excel software. The sonoSens® Analyzer software produced graphs of the spinal motion of the driver. These graphs were visually analyzed for motion trends and increased movement of the lumbar region.The sonoSens® Monitor is ideal for determining motion and posture of the lumbar region over periods of time. Acceleration and EMG data was found not to correlate with a driver’s movements while driving.

Peripheral Nerve Injury is one of the most common and serious traumatic injuries. About 300,000 cases of peripheral nerve injury are reported annually in Europe and another 50,000 procedures performed annually in the United States. Peripheral nerve injury can lead to lifelong disability and also death in severe cases. Repair techniques remain a major challenge because of lack of efficient regeneration. Though techniques like autografts are highly efficient, they face a lot of drawbacks. Alternate methods focus on the use of growth supporting and enhancement components in nerve guidance channels. Current research necessitates the use of a combination of compounds for enhanced nerve growth. In this thesis we study the combined effect of Nerve Growth Factor (NGF), laminin and Retinoic acid (RA) on nerve growth. This study is aimed at attaining higher efficiency in nerve growth by using a combination of compounds not tested together before. We expect to see an enhanced outgrowth, supposedly caused by the synergistic effect of these components, which will be greater than the outgrowth caused when these compounds are used individually or in combination with another compound. Chick dorsal root ganglion cells were cultured for 36 hrs in the presence of 50ng/ml NGF, 50μg/ml laminin and 1μM RA, either used individually, or in combination with another compound, or synergistically. The efficiency of each condition was determined based on neurite extension and area of outgrowth. The triple component system produced significantly greater neurite extension and outgrowth compared to all when the compounds are used individually or in combination with one another. The neurite length in the triple component system was 5 fold that of control whereas the area of outgrowth was as high as 60 times that of control. This enhanced neurite growth achieved will pave way for efficient nerve guidance channels development.

Cotton yield monitors are an important part of a precision agriculture program and are becoming widely used by cotton producers for making management decisions. Members of the cotton industry have shown interest in using cotton yield monitors for collecting data from production scale variety yield trials (experiments that test yield performance for numerous varieties). Weighing boll buggies are the current industry standard for measuring yield in variety trials. This process is time consuming and requires extra equipment and labor. The ability to use a yield monitor for measuring yield would streamline variety trial harvesting. Recommendations for the Ag Leader cotton yield monitor state that the monitor should be recalibrated when harvesting a new variety. This poses a problem for collecting yield data from a variety trial due to the numerous calibrations that would be required. The primary objective of this research is to evaluate and enhance monitor performance in order to use it for collecting variety trial data. This will be done using different calibration techniques and post-processing models developed using measured gin turnout and environmental variables.Data were collected in 2007 and 2008 at the Milan Research and Education Center in Milan, TN. Monitor weights were compared to boll buggy weights to determine variation between these two yield estimation techniques. This measured variation is defined as Yield Prediction Error (YPE). Before calibration, yield explained 44% of the variation in YPE. After post-calibration, moisture and yield explained 48% of the variation in YPE. Post-processing models were developed using these types of relationships but were unsuccessful as they introduced more variation into the data set. The relationship of YPE to moisture suggests that boll buggy weights should be adjusted to a common moisture content. The relationship of YPE to yield suggests that improvements could be made to the monitor. Post-processing the data using yield in the model was able to reduce the mean absolute error to 2.5% from 3.3% using only calibration C (recalibrating when weather or other events cause a multiple day stoppage in harvesting).Tukey’s mean separation test was used for both yield measurement techniques to determine differences in variety trial results. In both 2007 and 2008, the variety trial results returned the same differences for both yield estimation techniques. This dataset supports that with proper calibration, the yield monitor can be used to collect yield data for cotton variety trials.

The improvement in tracking a moving target for an extended period of time was measured on seven human subjects. Each subject was presented with a moving target for a few consecutive runs. The mean square error (MSE) between the eye position and the target position was measured for each run, also, the MSE between the eye velocity and the target velocity was measured. These MSEs were plotted versus time to obtain the learning curves. One subject did not show any improvement in MSEs. For four subjects the position MSE decreased with time. One of these four, the one who obtained the best results, also showed an improvement in his velocity MSE. Two subjects learned to adjust their eye velocity to the target's velocity, as well as to maintain small position mean square errors.