Saccharomyces cerevisiae–Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography

To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis

Constructive control of quantum systems using factorization of unitary operators

We demonstrate how structured decompositions of unitary operators can be employed to derive control schemes for finite-level quantum systems that require only sequences of simple control pulses such as square wave pulses with finite rise and decay times or Gaussian wavepackets. To illustrate the technique it is applied to find control schemes to achieve population transfers for pure-state systems, complete inversions of the ensemble populations for mixed-state systems, create arbitrary superposition states and optimize the ensemble average of dynamic observables.Comment: 28 pages, IoP LaTeX, principal author has moved to Cambridge University ([email protected]

Movements, distribution, and population dynamics of polar bears in the Beaufort Sea

Thesis (Ph.D.) University of Alaska Fairbanks, 1995I used mark and recapture, and radio telemetry to describe movements and population dynamics of polar bears of the Beaufort Sea. Rates of movement were lowest for females with cubs in spring, highest for females with yearlings in winter, and varied from 0.30-0.96 km/h. Total distances moved each month and year were 186-492 km and 1,454-6,203 km respectively. Highest and lowest levels of activity were in June and September. Activity levels were highest from mid-day to late evening. Females with cubs were more active than other bears. Annual home ranges varied from 12,730 km\sp2 to 596,800 km\sp2. The Beaufort Sea population occupied a 939,153 km\sp2 area extending 300 km offshore from Cape Bathurst, Canada, to Pt. Hope, Alaska. Maternal denning in the Beaufort Sea region was common, but 52% of discovered dens were on the drifting pack ice. Bears denning on pack ice drifted as far as 997 km (x = 385 km). Bears followed to >1 den did not reuse sites. Consecutive dens were 20-1,304 km apart, but radio-collared bears were faithful to substrate and locale of previous dens. Of 44 polar bears that denned along the Beaufort Sea coast, 80% were located between 137\sp\circ00'W and 146\sp\circ59'W. Of those 44, 20 (45%) were on the Arctic National Wildlife Refuge, including 15 (34%) in the 1002 coastal plain area, which may contain >9 billion barrels of recoverable oil. Data indicated, however, that spatial and temporal restrictions on developments could prevent most disruptions of denned bears. Survival of adult female polar bears was higher than previously thought ($\ S=0.96).$ Survival of cubs ($\ S=0.65)$ and yearlings ($\ S=0.86)$ was lower than for adults, but increased rapidly with age. Shooting accounted for 85% of the documented deaths of adult females. The population grew to ~1500 animals ($\ge$2% per year) from 1967-1992. Condition of adult females, survival of young, and litter sizes declined, while age of maturity and reproductive interval appeared to increase. The population may have approached carrying capacity by the end of the study

Integration of RTK GPS and IMU for Accurate UAV Positioning - Integrasjon av RTK GPS og IMU for nøyaktig UAV-posisjonering

Real-Time Kinematic GPS (RTK GPS) can provide centimeter accuracy and may be used for high accuracy positioning of Unmanned Aerial Vehicles (UAVs).In order to compute its position a Global Navigation Satellite System (GNSS) receiver processes measurements of signals from GNSS satellites. The processing results in a delay before the position measurement is available to the UAV. If the UAV is highly dynamic the delayed measurement will describe a past position, resulting in reduced accuracy. This thesis presents the hardware and software architecture of an integrated Real-Time Kinematic GPS/Inertial Navigation System (RTK GPS/INS) for UAV applications that compensates for the time-delay. The RTK GPS system is constructed using single-frequency GPS receivers and the open-source Real-Time Kinematic Library (RTKLIB). The RTK GPS/INS navigation system is implemented in the DUNE(DUNE:Unied Navigation Environment)-framework running on the embedded payload computer of the UAV. The integration of RTK GPS and ineartial measurements is performed by a nonlinear observer estimating the delayed states and recovering the current states using inertial measurements. Hardware-in-the-Loop simulations and experimental ights with the x8 Skywalker UAV are used to test the navigation system

Estimation of capture probabilities using generalized estimating equations and mixed effects approaches

Modeling individual heterogeneity in capture probabilities has been one of the most challenging tasks in capture-recapture studies. Heterogeneity in capture probabilities can be modeled as a function of individual covariates, but correlation structure among capture occasions should be taking into account. A proposed generalized estimating equations (GEE) and generalized linear mixed modeling (GLMM) approaches can be used to estimate capture probabilities and population size for capture-recapture closed population models. An example is used for an illustrative application and for comparison with currently used methodology. A simulation study is also conducted to show the performance of the estimation procedures. Our simulation results show that the proposed quasi-likelihood based on GEE approach provides lower SE than partial likelihood based on either generalized linear models (GLM) or GLMM approaches for estimating population size in a closed capture-recapture experiment. Estimator performance is good if a large proportion of individuals are captured. For cases where only a small proportion of individuals are captured, the estimates become unstable, but the GEE approach outperforms the other methods

Human Disturbances of Denning Polar Bears in Alaska

Polar bears (Ursus maritimus) give birth in dens of snow and ice. The altricial neonates cannot leave the den for >2 months post-partum and are potentially vulnerable to disturbances near dens. The coastal plain (1002) area of Alaska's Arctic National Wildlife Refuge (ANWR) lies in a region of known polar bear denning and also may contain >9 billion barrels of recoverable oil. Polar bears in dens could be affected in many ways by hydrocarbon development, but neither the distribution of dens nor the sensitivity of bears in dens has been known. I documented the distribution of dens on ANWR between 1981 and 1992 and observed responses of bears in dens to various anthropogenic disturbances. Of 44 dens located by radiotelemetry on the mainland coast of Alaska and Canada, 20 (45%) were on ANWR and 15 (34%) were within the 1002 area. Thus, development of ANWR will increase the potential that denning polar bears are disturbed by human activities. However, perturbations resulting from capture, marking, and radiotracking maternal bears did not affect litter sizes or stature of cubs produced. Likewise, 10 of 12 denned polar bears tolerated exposure to exceptional levels of activity. This tolerance and the fact that investment in the denning effort increases through the winter indicated that spatial and temporal restrictions on developments could prevent the potential for many disruptions of denned bears from being realized.Key words: Alaska, ANWR, Arctic, denning, disturbance, impact, oil development, polar bear, reproduction, Ursus maritimusLes ourses polaires (Ursus maritimus) donnent naissance à leurs petits dans des tanières de neige et de glace. Les nouveau-nés nidicoles ne peuvent quitter la tanière avant au moins deux mois suivant leur naissance, et des perturbations près de leur tanière sont susceptibles de les affecter. La région de la plaine côtière (1002) du Arctic National Wildlife Refuge (ANWR) en Alaska est située dans une zone connue comme site de mise bas pour l'ourse polaire et qui pourrait contenir plus de neuf milliards de barils de pétrole récupérable. Les ours polaires dans les tanières pourraient être affectés de bien des façons par l'exploitation des hydrocarbures, mais on n'a pas de données que ce soit sur la distribution des tanières ou sur la sensibilité des ours à l'intérieur de celles-ci. Nous avons documenté la distribution des tanières dans l'ANWR entre 1981 et 1992, et y avons observé la réponse des ours à diverses perturbations anthropiques. Parmi les 44 tanières localisées par radio-télémétrie sur la côte continentale de l'Alaska et du Canada, 20 (soit 45 p. cent) étaient situées dans l'ANWR et 15 (soit 34 p. cent) l'étaient dans la zone 1002. La mise en valeur de l'ANWR va donc accroître le potentiel de perturbation des ours polaires par l'activité humaine. Les perturbations résultant de la capture, du marquage et du pistage radio-électrique des mères ourses n'ont cependant pas affecté le nombre de petits dans une portée ni la taille des oursons. En outre, de 10 à 12 des ours polaires vivant dans leur tanière supportaient l'exposition à un niveau d'activité relativement intense. Cette tolérance et le fait que l'investissement dans la vie en tanière augmente au cours de l'hiver indiquent que des restrictions spatiales et temporelles imposées sur la mise en valeur pourraient empêcher que les ours en tanière soient soumis à d'éventuelles perturbations.Mots clés : Alaska, ANWR, Arctique, mise bas dans les tanières, perturbation, impact, exploitation pétrolière, ours polaire, reproduction, Ursus maritimu