Diversity and Activity of Roseobacters and Roseophage

Bacteria of the Roseobacter lineage are dominant bacterioplankton in coastal systems and contribute significantly to secondary production in oceanic environments. Generalities of Roseobacter ecology, diversity, and distributions are known, but the intraspecific differences between species and their dynamics over short temporal periods is not well understood. Bacteriophage that infect Roseobacters (‘roseophage’) have the potential to shunt secondary production into the dissolved carbon pool and through the process of infection alter Roseobacter physiology. Despite their significance, little effort was made prior to the onset of this study to characterize roseophage. Using culture dependent and independent approaches, I describe the diversity and activity of Roseobacters and roseophage from two distinct coastal environments. Chapter 2 describes the development of an alternative method to enumerate viruses using epifluorescence microscopy that not only reduces sample processing costs, but also the total volume of sample required. A novel species of the Roseobacter lineage (Marivita roseacus) is proposed in Chapter 3. M. roseacus is unique in its needle-like morphology, forming long, relatively inflexible chains of cells. The Marivita genus is characterized by a distinct ecology, being closely associated with algae, resistant to grazing, and present in numerous marine and saline environments. Chapter 4 details the use of deep-amplicon sequencing (16S rDNA) to describe bacterial succession patterns during a mesocosm algal bloom, revealing the temporal dynamics of ~100 distinct phylotypes. A multivariate analysis showed that temporal portioning amongst the bacterial community was occurring at both high and low taxonomic levels. Chapter 5 details the isolation and genomic characterization of roseophage and describes their ecology using publically available metagenomic databases collected from throughout the world. Four distinct phage were isolated and sequenced including an N4-like strain, a novel Siphoviridae, and two temperate Podoviridae. The two temperate phage were practically identical at the nucleotide level, except for a 3000 bp putative replication module, which showed no homology between the two. Overall, this dissertation suggests that ecological partitioning within the Roseobacter lineage is occurring at and arguably below traditional species level taxonomic classifications and microdiversity amongst closely related marine bacteria is likely the norm rather than the exception

Precision Linear Actuators for the Spherical Primary Optical Telescope Demonstration Mirror

The Spherical Primary Optical Telescope (SPOT) is an ongoing research effort at Goddard Space Flight Center developing wavefront sensing and control architectures for future space telescopes. The 03.5-m SPOT telescope primary mirror is comprise9 of six 0.86-m hexagonal mirror segments arranged in a single ring, with the central segment missing. The mirror segments are designed for laboratory use and are not lightweighted to reduce cost. Each primary mirror segment is actuated and has tip, tilt, and piston rigid-body motions. Additionally, the radius of curvature of each mirror segment may be varied mechanically. To provide these degrees of freedom, the SPOT mirror segment assembly requires linear actuators capable of <lO-nm resolution over a total stroke of 5 mm. These actuators must withstand high static loads as they must support the mirror segment, which has a mass of -100 kg. A stepper motor driving a differential satellite roller screw was designed to meet these demanding requirements. Initial testing showed that the actuator is capable of sub-micron repeatability over the entire 6-mm range, and was limited by 100-200 nm measurement noise levels present in the facility. Further testing must be accomplished in an isolated facility with a measurement noise floor of <5 nm. Such a facility should be ready for use at GSFC in the early summer of 2006, and will be used to better characterize this actuator

Graphite Composite Panel Polishing Fixture

The use of high-strength, lightweight composites for the fixture is the novel feature of this innovation. The main advantage is the light weight and high stiffness-to-mass ratio relative to aluminum. Meter-class optics require support during the grinding/polishing process with large tools. The use of aluminum as a polishing fixture is standard, with pitch providing a compliant layer to allow support without deformation. Unfortunately, with meter-scale optics, a meter-scale fixture weighs over 120 lb (.55 kg) and may distort the optics being fabricated by loading the mirror and/or tool used in fabrication. The use of composite structures that are lightweight yet stiff allows standard techniques to be used while providing for a decrease in fixture weight by almost 70 percent. Mounts classically used to support large mirrors during fabrication are especially heavy and difficult to handle. The mount must be especially stiff to avoid deformation during the optical fabrication process, where a very large and heavy lap often can distort the mount and optic being fabricated. If the optic is placed on top of the lapping tool, the weight of the optic and the fixture can distort the lap. Fixtures to support the mirror during fabrication are often very large plates of aluminum, often 2 in. (.5 cm) or more in thickness and weight upwards of 150 lb (68 kg). With the addition of a backing material such as pitch and the mirror itself, the assembly can often weigh over 250 lb (.113 kg) for a meter-class optic. This innovation is the use of a lightweight graphite panel with an aluminum honeycomb core for use as the polishing fixture. These materials have been used in the aerospace industry as structural members due to their light weight and high stiffness. The grinding polishing fixture consists of the graphite composite panel, fittings, and fixtures to allow interface to the polishing machine, and introduction of pitch buttons to support the optic under fabrication. In its operation, the grinding polishing fixture acts as a reaction structure to the polishing tool. It must be stiff enough to avoid imparting a distorted shape to the optic under fabrication and light enough to avoid self-deflection. The fixture must also withstand significant tangential loads from the polishing machine during operations

Geometric Manufacturability Analysis for Additive Manufacturing

During the development of a new product, it is difficult for designers to predict how their design decisions will impact manufacturability and manufacturing cost of the individual parts in their product. Additive manufacturing is increasingly becoming a viable option to produce high fidelity prototypes and even small-scale production part runs. However, as an emerging technology, there are few resources available to help designers make design decisions regarding quality and manufacturability for additive manufacturing. Most information developed to help designers ensure manufacturability is in the form of general guidelines that designers must interpret and then use their best judgment to scrutinize their design. Designers can only guess, based on previous experience, if the process can produce part features that meet their specified geometric tolerances. However, by using algorithms to analyze part geometry, it is possible to predict additive manufacturing outcomes. This thesis describes the development of two software tools to analyze part geometry in near real-time: one that predicts manufacturability, and another that predicts achievable quality. These tools are used to explore how automated part geometry analysis influences the effectiveness of design for additive manufacturing feedback. The research hypothesis of this thesis is that part geometry analysis improves the practicality, accuracy, and usefulness of design for additive manufacturing feedback. To test this hypothesis, three research thrusts were conducted: evaluating the performance of the newly developed tools relative to existing tools, experimental verification of the predictions of the tools, and a user study evaluating usage of the manufacturability tool during a design task. Comparison with existing tools indicated that both tools described in this thesis have similar computation time as existing solutions, while providing greater potential to allow designers to analyze manufacturing trade-offs, with a more comprehensive approach to modeling sources of errors in the manufacturing process. A range of parts were printed using fused deposition modeling and then inspected. The experimental results showed that the predictions of both tools were relatively accurate, and highlighted several additional process parameters that can be included in the modeling approach to improve accuracy. Lastly, a user study demonstrated that use of the software tool reduced the number of manufacturability problems in participants' designs while requiring a similar amount of time to use, compared with using a list of design heuristics. The findings of the thesis support the practicality, accuracy, and usefulness of geometry analysis software tools to support design for additive manufacturing

Wavefront Sensing and Control Architecture for the Spherical Primary Optical Telescope (SPOT)

Testbed results are presented demonstrating high-speed image-based wavefront sensing and control for a spherical primary optical telescope (SPOT). The testbed incorporates a phase retrieval camera coupled to a 3-Mirror Vertex testbed (3MV) at the NASA Goddard Space Flight Center. Actuator calibration based on the Hough transform is discussed as well as several supercomputing archtectures for image-based wavefront sensing. Timing results are also presented based on various algorithm implementations using a cluster of 64 TigerShare TSlOl DSP's (digital-signal processors)

Stationary Apparatus Would Apply Forces of Walking to Feet

A proposed apparatus would apply controlled cyclic forces to both feet for the purpose of preventing the loss of bone density in a human subject whose bones are not subjected daily to the mechanical loads of normal activity in normal Earth gravitation. The apparatus was conceived for use by astronauts on long missions in outer space; it could also be used by bedridden patients on Earth, including patients too weak to generate the necessary forces by their own efforts. The apparatus (see figure) would be a modified version of a bicycle-like exercise machine, called the cycle ergometer with vibration isolation system (CEVIS), now aboard the International Space Station. Attached to each CEVIS pedal would be a computer-controlled stress/ vibration exciter connected to the heel portion of a special-purpose pedal. The user would wear custom shoes that would amount to standard bicycle shoes equipped with cleats for secure attachment of the balls of the feet to the special- purpose pedals. If possible, prior to use of the apparatus, the human subject would wear a portable network of recording accelerometers, while walking, jogging, and running. The information thus gathered would be fed to the computer, wherein it would be used to make the exciters apply forces and vibrations closely approximating the forces and vibrations experienced by that individual during normal exercise. It is anticipated that like the forces applied to bones during natural exercise, these artificial forces would stimulate the production of osteoblasts (bone-forming cells), as needed to prevent or retard loss of bone mass. In addition to helping to prevent deterioration of bones, the apparatus could be used in treating a person already suffering from osteoporosis. For this purpose, the magnitude of the applied forces could be reduced, if necessary, to a level at which weak hip and leg bones would still be stimulated to produce osteoblasts without exposing them to the full stresses of walking and thereby risking fracture

A protocol for enumeration of aquatic viruses by epifluorescence microscopy using Anodisc™ 13 membranes

<p>Abstract</p> <p>Background</p> <p>Epifluorescence microscopy is a common method used to enumerate virus-like particles (VLP) from environmental samples and relies on the use of filter membranes with pore sizes < 0.02 μm; the most commonly used protocols employ 25 mm Anodisc™ membranes with a built-in support ring. Other filters with small pore sizes exist, including the 13 mm Anodisc™ membranes without a support ring. However, the use of these membranes for viral enumeration has not been previously reported.</p> <p>Results</p> <p>Here we describe a modified protocol for 13 mm Anodisc membranes that uses a custom filter holder that can be readily constructed in individual investigators' laboratories from commercially available Swinnex^®filter holders. We compared VLP concentrations obtained from phage lysates and seawater samples using both Anodisc membranes, as well as Nuclepore™ small pore-size membranes (0.015 or 0.030 μm). The 13 mm Anodisc membranes gave comparable estimates of VLP abundance to those obtained with the 25 mm Anodisc membranes when similar staining methods were employed. Both Nuclepore membranes typically gave an order of magnitude lower VLP abundance values for environmental samples.</p> <p>Conclusions</p> <p>The 13 mm Anodisc membranes are less costly and require smaller sample volumes than their 25 mm counterpart making them ideal for large-scale studies and sample replication. This method increases the options of reliable approaches available for quantifying VLP from environmental samples.</p

Genome Sequences of Two Temperate Phages, ΦCB2047-A and ΦCB2047-C, Infecting Sulfitobacter sp. Strain 2047

We announce the complete genome sequences of two temperate Podoviridae,Sulfitobacter phages ΦCB2047-A and ΦCB2047-C, which infect Sulfitobacter sp. strain 2047, a member of the Roseobacter clade. This is the first report of temperate podophage infecting members of the Sulfitobacter genus of theRoseobacter clade