Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers

The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75 % of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90 % of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the £j values for RCAI binding, internalization and efflux were 5, 18 and 13-14 min, respectively. By comparing efflux with and without galactose present, we found that 60 % of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4°C became localized within more caveolae within 2.5 min of warming to 37 °C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RCAI contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN

Immunogold Labeling to Enhance Contrast in Optical Coherence Microscopy of Tissue Engineered Corneal Constructs

Our lab has used an optical coherence microscope (OCM) to assess both the structure of tissue-engineered corneal constructs and their transparency. Currently, we are not able to resolve cells versus collagen matrix material in the images produced. We would like to distinguish cells in order to determine if they are viable while growing in culture and also if they are significantly contributing to the light scattering in the tissue. In order to do this, we are currently investigating the use of immunogold labeling. Gold nanoparticles are high scatterers and can create contrast in images. We have conjugated gold nanoparticles to antibodies specific to the α5 β1 integrins expressed in some corneal cells. This choice of target should allow assessment of the phenotypic behavior of the cells in the tissue, as different integrins are expressed in different phenotypes. This study applies the immunogold technique to study cultured corneal cells using the OCM with the ultimate goal of monitoring cellular behavior in engineered tissue and corroborating results from standard histological methods

SOF’S ROLE IN STRATEGIC COMPETITION: A WARGAME

Includes Supplementary MaterialCan a wargame effectively model how Special Operation Forces’ (SOF) irregular warfare capabilities influence strategic competition, and more broadly, do wargames have a place in the education of SOF professionals? SOF needs educational tools that stimulate critical thinking and intellectual creativity to address the challenges and complexities of irregular warfare and strategic competition so they can maintain their position as the DOD’s premier option for irregular warfare. Educational wargames have an extensive history in professional military organizations, and the wargame Tears of Tyranny is ready for use now to educate joint SOF on irregular warfare at the tactical and operational levels. Further development of Tears of Tyranny should include digitization and refinement through an additional capstone team.Outstanding ThesisMajor, United States ArmyMajor, United States ArmyMajor, United States ArmyMajor, United States ArmyApproved for public release. Distribution is unlimited

A dynamic Archean sulfur cycle

Many aspects of the Earth’s early sulfur cycle, from the origin of mass anomalous fractionations to the scale and degree of biological involvement, remain poorly understood. We have been studying the nature of multiple sulfur isotope (^(32)S, ^(33)S, and ^(34)S) signals using a novel combination of scanning high-resolution low-temperature superconductivity SQUID microscopy and secondary ion mass spectrometry (SIMS) techniques in a suite of samples from distal slope and basinal environments adjacent to a major Late Archean-age (~2.6-2.52 Ga) carbonate platform. Coupled with petrography, these techniques allow us to interrogate, at the same microscopic scale, the complex history of mineralization in samples containing diverse sulfide-bearing mineral components. Because of a general lack of Archean sulfate minerals, we focused our analyses on early diagenetic pyrite nodules, precipitated in surface sediments. This allows us to assay fractionations by controlling for isotope mass balance