Design, fabrication, and application of uniformly distributed RC networks for use in electronic circuits

In the design of electronic circuits, both linear and non-linear, one of the goals is the reduction of the number of elements needed to complete the design. By using distributed RC networks a reduction in the number of elements by at least 50% is usually possible. This dissertation discusses several distributed RC networks, develops detailed design procedures for each, and applies them in the design of electronic circuits. The indefinite admittance matrix (IAM) for the DRURC (double-resistive uniformly distributed RC network) and the URC (single-resistive uniformly distributed RC network) , which have been previously derived, are used as the starting points for the development of the IAM\u27s for the TURC (tapped URC) and the TDRURC (tapped DRURC) , a new distributed network. The development of the IAM for the TDRURC proceeds from the interconnection of two DRURC\u27s such that the IAM\u27s add. A similar procedure using URC\u27s yields the IAM for the TURC. These two IAM\u27s, which have not been previously published, allow the derivation of the design equations necessary to apply these networks to electronic circuits. These four distributed network elements (URC, TURC, DRURC, and TDRURC) are applied to the reduction in the numbers of elements needed for the following electronic circuits: RC-coupled amplifiers, multivibrator circuits, high-Q (10 to 150) band-pass amplifiers, and phase-shift oscillators. The application to RC-coupled amplifiers is new, while the applications to band-pass amplifiers and phase-shift oscillators are significant advances to work that has been previously suggested. A prototype of each of the four distributed networks was built by the author in 1972 using a thin-film deposition method which is described in Chapter VII. The phase-shift oscillator, band-pass amplifier, and multivibrator circuits were also built and tested. The results compared favorably with predicted results. The procedures for designing and fabricating uniformly distributed RC networks are reviewed in detail. A review of both thin-film fabrication by vacuum deposition and semiconductor fabrication by diffusion is included --Abstract, pages ii-iii

ASCT-1 Is a Neutral Amino Acid Exchanger with Chloride Channel Activity

The ubiquitous transport activity known as system ASC is characterized by a preference for small neutral amino acids including alanine, serine, and cysteine. ASCT-1 and ASCT-2, recently cloned transporters exhibiting system ASC-like selectivity, are members of a major amino acid transporter family that includes a number of glutamate transporters. Here we show that ASCT1 functions as an electroneutral exchanger that mediates negligible net amino acid flux. The electrical currents previously shown to be associated with ASCT1-mediated transport result from activation of a thermodynamically uncoupled chloride conductance with permeation properties similar to those described for the glutamate transporter subfamily. Like glutamate transporters, ASCT1 activity requires extracellular Na+. However, unlike glutamate transporters, which mediate net flux and complete a transport cycle by countertransport of K+, ASCT-1 mediates only homo- and heteroexchange of amino acids and is insensitive to K+. The properties of ASCT-1 suggest that it may function to equilibrate different pools of neutral amino acids and provide a mechanism to link amino acid concentration gradient

Isolation of Current Components and Partial Reaction Cycles in the Glial Glutamate Transporter EAAT2

The kinetic properties of the excitatory amino acid transporter EAAT2 were studied using rapid applications of l-glutamate to outside-out patches excised from transfected human embryonic kidney 293 cells. In the presence of the highly permeant anion SCN−, pulses of glutamate rapidly activated transient anion channel currents mediated by the transporter. In the presence of the impermeant anion gluconate, glutamate pulses activated smaller currents predicted to result from stoichiometric flux of cotransported ions. Both anion and stoichiometric currents displayed similar kinetics, suggesting that anion channel gating and stoichiometric charge movements are linked to early transitions in the transport cycle. Transporter-mediated anion currents were recorded with ion and glutamate gradients favoring either unidirectional influx or exchange. Analysis of deactivation and recovery kinetics in these two conditions suggests that, after binding, translocation of substrate is more likely than unbinding under physiological conditions. The kinetic properties of EAAT2, the dominant glutamate transporter in brain astrocytes, distinguish it as an efficient sink for synaptically released glutamate

Macroscopic and Microscopic Properties of a Cloned Glutamate Transporter/Chloride Channel

The behavior of a Cl− channel associated with a glutamate transporter was studied using intracellular and patch recording techniques in Xenopus oocytes injected with human EAAT1 cRNA. Channels could be activated by application of glutamate to either face of excised membrane patches. The channel exhibited strong selectivity for amphipathic anions and had a minimum pore diameter of ∼5Å. Glutamate flux exhibited a much greater temperature dependence than Cl− flux. Stationary and nonstationary noise analysis was consistent with a sub-femtosiemen Cl− conductance and a maximum channelPo ≪ 1. The glutamate binding rate was similar to estimates for receptor binding. After glutamate binding, channels activated rapidly followed by a relaxation phase. Differences in the macroscopic kinetics of channels activated by concentration jumps of l-glutamate or d-aspartate were correlated with differences in uptake kinetics, indicating a close correspondence of channel gating to state transitions in the transporter cycle

In vivo imaging of protease activity by Probody therapeutic activation.

Probody™ therapeutics are recombinant, proteolytically-activated antibody prodrugs, engineered to remain inert until activated locally by tumor-associated proteases. Probody therapeutics exploit the fundamental dysregulation of extracellular protease activity that exists in tumors relative to healthy tissue. Leveraging the ability of a Probody therapeutic to bind its target at the site of disease after proteolytic cleavage, we developed a novel method for profiling protease activity in living animals. Using NIR optical imaging, we demonstrated that a non-labeled anti-EGFR Probody therapeutic can become activated and compete for binding to tumor cells in vivo with a labeled anti-EGFR monoclonal antibody. Furthermore, by inhibiting matriptase activity in vivo with a blocking-matriptase antibody, we show that the ability of the Probody therapeutic to bind EGFR in vivo was dependent on protease activity. These results demonstrate that in vivo imaging of Probody therapeutic activation can be used for screening and characterization of protease activity in living animals, and provide a method that avoids some of the limitations of prior methods. This approach can improve our understanding of the activity of proteases in disease models and help to develop efficient strategies for cancer diagnosis and treatment