Aerospace Medicine and Biology: A continuing bibliography with indexes, supplement 172

This bibliography lists 132 reports, articles, and other documents introduced into the NASA scientific and technical information system in September 1977

FTIR difference and resonance raman spectroscopy of rhodopsins with applications to optogenetics

Thesis (Ph. D.)--Boston UniversityThe major aim of this thesis is to investigate the molecular basis for the function of several types of rhodopsins with special emphasis on their application to the new field of optogenetics. Rhodopsins are transmembrane biophotonic proteins with 7 a-helices and a retinal chromophore. Studies included Archaerhodopsin 3 (AR3), a light driven proton pump similar to the extensively studied bacteriorhodopsin (BR); channelrhodopsins 1 and 2, light-activated ion channels; sensory rhodopsin II (SRII), a light-sensing protein that modulates phototaxis used in archaebacteria; and squid rhodopsins (sRho), the major photopigment in squid vision and a model for human melanopsin, which controls circadian rythms. The primary techniques used in these studies were FTIR difference spectroscopy and resonance Raman spectroscopy. These techniques, in combination with site directed mutagenesis and other biochemical methodologies produced new knowledge regarding the structural changes of the retinal chromophore, the location and function of internal water molecules as well as specific amino acids and peptide backbone. Specialized techniques were developed that allowed rhodopsins to be studied in intact membrane environments and in some cases in vivo measurements were made on rhodopsin heterologously expressed in E. coli thus allowing the effects of interacting proteins and membrane potential to be investigated. Evidence was found that the local environment of one or more internal water molecules in SRII is altered by interaction with its cognate transducer, Htrii, and is also affected by the local lipid environment. In the case of AR3, many of the broad IR continuum absorption changes below 3000 cm-1, assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR, were found to be very similar to BR. Bands assigned to water molecules near the Schiff base postulated to be involved in proton transport were, however, shifted or absent. Structural changes of internal water molecules and possible bands associated with the interaction with ,8-arrestins were also detected in photoactivated squid rhodopsin when transformed to the acid Meta intermediate. Near-IR confocal resonance Raman measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. On the basis of these measurements, a model is proposed which provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential

Engineering derivatives from biological systems for advanced aerospace applications

The present study consisted of a literature survey, a survey of researchers, and a workshop on bionics. These tasks produced an extensive annotated bibliography of bionics research (282 citations), a directory of bionics researchers, and a workshop report on specific bionics research topics applicable to space technology. These deliverables are included as Appendix A, Appendix B, and Section 5.0, respectively. To provide organization to this highly interdisciplinary field and to serve as a guide for interested researchers, we have also prepared a taxonomy or classification of the various subelements of natural engineering systems. Finally, we have synthesized the results of the various components of this study into a discussion of the most promising opportunities for accelerated research, seeking solutions which apply engineering principles from natural systems to advanced aerospace problems. A discussion of opportunities within the areas of materials, structures, sensors, information processing, robotics, autonomous systems, life support systems, and aeronautics is given. Following the conclusions are six discipline summaries that highlight the potential benefits of research in these areas for NASA's space technology programs

Molecular Photochemistry

There have been various comprehensive and stand-alone text books on the introduction to Molecular Photochemistry which provide crystal clear concepts on fundamental issues. This book entitled "Molecular Photochemistry - Various Aspects" presents various advanced topics that inherently utilizes those core concepts/techniques to various advanced fields of photochemistry and are generally not available. The purpose of publication of this book is actually an effort to bring many such important topics clubbed together. The goal of this book is to familiarize both research scholars and post graduate students with recent advancement in various fields related to Photochemistry. The book is broadly divided in five parts: the photochemistry I) in solution, II) of metal oxides, III) in biology, IV) the computational aspects and V) applications. Each part provides unique aspect of photochemistry. These exciting chapters clearly indicate that the future of photochemistry like in any other burgeoning field is more exciting than the past

NASA/ASEE Summer Faculty Fellowship Program

This document is a collection of technical reports on research conducted by the participants in the 1996 NASA/ASEE Summer Faculty Fellowship Program at the Kennedy Space Center (KSC). This was the twelfth year that a NASA/ASEE program has been conducted at KSC. The 1996 program was administered by the University of Central Florida in cooperation with KSC. The program was operated under the auspices of the American Society for Engineering Education (ASEE) with sponsorship and funding from the Office of Educational Affairs, NASA Headquarters, Washington, DC and KSC. The KSC Program was one of nine such Aeronautics and Space Research Program funded by NASA in 1996. The NASA/ASEE Program is intended to be a two-year program to allow in-depth research by the University faculty member. The editors of this document were responsible for selecting appropriately qualified faculty to address some of the many problems of current interest to NASA/KSC

Improvements in optical techniques to investigate the behavior and neuronal network dynamics over long timescales

Developments in optical technology have produced an important shift in experimental neuroscience from electrophysiological methods for observation and stimulation to all-optical solutions. One expects this trend to continue as future developments continue to deliver, and improve upon, the original promises of the technology: 1) minimally invasive actuation and recording of neurons, and 2) a drastic increase in targets that can be treated simultaneously. Moreover, as the high costs of the technology are reduced, one may expect its larger-scale adoption in the neuroscience community. In this thesis, I describe the development and implementation of two alloptical solutions for the analysis of behavior, neuronal signaling, and stimulation, which improve on previous state-of-the-art: (1) A minimally-invasive, high signal-to-noise twophoton microscopy setup capable of simultaneous, live-imaging of a large subset of sensory neurons post activation, and (2) a low-cost tracking solution to stimulate and record behavior. I begin this thesis with a review of recent advances in optical neuroscience techniques for the study of neuronal networks with the focus on work done in Caenorhabditis elegans. Then, in chapter 2, I describe my implementation of a two-photon temporal focusing microscopy setup and show significant improvements through the use of a high power/ high pulse repetition rate excitation system, enabling live imaging with high resolution for extended periods of time. I model temperature increase during a physiological imaging scenario for different repetition rates at fixed peak intensities and find range centered around 1 MHz to be optimal. Lastly, I describe the low-cost tracking setup with the ability to stimulate and record behavior over the course of hours. The setup is capable of two-color stimulation of optogenetic proteins over the area of the behavioral arena in combination with volatile chemicals. To showcase the utility of the system, I demonstrate behavioral analysis of integration of contradictory cues. In summary, I present a set of techniques for the interrogation of neural networks from animal behavior to neuronal activity, over timescales of potentially hours and days. These techniques can be used to address a new dimension of scientific questions.Okinawa Institute of Science and Technology Graduate Universit

NASA Tech Briefs, July 1992

Topics include: New Product Ideas; Electronic Components and Circuits; Electronic Systems; Physical Sciences; Materials; Computer Programs; Mechanics; Machinery; Fabrication Technology; Mathematics and Information Sciences; Life Sciences

Optogenetics and biotechnology : production and in vitro characterization of Ab-Initio designed channelrhodopsin-2 mutants

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de CoimbraNos últimos anos têm sido desenvolvidas várias ferramentas para permitir o controlo de neurónios específicos, possibilitando o estudo da sua função. Estas novas ferramentas superam a falta de selectividade e o fraco controlo temporal proveniente do uso de estimulação eléctrica no controlo de actividade neuronal. A optogenética refere-se á integração de óptica e genética para obter um ganho ou perda de função em eventos bem definidos dentro de células específicas em tecido vivo. A capacidade de “ligar” e “desligar” neurónios utilizando luz é de facto uma tecnologia inovadora que oferece uma solução para limitações passadas. A optogenética, considerada por vários especialistas como ‘’método do ano’’ e ‘’inovação da década’’, em 2010, é utilizada para hiperpolarizar ou despolarizar neurónios alvo, de uma forma menos invasiva, utilizando luz e usufruindo de uma alta resolução espacial e escala temporal na ordem dos milissegundos. Esta técnica tem permitido o mapeamento e estudo de redes neuronais com uma grande eficácia. A ‘’Channelrhodopsin-2’’ (ChR2) é um canal catiónico sensível à luz, derivado da microalga Chlamydomonas reinhardtii. Na última década, a ChR2 tornou-se o arquétipo central e a principal ferramenta da optogenética. Actualmente, a caixa de ferramentas optogenética está em contínua actualização, com contribuições de estratégias de engenharia protéica, tais como mutagénese dirigida e a construção de quimeras com troca de domínios de diferentes espécies de channelrhodopsin. No entanto, alguns aspectos da forma ‘’wild-type’’ da ChR2 ainda requerem atenção e melhoramento. Estes incluem o seu espectro de acção, cinética, níveis de expressão, inactivação, condutância e exactidão de pico de absorção. Em termos de propriedades espectrais, poucas variantes desta proteína têm sido geradas e completamente caracterizadas com sucesso. No entanto, o aprimorar do espectro de activação da ChR2 e do formato do respectivo pico de absorção são algumas das propriedades mais desejadas. A ChR2 é excitada preferencialmente com comprimentos de onda de luz azul (470nm), o que limita o seu uso em material biológico de alta taxa de difusão, tal como o cérebro. Luz de excitação com maiores comprimentos de onda diminui a difusão de luz produzida por tecidos biológicos, e não é absorvida pela hemoglobina, assim, formas da ChR2 ‘’red-shifted’’, a absorver luz vermelha ou mesmo perto de infravermelha, são ferramentas desejáveis para a excitação de tecidos profundos. Alem disto, variantes ‘’blue-shifted’’ são também ferramentas atrativas para desenvolver, XXI dado que a combinação de várias ChR2 que apresentem sensibilidades a diversos comprimentos de onda permitiriam a estimulação de diferentes populações neuronais sem interferência entre si. Neste projecto, realizámos um desenho ab-initio para produzir quatro novas variantes de ChR2, usando uma abordagem de mutagénese dirigida no ambiente do cromófero da ChR2 alterando de forma radical os resíduos alvo. As mutações foram selecionadas com a aplicação de Time Dependent – Density Functional Theory (TDDFT) para prever o espectro de absorção dos mutantes selecionados da ChR2. O ‘’colour tuning’’ da ChR2 foi alcançado em quatro novas variantes criadas. Em particular, fomos capazes de gerar três variantes ‘’red-shifted’’ e uma ‘’blue-shifted’’. Após caracterização espectral, as variantes F217D e F269D apresentaram um ‘’red-shift’’ significativo de 90nm, a variante L221D apresentou um ‘’red-shift’’ de 180nm, a variante F269H apresentou um ‘’blue-shift’’ de 20nm. Apesar dos nossos resultados, é necessária uma caracterização protéica adicional, tal como a avaliação do tráfego membranar em neurónios e as características electrofisiológicas destes novos mutantes para determinar as proriedades cinéticas do canal. Neste trabalho, também conseguimos definir e descrever com sucesso a expressão e purificação da ChR2 ‘’wild-type’’ e de todas as quatro novas variantes no sistema eucariótico de expressão heteróloga - Pichia pastoris. Por fim, o nosso estudo valida as previsões de Time- Dependent Density Functional Theory e revela que abordagens de simulação biofísica podem ser utilizadas com vista à criação de variantes de ChR2 inteligentemente desenhadas. O desenho de novas variantes ChR2, seguindo a lógica racional aplicada, é uma abordagem poderosa e fiável para obter proteínas optimizadas para estratégias biotecnológicas. Os resultados originais obtidos com este trabalho demonstram potential para aplicações futuras, já que novas e melhoradas variantes de ChR2 continuarão a desempenhar um papel central no desenvolvimento e implementação da optogenéticaOver the last few years, several tools have been developed to allow the control over specific types of neuron to enable the study of their function. These novel tools aim to overcome the lack of selectivity and the poor temporal control that derives from trying to control neuronal activity with electrical stimulation. Optogenetics refers to the integration of optics and genetics to obtain gain or loss of function in well-defined events and within specific cells in living tissue. The capacity to turn neurons “on and off” using light is indeed a groundbreaking technology that has become a solution for past limitations. Considered by many, “method of the year” and “breakthrough of the decade”, in 2010, optogenetics is used to hyperpolarize or depolarize specific targeted neurons using light in a less invasive manner, with high spatial resolution and a temporal resolution on the scale of milliseconds. This technique has allowed the mapping and study of neuronal networks with demonstrated efficacy. Channelrhodopsin-2 (ChR2) is a light-gated cation channel, derived from the microalga Chlamydomonas reinhardtii. In the last decade, ChR2 has become the central archetype and the main tool of optogenetics. Presently, the optogenetic toolbox is under continuous update, with contributions from protein engineering strategies, such as site-directed mutagenesis and construction of chimeras with domain swaps between channelrhodopsins of different species. However, some aspects of the wild-type form of ChR2 still require attention and enhancement. These include its action spectra, kinetics, expression levels, inactivation, conductance and absorption peak sharpness. In terms of spectral properties, few variants of this protein have been successfully generated and fully characterized. Nevertheless, tuning of ChR2 activation spectra and absorption peak sharpness are one of the most sought after properties. ChR2 is optimally excitable at a wavelength of blue light (470nm), which limits its use in high light-scattering biologic material, such as the brain. However, long-wavelength excitation light decreases the scattering of light produced by biological tissues and is not absorbed by haemoglobin. Thus, a red-shifted form of ChR2, absorbing red or even near infrared light would be a desirable tool for the excitation of relatively deep tissues. Furthermore, blue-shifted variants would also be attractive tools to develop, since the combination of ChR2 proteins with well separate wavelength sensitivities, combined with multicoloured optics, would permit the stimulation of different neuronal populations with no XXIII interference between them. In this project, we performed ab-initio design to produce four new ChR2 variants, using a radical site-directed mutagenesis approach on target residues in the environment of the ChR2 chromophore. The mutations were selected with the application of Time Dependent – Density Functional Theory (TDDFT) to predict the absorption spectra of ChR2 selected mutants. We achieved successful colour tuning of ChR2 with our four newly created variants. In particular, we were able to generate three red-shifted and one blue–shifted variant. After spectral characterization, the F217D and F269D variants presented a significant 90nm red shift, the L221D variant had a 180nm red shift and the F269H variant presented a 20nm blue shift. Despite our results, additional protein characterization is needed, such as the assessment of membrane trafficking in neurons and an electrophysiological characterization to determine channel kinetic proprieties for each of the variants. In this work, we were also able to define and describe the successful expression and purification of wild type ChR2 and of all the new four variants using the eukaryotic Pichia pastoris heterologous expression system. Finally, our study validates Time-Dependent Density Functional Theory predictions and reveals that biophysical simulation approaches may be used towards the creation of intelligently designed ChR2 variants. The design of new ChR2 variants, following our applied rationale, is a powerful and reliable approach to obtain enhanced proteins for biotechnological strategies. The original output obtained here shows potential for future optogenetic application, as new and improved ChR2 variants will continue to play a central role in the development and implementation of optogenetic

Engineering an inducible NO pathway to facilitate cell-electronics communication

PhD ThesisTurning cells into useful devices to perform unnatural functions creates the potential to permit the interface between biological organisms and electronics. In this thesis cell-based devices were designed and constructed to respond to either light or a specific chemical stimulus. Design constraints were defined by the eventual application of the device, a biohybrid robot. The enzyme endothelial nitric oxide synthase (eNOS) was chosen as a target for genetic engineering. Prior to constructing the device a suitable host for the engineered construct was selected. CHO-K1 cells were transfected with nitric oxide synthase and expression levels were characterized via flow cytometry and inhibitor studies. A novel method for the effective delivery of inhibitors was developed and applied to demonstrate that transfected eNOS was sufficiently expressed to produce a measurable output. In addition, a balance between the native nitric oxide production machinery of the cells and the transfected endothelial nitric oxide synthase was observed. Two systems were designed and constructed for stimuli responsive nitric oxide production. The first system was designed to produce nitric oxide in response to the presence of the antibiotic rapamycin. Chemical induced dimerization would bring the two separated domains of endothelial nitric oxide synthase into close enough proximity to re-establish protein function. The separate oxygenase and reductase domains were successfully amplified and subsequently fused with components of the chemically induced dimerization system. The second system involved fusing a domain from the plant gene Nhp1 (Light Oxygen Voltage domain - LOV) capable of harvesting a photon, with mouse endothelial nitric oxide synthase. This strategy aimed to hijack the wild type protein’s native electron transfer pathway. Manipulation was carried out in bacteria with subsequent transfection into CHO-K1 cells. Subsequent testing of nitric oxide production the mutant cells confirmed the optical sensitivity of the mutant eNOS. Moreover both LOV mutant cell lines were capable of fast response times and switching behaviour. The findings of this thesis demonstrate that genetic engineering of endothelial nitric oxide synthase is a suitable strategy for the controlled release of nitric oxide upon optical stimulation. Moreover the potential of an engineered cell to respond quickly to stimuli has been realized, comparing favourably to genetically engineered systems that rely on gene expression to elicit an output