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

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

Aerospace Medicine and Biology: A continuing bibliography with indexes, supplement 267, January 1985

This publication is a cumulative index to the abstracts contained in the Supplements 255 through 266 of Aerospace Medicine and Biology: A Continuing Bibliography. It includes seven indexes--subject, personal author, corporate source, foreign technology, contract number, report number, and accession number

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

AlGaN/GaN sensors for direct monitoring of fluids and bioreactions

AlGaN/GaN based pH-sensors have been characterized and further developed for the in situ monitoring of cell reactions. Generally, good proliferation of different cell lines was observed on AlGaN and GaN surfaces without using any kind of thin films of organic material for improving of the cellular adhesion and biocompatibility. NG108-15 nerve cells were chosen for the investigation of the sensor response on cell activity. In an open setup with contact to normal atmosphere, the monitoring of the spontaneous cell activity (“breathing”) was recorded. By titration in complex electrolytes, it was demonstrated that these sensors are able to monitor complex cell reactions on different neuroinhibitors. Numerical simulations as well as simplified analytical calculations of ion fluxes give strong evidence that the signal in the cell-transistor coupling experiments is primarily generated by the Na+ flux. In conclusion, the AlGaN/GaN-ISFETs show stable operation under physiological conditions, exhibit a very good signal resolution and are suitable for long-time monitoring of cell reactions on different stimuli.In dieser Arbeit wurden AlGaN/GaN-Heterostrukturen, die ein hohes Potenzial für pH-Sensoren aufweisen, charakterisiert und weiterentwickelt für die elektronische Erfassung von Zellreaktionen. Dazu wurden NG108-15 Nervenzellen auf den Sensoroberflächen kultiviert und deren Antwort auf Stimulierung mit verschiedenen Neuroinhibitoren aufgezeichnet. Zunächst wurde ein Messaufbau für das Erfassen extrazellularer Potenzialänderungen entworfen und das bestehende Chipdesign sowie die Herstellungstechnologie weiterentwickelt. Für die Auswahl optimaler Sensoren für die Transistor-Zell-Kopplung wurden sowohl mittels PIMBE und MOCVD gewachsene Heterostrukturen charakterisiert bezüglich ihrer elektronischen Transporteigenschaften und ihres Verhaltens als pH-Sensor. Auf AlGaN- und GaN-Oberflächen konnte eine sehr gute Kultivierung verschiedener Zelllinien erzielt werden ohne die sie sonst übliche Verwendung organischer Zwischenschichten zur Erhöhung von Adhäsion (z.B. Fibroplasten). Der Einfluss verschiedener Technologie- und sensorrelevanter Behandlungsschritte auf die Oberflächeneigenschaften der AlGaN/GaN-Sensoren wurde untersucht und die Medienstabilität bzw. Wechselwirkungen wurden analysiert. In einem offenen Setup mit Gasaustausch zur Umgebung wurde eine spontane Zellaktivität erfasst ("Zellatmung"), die in einem abgeschlossenen Setup aufgrund des reduzierten Gasaustausches nicht auftrat. Weiterhin wurde die Empfindlichkeit des Sensors auf Potenzialänderungen durch Na+ and K+ Ionen und deren Reaktionen mit Neurotoxinen bestätigt. Durch Titration in komplexe Elektrolyte und durch Kultivierung von NG108-15 Nervenzellen auf der Sensoroberfläche wurde demonstriert, dass die Sensoren in der Lage sind, komplexe Zellreaktionen zu erfassen. Berechnungen mit Hilfe von Simulationen und vereinfachten analytischen Beschreibungen für die Ionenflüsse belegten, dass bei der Zell-Transistor-Kopplung das Sensorsignal im Wesentlichen durch die Na+ Flüsse erzeugt wird. Die experimentellen Beobachtungen und die theoretischen Modellierung zeigte dafür eine gute Übereinstimmung. Zusammenfassend wurde in dieser Arbeit gezeigt, dass AlGaN/GaN-ISFETs stabil unter physiologischen Bedingungen arbeiten, sehr gute Signalauflösung ermöglichen und für Langzeitmessungen mit lebenden Zellen geeignet sind

Aerospace Medicine and Biology: A continuing bibliography with indexes (supplement 259)

A bibliography containing 476 documents introduced into the NASA scientific and technical information system in May 1984 is presented. The primary subject categories included are: life sciences, aerospace medicine, behavioral sciences, man/system technology, life support, and planetary biology. Topics extensively represented were space flight stress, man machine systems, weightlessness, human performance, mental performance, and spacecraft environments. Abstracts for each citation are given

Inorganic micro/nanostructures-based high-performance flexible electronics for electronic skin application

Electronics in the future will be printed on diverse substrates, benefiting several emerging applications such as electronic skin (e-skin) for robotics/prosthetics, flexible displays, flexible/conformable biosensors, large area electronics, and implantable devices. For such applications, electronics based on inorganic micro/nanostructures (IMNSs) from high mobility materials such as single crystal silicon and compound semiconductors in the form of ultrathin chips, membranes, nanoribbons (NRs), nanowires (NWs) etc., offer promising high-performance solutions compared to conventional organic materials. This thesis presents an investigation of the various forms of IMNSs for high-performance electronics. Active components (from Silicon) and sensor components (from indium tin oxide (ITO), vanadium pentaoxide (V2O5), and zinc oxide (ZnO)) were realised based on the IMNS for application in artificial tactile skin for prosthetics/robotics. Inspired by human tactile sensing, a capacitive-piezoelectric tandem architecture was realised with indium tin oxide (ITO) on a flexible polymer sheet for achieving static (upto 0.25 kPa-1 sensitivity) and dynamic (2.28 kPa-1 sensitivity) tactile sensing. These passive tactile sensors were interfaced in extended gate mode with flexible high-performance metal oxide semiconductor field effect transistors (MOSFETs) fabricated through a scalable process. The developed process enabled wafer scale transfer of ultrathin chips (UTCs) of silicon with various devices (ultrathin chip resistive samples, metal oxide semiconductor (MOS) capacitors and n‐channel MOSFETs) on flexible substrates up to 4″ diameter. The devices were capable of bending upto 1.437 mm radius of curvature and exhibited surface mobility above 330 cm2/V-s, on-to-off current ratios above 4.32 decades, and a subthreshold slope above 0.98 V/decade, under various bending conditions. While UTCs are useful for realizing high-density high-performance micro-electronics on small areas, high-performance electronics on large area flexible substrates along with low-cost fabrication techniques are also important for realizing e-skin. In this regard, two other IMNS forms are investigated in this thesis, namely, NWs and NRs. The controlled selective source/drain doping needed to obtain transistors from such structure remains a bottleneck during post transfer printing. An attractive solution to address this challenge based on junctionless FETs (JLFETs), is investigated in this thesis via technology computer-aided design (TCAD) simulation and practical fabrication. The TCAD optimization implies a current of 3.36 mA for a 15 μm channel length, 40 μm channel width with an on-to-off ratio of 4.02x 107. Similar to the NRs, NWs are also suitable for realizing high performance e-skin. NWs of various sizes, distribution and length have been fabricated using various nano-patterning methods followed by metal assisted chemical etching (MACE). Synthesis of Si NWs of diameter as low as 10 nm and of aspect ratio more than 200:1 was achieved. Apart from Si NWs, V2O5 and ZnO NWs were also explored for sensor applications. Two approaches were investigated for printing NWs on flexible substrates namely (i) contact printing and (ii) large-area dielectrophoresis (DEP) assisted transfer printing. Both approaches were used to realize electronic layers with high NW density. The former approach resulted in 7 NWs/μm for bottom-up ZnO and 3 NWs/μm for top-down Si NWs while the latter approach resulted in 7 NWs/μm with simultaneous assembly on 30x30 electrode patterns in a 3 cm x 3 cm area. The contact-printing system was used to fabricate ZnO and Si NW-based ultraviolet (UV) photodetectors (PDs) with a Wheatstone bridge (WB) configuration. The assembled V2O5 NWs were used to realize temperature sensors with sensitivity of 0.03% /K. The sensor arrays are suitable for tactile e-skin application. While the above focuses on realizing conventional sensing and addressing elements for e-skin, processing of a large amount of data from e-skin has remained a challenge, especially in the case of large area skin. A Neural NW Field Effect Transistors (υ-NWFETs) based hardware-implementable neural network (HNN) approach for tactile data processing in e-skin is presented in the final part of this thesis. The concept is evaluated by interfacing with a fabricated kirigami-inspired e-skin. Apart from e-skin for prosthetics and robotics, the presented research will also be useful for obtaining high performance flexible circuits needed in many futuristic flexible electronics applications such as smart surgical tools, biosensors, implantable electronics/electroceuticals and flexible mobile phones

The Fifth National Technology Transfer Conference and Exposition

No abstract availabl

Aerospace medicine and biology: A continuing bibliography with indexes (supplement 405)

This bibliography lists 225 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during Sep. 1995. Subject coverage includes: aerospace medicine and physiology, life support systems and man/system technology, protective clothing, exobiology and extraterrestrial life, planetary biology, and flight crew behavior and performance