Multidisciplinary Design Optimization Application to Conceptual Design of University-class Microsatellite Projects

Esta tesis se ha realizado en el contexto del proyecto UPMSat-2, que es un microsatélite diseñado, construido y operado por el Instituto Universitario de Microgravedad "Ignacio Da Riva" (IDR / UPM) de la Universidad Politécnica de Madrid. Aplicación de la metodología Ingeniería Concurrente (Concurrent Engineering: CE) en el marco de la aplicación de diseño multidisciplinar (Multidisciplinary Design Optimization: MDO) es uno de los principales objetivos del presente trabajo. En los últimos años, ha habido un interés continuo en la participación de los grupos de investigación de las universidades en los estudios de la tecnología espacial a través de sus propios microsatélites. La participación en este tipo de proyectos tiene algunos desafíos inherentes, tales como presupuestos y servicios limitados. Además, debido al hecho de que el objetivo principal de estos proyectos es fundamentalmente educativo, por lo general hay incertidumbres en cuanto a su misión en órbita y cargas útiles en las primeras fases del proyecto. Por otro lado, existen limitaciones predeterminadas para sus presupuestos de masa, volumen y energía, debido al hecho de que la mayoría de ellos están considerados como una carga útil auxiliar para el lanzamiento. De este modo, el costo de lanzamiento se reduce considerablemente. En este contexto, el subsistema estructural del satélite es uno de los más afectados por las restricciones que impone el lanzador. Esto puede afectar a diferentes aspectos, incluyendo las dimensiones, la resistencia y los requisitos de frecuencia. En la primera parte de esta tesis, la atención se centra en el desarrollo de una herramienta de diseño del subsistema estructural que evalúa, no sólo las propiedades de la estructura primaria como variables, sino también algunas variables de nivel de sistema del satélite, como la masa de la carga útil y la masa y las dimensiones extremas de satélite. Este enfoque permite que el equipo de diseño obtenga una mejor visión del diseño en un espacio de diseño extendido. La herramienta de diseño estructural se basa en las fórmulas y los supuestos apropiados, incluyendo los modelos estáticos y dinámicos del satélite. Un algoritmo genético (Genetic Algorithm: GA) se aplica al espacio de diseño para optimizaciones de objetivo único y también multiobjetivo. El resultado de la optimización multiobjetivo es un Pareto-optimal basado en dos objetivo, la masa total de satélites mínimo y el máximo presupuesto de masa de carga útil. Por otro lado, la aplicación de los microsatélites en misiones espaciales es de interés por su menor coste y tiempo de desarrollo. La gran necesidad de las aplicaciones de teledetección es un fuerte impulsor de su popularidad en este tipo de misiones espaciales. Las misiones de tele-observación por satélite son esenciales para la investigación de los recursos de la tierra y el medio ambiente. En estas misiones existen interrelaciones estrechas entre diferentes requisitos como la altitud orbital, tiempo de revisita, el ciclo de vida y la resolución. Además, todos estos requisitos puede afectar a toda las características de diseño. Durante los últimos años la aplicación de CE en las misiones espaciales ha demostrado una gran ventaja para llegar al diseño óptimo, teniendo en cuenta tanto el rendimiento y el costo del proyecto. Un ejemplo bien conocido de la aplicación de CE es la CDF (Facilidad Diseño Concurrente) de la ESA (Agencia Espacial Europea). Está claro que para los proyectos de microsatélites universitarios tener o desarrollar una instalación de este tipo parece estar más allá de las capacidades del proyecto. Sin embargo, la práctica de la CE a cualquier escala puede ser beneficiosa para los microsatélites universitarios también. En la segunda parte de esta tesis, la atención se centra en el desarrollo de una estructura de optimización de diseño multidisciplinar (Multidisciplinary Design Optimization: MDO) aplicable a la fase de diseño conceptual de microsatélites de teledetección. Este enfoque permite que el equipo de diseño conozca la interacción entre las diferentes variables de diseño. El esquema MDO presentado no sólo incluye variables de nivel de sistema, tales como la masa total del satélite y la potencia total, sino también los requisitos de la misión como la resolución y tiempo de revisita. El proceso de diseño de microsatélites se divide en tres disciplinas; a) diseño de órbita, b) diseño de carga útil y c) diseño de plataforma. En primer lugar, se calculan diferentes parámetros de misión para un rango práctico de órbitas helio-síncronas (sun-synchronous orbits: SS-Os). Luego, según los parámetros orbitales y los datos de un instrumento como referencia, se calcula la masa y la potencia de la carga útil. El diseño de la plataforma del satélite se estima a partir de los datos de la masa y potencia de los diferentes subsistemas utilizando relaciones empíricas de diseño. El diseño del subsistema de potencia se realiza teniendo en cuenta variables de diseño más detalladas, como el escenario de la misión y diferentes tipos de células solares y baterías. El escenario se selecciona, de modo de obtener una banda de cobertura sobre la superficie terrestre paralelo al Ecuador después de cada intervalo de revisita. Con el objetivo de evaluar las interrelaciones entre las diferentes variables en el espacio de diseño, todas las disciplinas de diseño mencionados se combinan en un código unificado. Por último, una forma básica de MDO se ajusta a la herramienta de diseño de sistema de satélite. La optimización del diseño se realiza por medio de un GA con el único objetivo de minimizar la masa total de microsatélite. Según los resultados obtenidos de la aplicación del MDO, existen diferentes puntos de diseños óptimos, pero con diferentes variables de misión. Este análisis demuestra la aplicabilidad de MDO para los estudios de ingeniería de sistema en la fase de diseño conceptual en este tipo de proyectos. La principal conclusión de esta tesis, es que el diseño clásico de los satélites que por lo general comienza con la definición de la misión y la carga útil no es necesariamente la mejor metodología para todos los proyectos de satélites. Un microsatélite universitario, es un ejemplo de este tipo de proyectos. Por eso, se han desarrollado un conjunto de herramientas de diseño para encarar los estudios de la fase inicial de diseño. Este conjunto de herramientas incluye diferentes disciplinas de diseño centrados en el subsistema estructural y teniendo en cuenta una carga útil desconocida a priori. Los resultados demuestran que la mínima masa total del satélite y la máxima masa disponible para una carga útil desconocida a priori, son objetivos conflictivos. En este contexto para encontrar un Pareto-optimal se ha aplicado una optimización multiobjetivo. Según los resultados se concluye que la selección de la masa total por satélite en el rango de 40-60 kg puede considerarse como óptima para un proyecto de microsatélites universitario con carga útil desconocida a priori. También la metodología CE se ha aplicado al proceso de diseño conceptual de microsatélites de teledetección. Los resultados de la aplicación del CE proporcionan una clara comprensión de la interacción entre los requisitos de diseño de sistemas de satélites, tales como la masa total del microsatélite y la potencia y los requisitos de la misión como la resolución y el tiempo de revisita. La aplicación de MDO se hace con la minimización de la masa total de microsatélite. Los resultados de la aplicación de MDO aclaran la relación clara entre los diferentes requisitos de diseño del sistema y de misión, así como que permiten seleccionar las líneas de base para el diseño óptimo con el objetivo seleccionado en las primeras fase de diseño. ABSTRACT This thesis is done in the context of UPMSat-2 project, which is a microsatellite under design and manufacturing at the Instituto Universitario de Microgravedad “Ignacio Da Riva” (IDR/UPM) of the Universidad Politécnica de Madrid. Application of Concurrent Engineering (CE) methodology in the framework of Multidisciplinary Design application (MDO) is one of the main objectives of the present work. In recent years, there has been continuing interest in the participation of university research groups in space technology studies by means of their own microsatellites. The involvement in such projects has some inherent challenges, such as limited budget and facilities. Also, due to the fact that the main objective of these projects is for educational purposes, usually there are uncertainties regarding their in orbit mission and scientific payloads at the early phases of the project. On the other hand, there are predetermined limitations for their mass and volume budgets owing to the fact that most of them are launched as an auxiliary payload in which the launch cost is reduced considerably. The satellite structure subsystem is the one which is most affected by the launcher constraints. This can affect different aspects, including dimensions, strength and frequency requirements. In the first part of this thesis, the main focus is on developing a structural design sizing tool containing not only the primary structures properties as variables but also the satellite system level variables such as payload mass budget and satellite total mass and dimensions. This approach enables the design team to obtain better insight into the design in an extended design envelope. The structural design sizing tool is based on the analytical structural design formulas and appropriate assumptions including both static and dynamic models of the satellite. A Genetic Algorithm (GA) is applied to the design space for both single and multiobejective optimizations. The result of the multiobjective optimization is a Pareto-optimal based on two objectives, minimum satellite total mass and maximum payload mass budget. On the other hand, the application of the microsatellites is of interest for their less cost and response time. The high need for the remote sensing applications is a strong driver of their popularity in space missions. The satellite remote sensing missions are essential for long term research around the condition of the earth resources and environment. In remote sensing missions there are tight interrelations between different requirements such as orbital altitude, revisit time, mission cycle life and spatial resolution. Also, all of these requirements can affect the whole design characteristics. During the last years application of the CE in the space missions has demonstrated a great advantage to reach the optimum design base lines considering both the performance and the cost of the project. A well-known example of CE application is ESA (European Space Agency) CDF (Concurrent Design Facility). It is clear that for the university-class microsatellite projects having or developing such a facility seems beyond the project capabilities. Nevertheless practicing CE at any scale can be beneficiary for the university-class microsatellite projects. In the second part of this thesis, the main focus is on developing a MDO framework applicable to the conceptual design phase of the remote sensing microsatellites. This approach enables the design team to evaluate the interaction between the different system design variables. The presented MDO framework contains not only the system level variables such as the satellite total mass and total power, but also the mission requirements like the spatial resolution and the revisit time. The microsatellite sizing process is divided into the three major design disciplines; a) orbit design, b) payload sizing and c) bus sizing. First, different mission parameters for a practical range of sun-synchronous orbits (SS-Os) are calculated. Then, according to the orbital parameters and a reference remote sensing instrument, mass and power of the payload are calculated. Satellite bus sizing is done based on mass and power calculation of the different subsystems using design estimation relationships. In the satellite bus sizing, the power subsystem design is realized by considering more detailed design variables including a mission scenario and different types of solar cells and batteries. The mission scenario is selected in order to obtain a coverage belt on the earth surface parallel to the earth equatorial after each revisit time. In order to evaluate the interrelations between the different variables inside the design space all the mentioned design disciplines are combined in a unified code. The integrated satellite system sizing tool developed in this section is considered as an application of the CE to the conceptual design of the remote sensing microsatellite projects. Finally, in order to apply the MDO methodology to the design problem, a basic MDO framework is adjusted to the developed satellite system design tool. Design optimization is done by means of a GA single objective algorithm with the objective function as minimizing the microsatellite total mass. According to the results of MDO application, there exist different optimum design points all with the minimum satellite total mass but with different mission variables. This output demonstrates the successful applicability of MDO approach for system engineering trade-off studies at the conceptual design phase of the design in such projects. The main conclusion of this thesis is that the classical design approach for the satellite design which usually starts with the mission and payload definition is not necessarily the best approach for all of the satellite projects. The university-class microsatellite is an example for such projects. Due to this fact an integrated satellite sizing tool including different design disciplines focusing on the structural subsystem and considering unknown payload is developed. According to the results the satellite total mass and available mass for the unknown payload are conflictive objectives. In order to find the Pareto-optimal a multiobjective GA optimization is conducted. Based on the optimization results it is concluded that selecting the satellite total mass in the range of 40-60 kg can be considered as an optimum approach for a university-class microsatellite project with unknown payload(s). Also, the CE methodology is applied to the remote sensing microsatellites conceptual design process. The results of CE application provide a clear understanding of the interaction between satellite system design requirements such as satellite total mass and power and the satellite mission variables such as revisit time and spatial resolution. The MDO application is done with the total mass minimization of a remote sensing satellite. The results from the MDO application clarify the unclear relationship between different system and mission design variables as well as the optimum design base lines according to the selected objective during the initial design phases.

Characterization of the manufacturing processes to grow triple-junction solar cells

A number of important but little-investigated problems connected with III-V/Ge heterostructure in the GaInP/GaInAs/Ge multijunction solar cells grown by MOVPE are considered in the paper. The opportunity for successfully applying the combination of reflectance and reflectance anisotropy spectroscopy in situ methods for investigating III-V structure growth on a Ge substrate has been demonstrated. Photovoltaic properties of the III-V/Ge narrow-band subcell of the triple-junction solar cells have been investigated. It has been shown that there are excess currents in the Ge photovoltaic p-n junctions, and they have the tunneling or thermotunneling character. The values of the diode parameters for these current flow mechanisms have been determined. The potential barrier at the III-V/Ge interface was determined and the origin of this barrier formation during MOVPE heterogrowth was suggested.

Translocation and Functional Analysis of Pseudomonas savastanoi pv. savastanoi NCPPB 3335 Type III Secretion System Effectors Reveals Two Novel Effector Families of the Pseudomonas syringae Complex

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 causes olive knot disease and is a model pathogen for exploring bacterial infection of woody hosts. The type III secretion system (T3SS) effector repertoire of this strain includes 31 effector candidates plus two novel candidates identified in this study which have not been reported to translocate into plant cells. In this work, we demonstrate the delivery of seven NCPPB 3335 effectors into Nicotiana tabacum leaves, including three proteins from two novel families of the P. syringae complex effector super-repertoire (HopBK and HopBL), one of which comprises two proteins (HopBL1 and HopBL2) that harbor a SUMO protease domain. When delivered by P. fluorescens heterologously expressing a P. syringae T3SS, all seven effectors were found to suppress the production of defense-associated reactive oxygen species. Moreover, six of these effectors, including the truncated versions of HopAA1 and HopAZ1 encoded by NCPPB 3335, suppressed callose deposition. The expression of HopAZ1 and HopBL1 by functionally effectorless P. syringae pv. tomato DC3000D28E inhibited the hypersensitive response in tobacco and, additionally, expression of HopBL2 by this strain significantly increased its competitiveness in N. benthamiana. DNA sequences encoding HopBL1 and HopBL2 were uniquely detected in a collection of 31 P. savastanoi pv. savastanoi strains and other P. syringae strains isolated from woody hosts, suggesting a relevant role of these two effectors in bacterial interactions with olive and other woody plants.

A simple mathematical model that describes the growth of the area and the number of total and viable cells in yeast colonies

We propose a model, based on the Gompertz equation, to describe the growth of yeasts colonies on agar medium. This model presents several advantages: (i) one equation describes the colony growth, which previously needed two separate ones (linear increase of radius and of the squared radius); (ii) a similar equation can be applied to total and viable cells, colony area or colony radius, because the number of total cells in mature colonies is proportional to their area; and (iii) its parameters estimate the cell yield, the cell concentration that triggers growth limitation and the effect of this limitation on the specific growth rate. To elaborate the model, area, total and viable cells of 600 colonies of Saccharomyces cerevisiae, Debaryomyces fabryi, Zygosaccharomyces rouxii and Rhodotorula glutinis have been measured. With low inocula, viable cells showed an initial short exponential phase when colonies were not visible. This phase was shortened with higher inocula. In visible or mature colonies, cell growth displayed Gompertz-type kinetics. It was concluded that the cells growth in colonies is similar to liquid cultures only during the first hours, the rest of the time they grow, with near-zero specific growth rates, at least for 3 weeks.

Highly conductive p++-AlGaAs/n++-GaInP tunnel junctions for operation up to 15,000 suns in concentrator solar cells

In the last few decades there has been great interest in III-V multijunction solar cells (MJSC) for concentrator applications due to their promise to significantly reduce the cost of electricity. Being formed by series connection of several solar cells with different bandgaps, a key role in a MJSC structure is played by the tunnel junctions (TJ) aimed to implement such series connection. Essentially, tunnel junctions (tunnel diodes or Esaki diodes) are thin, heavily doped p-n junctions where quantum tunneling plays a key role as a conduction mechanism. Such devices were discovered by Nobel laureate Leo Esaki at the end of 1950. The key feature of tunnel junctions for their application in MJSC is that, as long as quantum tunneling is the dominant conduction mechanism, they exhibit a linear I-V dependence until the peak tunneling current (Jp) is reached. This initial ohmic region in the I-V curve is ideal for implementing low-loss interconnections between the subcells with different energy bandgaps that constitute a MJSC.

Impact of metal-organic vapor phase epitaxy environment on silicon bulk lifetime for III–V-on-Si multijunction solar cells

With the final goal of integrating III-V materials on silicon substrates for tandem solar cells, the influence of the Metal-Organic Vapor Phase Epitaxy (MOVPE) environment on the minority carrier properties of silicon wafers has been evaluated. These properties will essentially determine the photovoltaic performance of the bottom cell in a III-V-on-Si tandem solar cell. A comparison of the base minority carrier lifetimes obtained for different thermal processes carried out in a MOVPE reactor on Czochralski silicon wafers has been carried out. An important degradation of minority carrier lifetime during the surface preparation (i.e. H2 anneal) has been observed. Three different mechanisms have been proposed for explaining this behavior: 1) the introduction of extrinsic impurities coming from the reactor; 2) the activation of intrinsic lifetime killing impurities coming from the wafer itself; and finally, 3) the formation of crystal defects, which eventually become recombination centers. The effect of the emitter formation by phosphorus diffusion has also been evaluated. In this sense, it has been reported that lifetime can be recovered during the emitter formation either by the effect of the P on extracting impurities, or by the role of the atomic hydrogen on passivating the defects.

New strategies in laser processing of TCOs for light management improvement in thin-film silicon solar cells

Light confinement strategies play a crucial role in the performance of thin-film (TF) silicon solar cells. One way to reduce the optical losses is the texturing of the transparent conductive oxide (TCO) that acts as the front contact. Other losses arise from the mismatch between the incident light spectrum and the spectral properties of the absorbent material that imply that low energy photons (below the bandgap value) are not absorbed, and therefore can not generate photocurrent. Up-conversion techniques, in which two sub-bandgap photons are combined to give one photon with a better matching with the bandgap, were proposed to overcome this problem. In particular, this work studies two strategies to improve light management in thin film silicon solar cells using laser technology. The first one addresses the problem of TCO surface texturing using fully commercial fast and ultrafast solid state laser sources. Aluminum doped Zinc Oxide (AZO) samples were laser processed and the results were optically evaluated by measuring the haze factor of the treated samples. As a second strategy, laser annealing experiments of TCOs doped with rare earth ions are presented as a potential process to produce layers with up-conversion properties, opening the possibility of its potential use in high efficiency solar cells.

Design and demonstration of an electronically scanned reflectarray antenna at 100 GHz using multiresonant cells based on liquid crystals

The design, fabrication and measured results are presented for a reconfigurable reflectarray antenna based on liquid crystals (LC) which operates above 100 GHz. The antenna has been designed to provide beam scanning capabilities over a wide angular range, a large bandwidth and reduced Side-Lobe Level. Measured radiation patterns are in good agreement with simulations, and show that the antenna generates an electronically steerable beam in one plane over an angular range of 55º in the frequency band from 96 to 104 GHz. The Side Lobes Level is lower than -13 dB for all the scan angles and -18 dB is obtained over 16% of the scan range. The measured performance is significantly better than previously published results for this class of electronically tunable antenna, and moreover verifies the accuracy of the proposed procedure for LC modeling and antenna design.

Improved modeling of photoluminescent and electroluminescent coupling in multijunction solar cells

The performance of tandem stacks of Group III?V multijunction solar cells continues to improve rapidly, both through improved performance of the individual cells in the stack and throughi ncrease in the number of stacked cells. As the radiative efficiency of these individual cells increases, radiative coupling between the stacked cells becomes an increasingly important factor not only in cell design, but also in accurate efficiency measurement and in determining performance of cells and systems under varying spectral conditions in the field. Past modeling has concentrated on electroluminescent coupling between the cells, although photoluminescent coupling is shown to be important for cells operating near their maximum power point voltage or below or when junction defect recombination is significant. Extension of earlier models i sproposed to allow this non-negligible component of luminescent coupling to be included. Therefined model is validated by measurement of the closely related external emission from both single and double junction cells.

Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigens in vitro and experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B−/− or Cat D−/− antigen-presenting cells was unaffected. Degradation of Ii proceeded normally in Cat B−/− splenocytes, as it did in Cat D−/− cells. We conclude that neither Cat B nor Cat D are essential for MHC class II-mediated antigen presentation.

Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)–Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2q was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+ BMDMs for 5–8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.

Antisense oligonucleotides against α1E reduce R-type calcium currents in cerebellar granule cells

Many neurons of the central nervous system display multiple high voltage-activated Ca2+ currents, pharmacologically classified as L-, N-, P-, Q-, and R-type. Of these current types, the R-type is the least understood. The leading candidate for the molecular correlate of R-type currents in cerebellar granule cells is the α1E subunit, which yields Ca2+ currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against α1E could decrease the expression of R-type current in rat cerebellar granule neurons in culture. Cells were supplemented with either antisense or sense oligonucleotides and whole-cell patch clamp recordings were obtained after 6–8 days in vitro. Incubation with α1E antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from −10 ± 0.6 picoamperes/picofarad (pA/pF) (n = 6) in the untreated controls to −4.8 ± 0.8 pA/pF (n = 11) (P < 0.01). In contrast, no significant changes in the current expression were seen in sense oligonucleotide-treated cells (−11.3 ± 3.2 pA/pF). The specificity of the α1E antisense oligonucleotides was supported by the lack of change in estimates of the P/Q current amplitude. Furthermore, antisense and sense oligonucleotides against α1A did not affect R-type current expression (−11.5 ± 1.7 and −11.7 ± 1.7 pA/pF, respectively), whereas the α1A antisense oligonucleotide significantly reduced whole cell currents under conditions in which P/Q current is dominant. Our results support the hypothesis that members of the E class of α1 subunits support the high voltage-activated R-type current in cerebellar granule cells.

Diversification, expression, and γδ T cell recognition of evolutionarily distant members of the MIC family of major histocompatibility complex class I-related molecules

Distant relatives of major histocompatibility complex (MHC) class I molecules, human MICA and MICB, function as stress-induced antigens that are broadly recognized by intestinal epithelial γδ T cells. They may thus play a central role in the immune surveillance of damaged, infected, or otherwise stressed intestinal epithelial cells. However, the generality of this system in evolution and the mode of recognition of MICA and MICB are undefined. Analysis of cDNA sequences from various primate species defined translation products that are homologous to MICA and MICB. All of the MIC polypeptides have common characteristics, although they are extraordinarily diverse. The most notable alterations are several deletions and frequent amino acid substitutions in the putative α-helical regions of the α1α2 domains. However, the primate MIC molecules were expressed on the surfaces of normal and transfected cells. Moreover, despite their sharing of relatively few identical amino acids in potentially accessible regions of their α1α2 domains, they were recognized by diverse human intestinal epithelial γδ T cells that are restricted by MICA and MICB. Thus, MIC molecules represent a family of MHC proteins that are structurally diverse yet appear to be functionally conserved. The promiscuous mode of γδ T cell recognition of these antigens may be explained by their sharing of a single conserved interaction site.

Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: Implication for the in vivo delivery of CD8+ T cell epitopes into antigen-presenting cells

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8+ cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11–25 amino acids, corresponding to defined CD8+ T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (−1 or −2) decreased or completely blocked (charge of −4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.