High Efficiency Envelope Amplifier based on a Ripple Cancellation Buck Converter. Design, Optimization and Integration in an EER RFPA

Durante los últimos años la tendencia en el sector de las telecomunicaciones ha sido un aumento y diversificación en la transmisión de voz, video y fundamentalmente de datos. Para conseguir alcanzar las tasas de transmisión requeridas, los nuevos estándares de comunicaciones requieren un mayor ancho de banda y tienen un mayor factor de pico, lo cual influye en el bajo rendimiento del amplificador de radiofrecuencia (RFPA). Otro factor que ha influido en el bajo rendimiento es el diseño del amplificador de radiofrecuencia. Tradicionalmente se han utilizado amplificadores lineales por su buen funcionamiento. Sin embargo, debido al elevado factor de pico de las señales transmitidas, el rendimiento de este tipo de amplificadores es bajo. El bajo rendimiento del sistema conlleva desventajas adicionales como el aumento del coste y del tamaño del sistema de refrigeración, como en el caso de una estación base, o como la reducción del tiempo de uso y un mayor calentamiento del equipo para sistemas portátiles alimentados con baterías. Debido a estos factores, se han desarrollado durante las últimas décadas varias soluciones para aumentar el rendimiento del RFPA como la técnica de Outphasing, combinadores de potencia o la técnica de Doherty. Estas soluciones mejoran las prestaciones del RFPA y en algún caso han sido ampliamente utilizados comercialmente como la técnica de Doherty, que alcanza rendimientos hasta del 50% para el sistema completo para anchos de banda de hasta 20MHz. Pese a las mejoras obtenidas con estas soluciones, los mayores rendimientos del sistema se obtienen para soluciones basadas en la modulación de la tensión de alimentación del amplificador de potencia como “Envelope Tracking” o “EER”. La técnica de seguimiento de envolvente o “Envelope Tracking” está basada en la modulación de la tensión de alimentación de un amplificador lineal de potencia para obtener una mejora en el rendimiento en el sistema comparado a una solución con una tensión de alimentación constante. Para la implementación de esta técnica se necesita una etapa adicional, el amplificador de envolvente, que añade complejidad al amplificador de radiofrecuencia. En un amplificador diseñado con esta técnica, se aumentan las pérdidas debido a la etapa adicional que supone el amplificador de envolvente pero a su vez disminuyen las pérdidas en el amplificador de potencia. Si el diseño se optimiza adecuadamente, puede conseguirse un aumento global en el rendimiento del sistema superior al conseguido con las técnicas mencionadas anteriormente. Esta técnica presenta ventajas en el diseño del amplificador de envolvente, ya que el ancho de banda requerido puede ser menor que el ancho de banda de la señal de envolvente si se optimiza adecuadamente el diseño. Adicionalmente, debido a que la sincronización entre la señal de envolvente y de fase no tiene que ser perfecta, el proceso de integración conlleva ciertas ventajas respecto a otras técnicas como EER. La técnica de eliminación y restauración de envolvente, llamada EER o técnica de Kahn está basada en modulación simultánea de la envolvente y la fase de la señal usando un amplificador de potencia conmutado, no lineal y que permite obtener un elevado rendimiento. Esta solución fue propuesta en el año 1952, pero no ha sido implementada con éxito durante muchos años debido a los exigentes requerimientos en cuanto a la sincronización entre fase y envolvente, a las técnicas de control y de corrección de los errores y no linealidades de cada una de las etapas así como de los equipos para poder implementar estas técnicas, que tienen unos requerimientos exigentes en capacidad de cálculo y procesamiento. Dentro del diseño de un RFPA, el amplificador de envolvente tiene una gran importancia debido a su influencia en el rendimiento y ancho de banda del sistema completo. Adicionalmente, la linealidad y la calidad de la señal de transmitida deben ser elevados para poder cumplir con los diferentes estándares de telecomunicaciones. Esta tesis se centra en el amplificador de envolvente y el objetivo principal es el desarrollo de soluciones que permitan el aumento del rendimiento total del sistema a la vez que satisfagan los requerimientos de ancho de banda, calidad de la señal transmitida y de linealidad. Debido al elevado rendimiento que potencialmente puede alcanzarse con la técnica de EER, esta técnica ha sido objeto de análisis y en el estado del arte pueden encontrarse numerosas referencias que analizan el diseño y proponen diversas implementaciones. En una clasificación de alto nivel, podemos agrupar las soluciones propuestas del amplificador de envolvente según estén compuestas de una o múltiples etapas. Las soluciones para el amplificador de envolvente en una configuración multietapa se basan en la combinación de un convertidor conmutado, de elevado rendimiento con un regulador lineal, de alto ancho de banda, en una combinación serie o paralelo. Estas soluciones, debido a la combinación de las características de ambas etapas, proporcionan un buen compromiso entre rendimiento y buen funcionamiento del amplificador de RF. Por otro lado, la complejidad del sistema aumenta debido al mayor número de componentes y de señales de control necesarias y el aumento de rendimiento que se consigue con estas soluciones es limitado. Una configuración en una etapa tiene las ventajas de una mayor simplicidad, pero debido al elevado ancho de banda necesario, la frecuencia de conmutación debe aumentarse en gran medida. Esto implicará un bajo rendimiento y un peor funcionamiento del amplificador de envolvente. En el estado del arte pueden encontrarse diversas soluciones para un amplificador de envolvente en una etapa, como aumentar la frecuencia de conmutación y realizar la implementación en un circuito integrado, que tendrá mejor funcionamiento a altas frecuencias o utilizar técnicas topológicas y/o filtros de orden elevado, que permiten una reducción de la frecuencia de conmutación. En esta tesis se propone de manera original el uso de la técnica de cancelación de rizado, aplicado al convertidor reductor síncrono, para reducir la frecuencia de conmutación comparado con diseño equivalente del convertidor reductor convencional. Adicionalmente se han desarrollado dos variantes topológicas basadas en esta solución para aumentar la robustez y las prestaciones de la misma. Otro punto de interés en el diseño de un RFPA es la dificultad de poder estimar la influencia de los parámetros de diseño del amplificador de envolvente en el amplificador final integrado. En esta tesis se ha abordado este problema y se ha desarrollado una herramienta de diseño que permite obtener las principales figuras de mérito del amplificador integrado para la técnica de EER a partir del diseño del amplificador de envolvente. Mediante el uso de esta herramienta pueden validarse el efecto del ancho de banda, el rizado de tensión de salida o las no linealidades del diseño del amplificador de envolvente para varias modulaciones digitales. Las principales contribuciones originales de esta tesis son las siguientes: La aplicación de la técnica de cancelación de rizado a un convertidor reductor síncrono para un amplificador de envolvente de alto rendimiento para un RFPA linealizado mediante la técnica de EER. Una reducción del 66% en la frecuencia de conmutación, comparado con el reductor convencional equivalente. Esta reducción se ha validado experimentalmente obteniéndose una mejora en el rendimiento de entre el 12.4% y el 16% para las especificaciones de este trabajo. La topología y el diseño del convertidor reductor con dos redes de cancelación de rizado en cascada para mejorar el funcionamiento y robustez de la solución con una red de cancelación. La combinación de un convertidor redactor multifase con la técnica de cancelación de rizado para obtener una topología que proporciona una reducción del cociente entre frecuencia de conmutación y ancho de banda de la señal. El proceso de optimización del control del amplificador de envolvente en lazo cerrado para mejorar el funcionamiento respecto a la solución en lazo abierto del convertidor reductor con red de cancelación de rizado. Una herramienta de simulación para optimizar el proceso de diseño del amplificador de envolvente mediante la estimación de las figuras de mérito del RFPA, implementado mediante EER, basada en el diseño del amplificador de envolvente. La integración y caracterización del amplificador de envolvente basado en un convertidor reductor con red de cancelación de rizado en el transmisor de radiofrecuencia completo consiguiendo un elevado rendimiento, entre 57% y 70.6% para potencias de salida de 14.4W y 40.7W respectivamente. Esta tesis se divide en seis capítulos. El primer capítulo aborda la introducción enfocada en la aplicación, los amplificadores de potencia de radiofrecuencia, así como los principales problemas, retos y soluciones existentes. En el capítulo dos se desarrolla el estado del arte de amplificadores de potencia de RF, describiéndose las principales técnicas de diseño, las causas de no linealidad y las técnicas de optimización. El capítulo tres está centrado en las soluciones propuestas para el amplificador de envolvente. El modo de control se ha abordado en este capítulo y se ha presentado una optimización del diseño en lazo cerrado para el convertidor reductor convencional y para el convertidor reductor con red de cancelación de rizado. El capítulo cuatro se centra en el proceso de diseño del amplificador de envolvente. Se ha desarrollado una herramienta de diseño para evaluar la influencia del amplificador de envolvente en las figuras de mérito del RFPA. En el capítulo cinco se presenta el proceso de integración realizado y las pruebas realizadas para las diversas modulaciones, así como la completa caracterización y análisis del amplificador de RF. El capítulo seis describe las principales conclusiones de la tesis y las líneas futuras. ABSTRACT The trend in the telecommunications sector during the last years follow a high increase in the transmission rate of voice, video and mainly in data. To achieve the required levels of data rates, the new modulation standards demand higher bandwidths and have a higher peak to average power ratio (PAPR). These specifications have a direct impact in the low efficiency of the RFPA. An additional factor for the low efficiency of the RFPA is in the power amplifier design. Traditionally, linear classes have been used for the implementation of the power amplifier as they comply with the technical requirements. However, they have a low efficiency, especially in the operating range of signals with a high PAPR. The low efficiency of the transmitter has additional disadvantages as an increase in the cost and size as the cooling system needs to be increased for a base station and a temperature increase and a lower use time for portable devices. Several solutions have been proposed in the state of the art to improve the efficiency of the transmitter as Outphasing, power combiners or Doherty technique. However, the highest potential of efficiency improvement can be obtained using a modulated power supply for the power amplifier, as in the Envelope Tracking and EER techniques. The Envelope Tracking technique is based on the modulation of the power supply of a linear power amplifier to improve the overall efficiency compared to a fixed voltage supply. In the implementation of this technique an additional stage is needed, the envelope amplifier, that will increase the complexity of the RFPA. However, the efficiency of the linear power amplifier will increase and, if designed properly, the RFPA efficiency will be improved. The advantages of this technique are that the envelope amplifier design does not require such a high bandwidth as the envelope signal and that in the integration process a perfect synchronization between envelope and phase is not required. The Envelope Elimination and Restoration (EER) technique, known also as Kahn’s technique, is based on the simultaneous modulation of envelope and phase using a high efficiency switched power amplifier. This solution has the highest potential in terms of the efficiency improvement but also has the most challenging specifications. This solution, proposed in 1952, has not been successfully implemented until the last two decades due to the high demanding requirements for each of the stages as well as for the highly demanding processing and computation capabilities needed. At the system level, a very precise synchronization is required between the envelope and phase paths to avoid a linearity decrease of the system. Several techniques are used to compensate the non-linear effects in amplitude and phase and to improve the rejection of the out of band noise as predistortion, feedback and feed-forward. In order to obtain a high bandwidth and efficient RFPA using either ET or EER, the envelope amplifier stage will have a critical importance. The requirements for this stage are very demanding in terms of bandwidth, linearity and quality of the transmitted signal. Additionally the efficiency should be as high as possible, as the envelope amplifier has a direct impact in the efficiency of the overall system. This thesis is focused on the envelope amplifier stage and the main objective will be the development of high efficiency envelope amplifier solutions that comply with the requirements of the RFPA application. The design and optimization of an envelope amplifier for a RFPA application is a highly referenced research topic, and many solutions that address the envelope amplifier and the RFPA design and optimization can be found in the state of the art. From a high level classification, multiple and single stage envelope amplifiers can be identified. Envelope amplifiers for EER based on multiple stage architecture combine a linear assisted stage and a switched-mode stage, either in a series or parallel configuration, to achieve a very high performance RFPA. However, the complexity of the system increases and the efficiency improvement is limited. A single-stage envelope amplifier has the advantage of a lower complexity but in order to achieve the required bandwidth the switching frequency has to be highly increased, and therefore the performance and the efficiency are degraded. Several techniques are used to overcome this limitation, as the design of integrated circuits that are capable of switching at very high rates or the use of topological solutions, high order filters or a combination of both to reduce the switching frequency requirements. In this thesis it is originally proposed the use of the ripple cancellation technique, applied to a synchronous buck converter, to reduce the switching frequency requirements compared to a conventional buck converter for an envelope amplifier application. Three original proposals for the envelope amplifier stage, based on the ripple cancellation technique, are presented and one of the solutions has been experimentally validated and integrated in the complete amplifier, showing a high total efficiency increase compared to other solutions of the state of the art. Additionally, the proposed envelope amplifier has been integrated in the complete RFPA achieving a high total efficiency. The design process optimization has also been analyzed in this thesis. Due to the different figures of merit between the envelope amplifier and the complete RFPA it is very difficult to obtain an optimized design for the envelope amplifier. To reduce the design uncertainties, a design tool has been developed to provide an estimation of the RFPA figures of merit based on the design of the envelope amplifier. The main contributions of this thesis are: The application of the ripple cancellation technique to a synchronous buck converter for an envelope amplifier application to achieve a high efficiency and high bandwidth EER RFPA. A 66% reduction of the switching frequency, validated experimentally, compared to the equivalent conventional buck converter. This reduction has been reflected in an improvement in the efficiency between 12.4% and 16%, validated for the specifications of this work. The synchronous buck converter with two cascaded ripple cancellation networks (RCNs) topology and design to improve the robustness and the performance of the envelope amplifier. The combination of a phase-shifted multi-phase buck converter with the ripple cancellation technique to improve the envelope amplifier switching frequency to signal bandwidth ratio. The optimization of the control loop of an envelope amplifier to improve the performance of the open loop design for the conventional and ripple cancellation buck converter. A simulation tool to optimize the envelope amplifier design process. Using the envelope amplifier design as the input data, the main figures of merit of the complete RFPA for an EER application are obtained for several digital modulations. The successful integration of the envelope amplifier based on a RCN buck converter in the complete RFPA obtaining a high efficiency integrated amplifier. The efficiency obtained is between 57% and 70.6% for an output power of 14.4W and 40.7W respectively. The main figures of merit for the different modulations have been characterized and analyzed. This thesis is organized in six chapters. In Chapter 1 is provided an introduction of the RFPA application, where the main problems, challenges and solutions are described. In Chapter 2 the technical background for radiofrequency power amplifiers (RF) is presented. The main techniques to implement an RFPA are described and analyzed. The state of the art techniques to improve performance of the RFPA are identified as well as the main sources of no-linearities for the RFPA. Chapter 3 is focused on the envelope amplifier stage. The three different solutions proposed originally in this thesis for the envelope amplifier are presented and analyzed. The control stage design is analyzed and an optimization is proposed both for the conventional and the RCN buck converter. Chapter 4 is focused in the design and optimization process of the envelope amplifier and a design tool to evaluate the envelope amplifier design impact in the RFPA is presented. Chapter 5 shows the integration process of the complete amplifier. Chapter 6 addresses the main conclusions of the thesis and the future work.

Design and integration of lower ports for ITER diagnostic systems

All around the ITER vacuum vessel, forty-four ports will provide access to the vacuum vessel for remotehandling operations, diagnostic systems, heating, and vacuum systems: 18 upper ports, 17 equatorialports, and 9 lower ports. Among the lower ports, three of them will be used for the remote handlinginstallation of the ITER divertor. Once the divertor is in place, these ports will host various diagnosticsystems mounted in the so-called diagnostic racks. The diagnostic racks must allow the support andcooling of the diagnostics, extraction of the required diagnostic signals, and providing access and main-tainability while minimizing the leakage of radiation toward the back of the port where the humans areallowed to enter. A fully integrated inner rack, carrying the near plasma diagnostic components, will bean stainless steel structure, 4.2 m long, with a maximum weight of 10 t. This structure brings water forcooling and baking at maximum temperature of 240?C and provides connection with gas, vacuum andelectric services. Additional racks (placed away from plasma and not requiring cooling) may be requiredfor the support of some particular diagnostic components. The diagnostics racks and its associated exvessel structures, which are in its conceptual design phase, are being designed to survive the lifetimeof ITER of 20 years. This paper presents the current state of development including interfaces, diagnos-tic integration, operation and maintenance, shielding requirements, remote handling, loads cases anddiscussion of the main challenges coming from the severe environment and engineering requirements.

Overexpression of the Matrix Metalloproteinase Matrilysin Results in Premature Mammary Gland Differentiation and Male Infertility

To examine the role of matrilysin (MAT), an epithelial cell-specific matrix metalloproteinase, in the normal development and function of reproductive tissues, we generated transgenic animals that overexpress MAT in several reproductive organs. Three distinct forms of human MAT (wild-type, active, and inactive) were placed under the control of the murine mammary tumor virus promoter/enhancer. Although wild-type, active, and inactive forms of the human MAT protein could be produced in an in vitro culture system, mutations of the MAT cDNA significantly decreased the efficiency with which the MAT protein was produced in vivo. Therefore, animals carrying the wild-type MAT transgene that expressed high levels of human MAT in vivo were further examined. Mammary glands from female transgenic animals were morphologically normal throughout mammary development, but displayed an increased ability to produce β-casein protein in virgin animals. In addition, beginning at approximately 8 mo of age, the testes of male transgenic animals became disorganized with apparent disintegration of interstitial tissue that normally surrounds the seminiferous tubules. The disruption of testis morphology was concurrent with the onset of infertility. These results suggest that overexpression of the matrix-degrading enzyme MAT alters the integrity of the extracellular matrix and thereby induces cellular differentiation and cellular destruction in a tissue-specific manner.

Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells

Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin. mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba/F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively. In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.

Biodiversity of Costa Rican salamanders: Implications of high levels of genetic differentiation and phylogeographic structure for species formation

Although salamanders are characteristic amphibians in Holarctic temperate habitats, in tropical regions they have diversified evolutionarily only in tropical America. An adaptive radiation centered in Middle America occurred late in the history of a single clade, the supergenus Bolitoglossa (Plethodontidae), and large numbers of species now occur in diverse habitats. Sublineages within this clade decrease in number from the northern to southern parts of Middle America, and in Costa Rica, there are but three. Despite this phylogenetic constraint, Costa Rica has many species; the number of salamander species on one local elevational transect in the Cordillera de Talamanca may be the largest for any such transect in the world. Extraordinary variation in sequences of the mitochondrial gene cytochrome b within a clade of the genus Bolitoglossa in Costa Rica reveals strong phylogeographic structure within a single species, Bolitoglossa pesrubra. Allozymic variation in 19 proteins reveals a pattern largely concordant with the mitochondrial DNA phylogeography. More species exist than are currently recognized. Diversification occurs in restricted geographic areas and involves sharp geographic and elevational differentiation and zonation. In their degree of genetic differentiation at a local scale, these species of the deep tropics exceed the known variation of extratropical salamanders, which also differ in being less restricted in elevational range. Salamanders display “tropicality” in that although speciose, they are usually local in distribution and rare. They display strong ecological and physiological differentiation that may contribute importantly to morphological divergence and species formation.

Interaction of the E1A Oncoprotein with Yak1p, a Novel Regulator of Yeast Pseudohyphal Differentiation, and Related Mammalian Kinases

The C-terminal portion of adenovirus E1A suppresses ras-induced metastasis and tumorigenicity in mammalian cells; however, little is known about the mechanisms by which this occurs. In the simple eukaryote Saccharomyces cerevisiae, Ras2p, the homolog of mammalian h-ras, regulates mitogen-activated protein kinase (MAPK) and cyclic AMP-dependent protein kinase A (cAMP/PKA) signaling pathways to control differentiation from the yeast form to the pseudohyphal form. When expressed in yeast, the C-terminal region of E1A induced pseudohyphal differentiation, and this was independent of both the MAPK and cAMP/PKA signaling pathways. Using the yeast two-hybrid system, we identified an interaction between the C-terminal region of E1A and Yak1p, a yeast dual-specificity serine/threonine protein kinase that functions as a negative regulator of growth. E1A also physically interacts with Dyrk1A and Dyrk1B, two mammalian homologs of Yak1p, and stimulates their kinase activity in vitro. We further demonstrate that Yak1p is required in yeast to mediate pseudohyphal differentiation induced by Ras2p-regulated signaling pathways. However, pseudohyphal differentiation induced by the C-terminal region of E1A is largely independent of Yak1p. These data suggest that mammalian Yak1p-related kinases may be targeted by the E1A oncogene to modulate cell growth.

Auditory neuroscience: Development, transduction, and integration

Hearing underlies our ability to locate sound sources in the environment, our appreciation of music, and our ability to communicate. Participants in the National Academy of Sciences colloquium on Auditory Neuroscience: Development, Transduction, and Integration presented research results bearing on four key issues in auditory research. How does the complex inner ear develop? How does the cochlea transduce sounds into electrical signals? How does the brain's ability to compute the location of a sound source develop? How does the forebrain analyze complex sounds, particularly species-specific communications? This article provides an introduction to the papers stemming from the meeting.

Saline Stress Alters the Temporal Patterns of Xylem Differentiation and Alternative Oxidase Expression in Developing Soybean Roots1

We conducted a coordinated biochemical and morphometric analysis of the effect of saline conditions on the differentiation zone of developing soybean (Glycine max L.) roots. Between d 3 and d 14 for seedlings grown in control or NaCl-supplemented medium, we studied (a) the temporal evolution of the respiratory alternative oxidase (AOX) capacity in correlation with the expression and localization of AOX protein analyzed by tissue-print immunoblotting; (b) the temporal evolution and tissue localization of a peroxidase activity involved in lignification; and (c) the structural changes, visualized by light microscopy and quantified by image digitization. The results revealed that saline stress retards primary xylem differentiation. There is a corresponding delay in the temporal pattern of AOX expression, which is consistent with the xylem-specific localization of AOX protein and the idea that this enzyme is linked to xylem development. An NaCl-induced acceleration of the development of secondary xylem was also observed. However, the temporal pattern of a peroxidase activity localized in the primary and secondary xylem was unaltered by NaCl treatment. Thus, the NaCl-stressed root was specifically affected in the temporal patterns of AOX expression and xylem development.

C/EBPɛ mediates myeloid differentiation and is regulated by the CCAAT displacement protein (CDP/cut)

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBPɛ) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C/EBPɛ activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. We have previously demonstrated that overexpression of CCAAT displacement protein (CDP/cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C/EBPɛ−/− mice and in SGD patients. Based on these observations we investigated potential interactions between C/EBPɛ and CDP/cut during neutrophil maturation. In this study, we demonstrate that inducible expression of C/EBPɛ in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells revealed absence of C/EBPɛ mRNA. We therefore hypothesize that C/EBPɛ positively regulates SGP gene expression, and that C/EBPɛ is itself negatively regulated by CDP/cut during neutrophil maturation. We further demonstrate that the C/EBPɛ promoter is regulated by CDP/cut during myeloid differentiation.

Magnetic resonance imaging (MRI) detection of the murine brain response to light: temporal differentiation and negative functional MRI changes.

Using a 9.4 T MRI instrument, we have obtained images of the mouse brain response to photic stimulation during a period between deep anesthesia and the early stages of arousal. The large image enhancements we observe (often >30%) are consistent with literature results extrapolated to 9.4 T. However, there are also two unusual aspects to our findings. (i) The visual area of the brain responds only to changes in stimulus intensity, suggesting that we directly detect operations of the M visual system pathway. Such a channel has been observed in mice by invasive electrophysiology, and described in detail for primates. (ii) Along with the typical positive response in the area of the occipital portion of the brain containing the visual cortex, another area displays decreased signal intensity upon stimulation.

Activated Ras signals differentiation and expansion of CD4+8+ thymocytes.

We describe a novel approach to assay the ability of particular gene products to signal transitions in lymphocyte differentiation in vivo. The method involves transfection of test expression constructs into RAG-1-deficient embryonic stem cells, which are subsequently assayed by the RAG-2-deficient blastocyst complementation approach. We have used this method to demonstrate that expression of activated Ras in CD4-8- (double negative, DN) prothymocytes in vivo induces their differentiation into small CD4+8+ (double positive, DP) cortical thymocytes with accompanying expansion to normal thymocyte numbers. However, activated Ras expression in DP cells does not cause proliferation or maturation to CD4+8- or CD4-8+ (single positive) thymocytes. Therefore, signaling through Ras is sufficient for promoting differentiation of DN to DP cells, but further differentiation requires the activity of additional signaling pathways.

STAT3 activation is a critical step in gp130-mediated terminal differentiation and growth arrest of a myeloid cell line.

Myeloid leukemia M1 cells can be induced for growth arrest and terminal differentiation into macrophages in response to interleukin 6 (IL-6) or leukemia inhibitory factor (LIF). Recently, a large number of cytokines and growth factors have been shown to activate the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In the case of IL-6 and LIF, which share a signal transducing receptor gp130, STAT3 is specifically tyrosine-phosphorylated and activated by stimulation with each cytokine in various cell types. To know the role of JAK-STAT pathway in M1 differentiation, we have constructed dominant negative forms of STAT3 and established M1 cell lines that constitutively express them. These M1 cells that overexpressed dominant negative forms showed no induction of differentiation-associated markers including Fc gamma receptors, ferritin light chain, and lysozyme after treatment with IL-6. Expression of either c-myb or c-myc was not downregulated. Furthermore, IL-6- and LIF-mediated growth arrest and apoptosis were completely blocked. Thus these findings demonstrate that STAT3 activation is the critical step in a cascade of events that leads to terminal differentiation of M1 cells.

Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo.

A genetic approach has been established that combines the advantages of blastocyst complementation with the experimental attributes of the developing lens for the functional analysis of genes governing cellular proliferation, terminal differentiation, and apoptosis. This lens complementation system (LCS) makes use of a mutant mouse strain, aphakia (ak), homozygotes of which fail to develop an ocular lens. We demonstrate that microinjection of wild-type embryonic stem (ES) cells into ak/ak blastocysts produces chimeras with normal ES-cell-derived lenses and that microinjection of Rb-/- ES cells generates an aberrant lens phenotype identical to that obtained through conventional gene targeting methodology. Our determination that a cell autonomous defect underlies the aphakia condition assures that lenses generated through LCS are necessarily ES-cell-derived. LCS provides for the rapid phenotypic analysis of loss-of-function mutations, circumvents the need for germ-line transmission of null alleles, and, most significantly, facilitates the study of essential genes whose inactivation is associated with early lethal phenotypes.

Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts.

The mechanisms by which insulin-like growth factors (IGFs) can be both mitogenic and differentiation-promoting in skeletal myoblasts are unclear because these two processes are believed to be mutually exclusive in this tissue. The phosphorylation state of the ubiquitous nuclear retinoblastoma protein (Rb) plays an important role in determining whether myoblasts proliferate or differentiate: Phosphorylated Rb promotes mitogenesis, whereas un- (or hypo-) phosphorylated Rb promotes cell cycle exit and differentiation. We hypothesized that IGFs might affect the fate of myoblasts by regulating the phosphorylation of Rb. Although long-term IGF treatment is known to stimulate differentiation, we find that IGFs act initially to inhibit differentiation and are exclusively mitogenic. These early effects of IGFs are associated with maintenance of Rb phosphorylation typical of proliferating cells; upregulation of the gene expression of cyclin-dependent kinase 4 and cyclin D1, components of a holoenzyme that plays a principal role in mediating Rb phosphorylation; and marked inhibition of the gene expression of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that is essential in muscle differentiation. We also find that IGF-induced inhibition of differentiation occurs through a process that is independent of its mitogenic effects. We demonstrate, thus, that IGFs regulate Rb phosphorylation and cyclin D1 and cyclin-dependent kinase 4 gene expression; together with their biphasic effects on myogenin expression, these results suggest a mechanism by which IGFs are initially mitogenic and subsequently differentiation-promoting in skeletal muscle.

Overexpression of DR-nm23, a protein encoded by a member of the nm23 gene family, inhibits granulocyte differentiation and induces apoptosis in 32Dc13 myeloid cells.

Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.