Contribución al diseño de lazos de realimentación electrónica para microsistemas electromecánicos (MEMS) resonantes: ruido de fase generado en lazos osciladores por sus realimentaciones

En 1966, D. B. Leeson publicó el artículo titulado “A simple model of feedback oscillator noise spectrum” en el que, mediante una ecuación obtenida de forma heurística y basada en parámetros conocidos de los osciladores, proponía un modelo para estimar el espectro de potencia que cuantifica el Ruido de Fase de estos osciladores. Este Ruido de Fase pone de manifiesto las fluctuaciones aleatorias que se producen en la fase de la señal de salida de cualquier oscilador de frecuencia f_0. Desde entonces, los adelantos tecnológicos han permitido grandes progresos en cuanto a la medida del Ruido de Fase, llegando a encontrar una estrecha “zona plana”, alrededor de f_0, conocida con el nombre de Ensanchamiento de Línea (EL) que Leeson no llegó a observar y que su modelo empírico no recogía. Paralelamente han ido surgiendo teorías que han tratado de explicar el Ruido de Fase con mayor o menor éxito. En esta Tesis se propone una nueva teoría para explicar el espectro de potencia del Ruido de Fase de un oscilador realimentado y basado en resonador L-C (Inductancia-Capacidad). Al igual que otras teorías, la nuestra también relaciona el Ruido de Fase del oscilador con el ruido térmico del circuito que lo implementa pero, a diferencia de aquellas, nuestra teoría se basa en un Modelo Complejo de ruido eléctrico que considera tanto las Fluctuaciones de energía eléctrica asociadas a la susceptancia capacitiva del resonador como las Disipaciones de energía eléctrica asociadas a su inevitable conductancia G=1⁄R, que dan cuenta del contacto térmico entre el resonador y el entorno térmico que le rodea. En concreto, la nueva teoría que proponemos explica tanto la parte del espectro del Ruido de Fase centrada alrededor de la frecuencia portadora f_0 que hemos llamado EL y su posterior caída proporcional a 〖∆f〗^(-2) al alejarnos de f_0, como la zona plana o pedestal que aparece en el espectro de Ruido de Fase lejos de esa f_0. Además, al saber cuantificar el EL y su origen, podemos explicar con facilidad la aparición de zonas del espectro de Ruido de Fase con caída 〖∆f〗^(-3) cercanas a la portadora y que provienen del denominado “exceso de ruido 1⁄f” de dispositivos de Estado Sólido y del ruido “flicker” de espectro 1⁄f^β (0,8≤β≤1,2) que aparece en dispositivos de vacío como las válvulas termoiónicas. Habiendo mostrado que una parte del Ruido de Fase de osciladores L-C realimentados que hemos denominado Ruido de Fase Térmico, se debe al ruido eléctrico de origen térmico de la electrónica que forma ese oscilador, proponemos en esta Tesis una nueva fuente de Ruido de Fase que hemos llamado Ruido de Fase Técnico, que se añadirá al Térmico y que aparecerá cuando el desfase del lazo a la frecuencia de resonancia f_0 del resonador no sea 0° o múltiplo entero de 360° (Condición Barkhausen de Fase, CBF). En estos casos, la modulación aleatoria de ganancia de lazo que realiza el Control Automático de Amplitud en su lucha contra ruidos que traten de variar la amplitud de la señal oscilante del lazo, producirá a su vez una modulación aleatoria de la frecuencia de tal señal que se observará como más Ruido de Fase añadido al Térmico. Para dar una prueba empírica sobre la existencia de esta nueva fuente de Ruido de Fase, se diseñó y construyó un oscilador en torno a un resonador mecánico “grande” para tener un Ruido de Fase Térmico despreciable a efectos prácticos. En este oscilador se midió su Ruido de Fase Técnico tanto en función del valor del desfase añadido al lazo de realimentación para apartarlo de su CBF, como en función de la perturbación de amplitud inyectada para mostrar sin ambigüedad la aparición de este Ruido de Fase Técnico cuando el lazo tiene este fallo técnico: que no cumple la Condición Barkhausen de Fase a la frecuencia de resonancia f_0 del resonador, por lo que oscila a otra frecuencia. ABSTRACT In 1966, D. B. Leeson published the article titled “A simple model of feedback oscillator noise spectrum” in which, by means of an equation obtained heuristically and based on known parameters of the oscillators, a model was proposed to estimate the power spectrum that quantifies the Phase Noise of these oscillators. This Phase Noise reveals the random fluctuations that are produced in the phase of the output signal from any oscillator of frequencyf_0. Since then, technological advances have allowed significant progress regarding the measurement of Phase Noise. This way, the narrow flat region that has been found around f_(0 ), is known as Line Widening (LW). This region that Leeson could not detect at that time does not appear in his empirical model. After Leeson’s work, different theories have appeared trying to explain the Phase Noise of oscillators. This Thesis proposes a new theory that explains the Phase Noise power spectrum of a feedback oscillator around a resonator L-C (Inductance-Capacity). Like other theories, ours also relates the oscillator Phase Noise to the thermal noise of the feedback circuitry, but departing from them, our theory uses a new, Complex Model for electrical noise that considers both Fluctuations of electrical energy associated with the capacitive susceptance of the resonator and Dissipations of electrical energy associated with its unavoidable conductance G=1/R, which accounts for the thermal contact between the resonator and its surrounding environment (thermal bath). More specifically, the new theory we propose explains both the Phase Noise region of the spectrum centered at the carrier frequency f_0 that we have called LW and shows a region falling as 〖∆f〗^(-2) as we depart from f_0, and the flat zone or pedestal that appears in the Phase Noise spectrum far from f_0. Being able to quantify the LW and its origin, we can easily explain the appearance of Phase Noise spectrum zones with 〖∆f〗^(-3) slope near the carrier that come from the so called “1/f excess noise” in Solid-State devices and “flicker noise” with 1⁄f^β (0,8≤β≤1,2) spectrum that appears in vacuum devices such as thermoionic valves. Having shown that the part of the Phase Noise of L-C oscillators that we have called Thermal Phase Noise is due to the electrical noise of the electronics used in the oscillator, this Thesis can propose a new source of Phase Noise that we have called Technical Phase Noise, which will appear when the loop phase shift to the resonance frequency f_0 is not 0° or an integer multiple of 360° (Barkhausen Phase Condition, BPC). This Phase Noise that will add to the Thermal one, comes from the random modulation of the loop gain carried out by the Amplitude Automatic Control fighting against noises trying to change the amplitude of the oscillating signal in the loop. In this case, the BPC failure gives rise to a random modulation of the frequency of the output signal that will be observed as more Phase Noise added to the Thermal one. To give an empirical proof on the existence of this new source of Phase Noise, an oscillator was designed and constructed around a “big” mechanical resonator whose Thermal Phase Noise is negligible for practical effects. The Technical Phase Noise of this oscillator has been measured with regard to the phase lag added to the feedback loop to separate it from its BPC, and with regard to the amplitude disturbance injected to show without ambiguity the appearance of this Technical Phase Noise that appears when the loop has this technical failure: that it does not fulfill the Barkhausen Phase Condition at f_0, the resonance frequency of the resonator and therefore it is oscillating at a frequency other than f_0.

Engineering Computational Emotion - A Reference Model for Emotion in Artificial Systems

Emotion is generally argued to be an influence on the behavior of life systems, largely concerning flexibility and adaptivity. The way in which life systems acts in response to a particular situations of the environment, has revealed the decisive and crucial importance of this feature in the success of behaviors. And this source of inspiration has influenced the way of thinking artificial systems. During the last decades, artificial systems have undergone such an evolution that each day more are integrated in our daily life. They have become greater in complexity, and the subsequent effects are related to an increased demand of systems that ensure resilience, robustness, availability, security or safety among others. All of them questions that raise quite a fundamental challenges in control design. This thesis has been developed under the framework of the Autonomous System project, a.k.a the ASys-Project. Short-term objectives of immediate application are focused on to design improved systems, and the approaching of intelligence in control strategies. Besides this, long-term objectives underlying ASys-Project concentrate on high order capabilities such as cognition, awareness and autonomy. This thesis is placed within the general fields of Engineery and Emotion science, and provides a theoretical foundation for engineering and designing computational emotion for artificial systems. The starting question that has grounded this thesis aims the problem of emotion--based autonomy. And how to feedback systems with valuable meaning has conformed the general objective. Both the starting question and the general objective, have underlaid the study of emotion, the influence on systems behavior, the key foundations that justify this feature in life systems, how emotion is integrated within the normal operation, and how this entire problem of emotion can be explained in artificial systems. By assuming essential differences concerning structure, purpose and operation between life and artificial systems, the essential motivation has been the exploration of what emotion solves in nature to afterwards analyze analogies for man--made systems. This work provides a reference model in which a collection of entities, relationships, models, functions and informational artifacts, are all interacting to provide the system with non-explicit knowledge under the form of emotion-like relevances. This solution aims to provide a reference model under which to design solutions for emotional operation, but related to the real needs of artificial systems. The proposal consists of a multi-purpose architecture that implement two broad modules in order to attend: (a) the range of processes related to the environment affectation, and (b) the range or processes related to the emotion perception-like and the higher levels of reasoning. This has required an intense and critical analysis beyond the state of the art around the most relevant theories of emotion and technical systems, in order to obtain the required support for those foundations that sustain each model. The problem has been interpreted and is described on the basis of AGSys, an agent assumed with the minimum rationality as to provide the capability to perform emotional assessment. AGSys is a conceptualization of a Model-based Cognitive agent that embodies an inner agent ESys, the responsible of performing the emotional operation inside of AGSys. The solution consists of multiple computational modules working federated, and aimed at conforming a mutual feedback loop between AGSys and ESys. Throughout this solution, the environment and the effects that might influence over the system are described as different problems. While AGSys operates as a common system within the external environment, ESys is designed to operate within a conceptualized inner environment. And this inner environment is built on the basis of those relevances that might occur inside of AGSys in the interaction with the external environment. This allows for a high-quality separate reasoning concerning mission goals defined in AGSys, and emotional goals defined in ESys. This way, it is provided a possible path for high-level reasoning under the influence of goals congruence. High-level reasoning model uses knowledge about emotional goals stability, letting this way new directions in which mission goals might be assessed under the situational state of this stability. This high-level reasoning is grounded by the work of MEP, a model of emotion perception that is thought as an analogy of a well-known theory in emotion science. The work of this model is described under the operation of a recursive-like process labeled as R-Loop, together with a system of emotional goals that are assumed as individual agents. This way, AGSys integrates knowledge that concerns the relation between a perceived object, and the effect which this perception induces on the situational state of the emotional goals. This knowledge enables a high-order system of information that provides the sustain for a high-level reasoning. The extent to which this reasoning might be approached is just delineated and assumed as future work. This thesis has been studied beyond a long range of fields of knowledge. This knowledge can be structured into two main objectives: (a) the fields of psychology, cognitive science, neurology and biological sciences in order to obtain understanding concerning the problem of the emotional phenomena, and (b) a large amount of computer science branches such as Autonomic Computing (AC), Self-adaptive software, Self-X systems, Model Integrated Computing (MIC) or the paradigm of models@runtime among others, in order to obtain knowledge about tools for designing each part of the solution. The final approach has been mainly performed on the basis of the entire acquired knowledge, and described under the fields of Artificial Intelligence, Model-Based Systems (MBS), and additional mathematical formalizations to provide punctual understanding in those cases that it has been required. This approach describes a reference model to feedback systems with valuable meaning, allowing for reasoning with regard to (a) the relationship between the environment and the relevance of the effects on the system, and (b) dynamical evaluations concerning the inner situational state of the system as a result of those effects. And this reasoning provides a framework of distinguishable states of AGSys derived from its own circumstances, that can be assumed as artificial emotion.

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

Physiological response to long-term peripheral and central leptin infusion in lean and obese mice

Recent data have identified leptin as an afferent signal in a negative-feedback loop regulating the mass of the adipose tissue. High leptin levels are observed in obese humans and rodents, suggesting that, in some cases, obesity is the result of leptin insensitivity. This hypothesis was tested by comparing the response to peripherally and centrally administered leptin among lean and three obese strains of mice: diet-induced obese AKR/J, New Zealand Obese (NZO), and Ay. Subcutaneous leptin infusion to lean mice resulted in a dose-dependent loss of body weight at physiologic plasma levels. Chronic infusions of leptin intracerebroventricularly (i.c.v.) at doses of 3 ng/hr or greater resulted in complete depletion of visible adipose tissue, which was maintained throughout 30 days of continuous i.c.v. infusion. Direct measurement of energy balance indicated that leptin treatment did not increase total energy expenditure but prevented the decrease that follows reduced food intake. Diet-induced obese mice lost weight in response to peripheral leptin but were less sensitive than lean mice. NZO mice were unresponsive to peripheral leptin but were responsive to i.c.v. leptin. Ay mice did not respond to subcutaneous leptin and were 1/100 as sensitive to i.c.v. leptin. The decreased response to leptin in diet-induced obese, NZO, and Ay mice suggests that obesity in these strains is the result of leptin resistance. In NZO mice, leptin resistance may be the result of decreased transport of leptin into the cerebrospinal fluid, whereas in Ay mice, leptin resistance probably results from defects downstream of the leptin receptor in the hypothalamus.

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1−/− mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1−/− mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage

The p53 tumor-suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. This process is associated with posttranslational modifications of p53, some of which are mediated by the ATM protein kinase. However, these modifications alone may not account in full for p53 stabilization. p53's stability and activity are negatively regulated by the oncoprotein MDM2, whose gene is activated by p53. Conceivably, p53 function may be modulated by modifications of MDM2 as well. We show here that after treatment of cells with ionizing radiation or a radiomimetic chemical, but not UV radiation, MDM2 is phosphorylated rapidly in an ATM-dependent manner. This phosphorylation is independent of p53 and the DNA-dependent protein kinase. Furthermore, MDM2 is directly phosphorylated by ATM in vitro. These findings suggest that in response to DNA strand breaks, ATM may promote p53 activity and stability by mediating simultaneous phosphorylation of both partners of the p53-MDM2 autoregulatory feedback loop.

Rapid down-regulation of mammalian Period genes during behavioral resetting of the circadian clock

The pervasive role of circadian clocks in regulating physiology and behavior is widely recognized. Their adaptive value is their ability to be entrained by environmental cues such that the internal circadian phase is a reliable predictor of solar time. In mammals, both light and nonphotic behavioral cues can entrain the principal oscillator of the hypothalamic suprachiasmatic nuclei (SCN). However, although light can advance or delay the clock during circadian night, behavioral events trigger phase advances during the subjective day, when the clock is insensitive to light. The recent identification of Period (Per) genes in mammals, homologues of dperiod, which encodes a core element of the circadian clockwork in Drosophila, now provides the opportunity to explain circadian timing and entrainment at a molecular level. In mice, expression of mPer1 and mPer2 in the SCN is rhythmic and acutely up-regulated by light. Moreover, the temporal relations between mRNA and protein cycles are consistent with a clock based on a transcriptional/translational feedback loop. Here we describe circadian oscillations of Per1 and Per2 in the SCN of the Syrian hamster, showing that PER1 protein and mRNA cycles again behave in a manner consistent with a negative-feedback oscillator. Furthermore, we demonstrate that nonphotic resetting has the opposite effect to light: acutely down-regulating these genes. Their sensitivity to nonphotic resetting cues supports their proposed role as core elements of the circadian oscillator. Moreover, this study provides an explanation at the molecular level for the contrasting but convergent effects of photic and nonphotic cues on the clock.

Parasite-mediated nuclear factor κB regulation in lymphoproliferation caused by Theileria parva infection

Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor κB (NFκB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NFκB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IκB molecules which normally sequester NFκB in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IκBα. However, IκBα reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NFκB-mediated positive feedback loop which restores cytoplasmic IκBα. In contrast, T. parva mediated continuous degradation of IκBβ resulting in persistently low cytoplasmic IκBβ levels. Normal IκBβ levels were only restored following T. parva killing, indicating that viable parasites are required for IκBβ degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IκB degradation and consequent enhanced expression of NFκB dependent genes. However treatment using the antioxidant N-acetylcysteine had no effect on either IκB levels or NFκB activation, indicating that the parasite subverts the normal IκB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function.

Cyclin E, a redundant cyclin in breast cancer

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.

The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation

The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.

The Arabidopsis thaliana RPM1 disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response

Disease resistance in plants is often controlled by a gene-for-gene mechanism in which avirulence (avr) gene products encoded by pathogens are specifically recognized, either directly or indirectly, by plant disease resistance (R) gene products. Members of the NBS-LRR class of R genes encode proteins containing a putative nucleotide binding site (NBS) and carboxyl-terminal leucine-rich repeats (LRRs). Generally, NBS-LRR proteins do not contain predicted transmembrane segments or signal peptides, suggesting they are soluble cytoplasmic proteins. RPM1 is an NBS-LRR protein from Arabidopsis thaliana that confers resistance to Pseudomonas syringae expressing either avrRpm1 or avrB. RPM1 protein was localized by using an epitope tag. In contrast to previous suggestions, RPM1 is a peripheral membrane protein that likely resides on the cytoplasmic face of the plasma membrane. Furthermore, RPM1 is degraded coincident with the onset of the hypersensitive response, suggesting a negative feedback loop controlling the extent of cell death and overall resistance response at the site of infection.

The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development

During retinogenesis, the Xenopus basic helix–loop–helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, we analyzed the relationship between Ath5 and Brn3 genes by using a gain-of-function approach in the chicken embryo. We found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, we found that forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. We further found that Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells.

Stathmin/Op18 Phosphorylation Is Regulated by Microtubule Assembly

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of “mitotic chromatin.” Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.

The Patient Informatics Consult Service (PICS): an approach for a patient-centered service

The Patient Informatics Consult Service (PICS) at the Eskind Biomedical Library at Vanderbilt University Medical Center (VUMC) provides patients with consumer-friendly information by using an information prescription mechanism. Clinicians refer patients to the PICS by completing the prescription and noting the patient's condition and any relevant factors. In response, PICS librarians critically appraise and summarize consumer-friendly materials into a targeted information report. Copies of the report are given to both patient and clinician, thus facilitating doctor-patient communication and closing the clinician-librarian feedback loop. Moreover, the prescription form also circumvents many of the usual barriers for patients in locating information, namely, patients' unfamiliarity with medical terminology and lack of knowledge of authoritative sources. PICS librarians capture the time and expertise put into these reports by creating Web-based pathfinders on prescription topics. Pathfinders contain librarian-created disease overviews and links to authoritative resources and seek to minimize the consumer's exposure to unreliable information. Pathfinders also adhere to strict guidelines that act as a model for locating, appraising, and summarizing information for consumers. These mechanisms—the information prescription, research reports, and pathfinders—serve as steps toward the long-term goal of full integration of consumer health information into patient care at VUMC.

Directed evolution of polymerase function by compartmentalized self-replication

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.