A digital framework to build, visualize and analyze a gene expression atlas with cellular resolution in zebrafish early embryogenesis

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages.

An interactive web-based learning assistant for structural analysis of continuous beams

There are significant levels of concern about the relevance and the difficulty of learning some issues on Strength of Materials and Structural Analysis. Most students of Continuum Mechanics and Structural Analysis in Civil Engineering usually point out some key learning aspects as especially difficult for acquiring specific skills. These key concepts entail comprehension difficulties but ease access and applicability to structural analysis in more advanced subjects. Likewise, some elusive but basic structural concepts, such as flexibility, stiffness or influence lines, are paramount for developing further skills required for advanced structural design: tall buildings, arch-type structures as well as bridges. As new curricular itineraries are currently being implemented, it appears appropriate to devise a repository of interactive web-based applications for training in those basic concepts. That will hopefully train the student to understand the complexity of such concepts, to develop intuitive knowledge on actual structural response and to improve their preparation for exams. In this work, a web-based learning assistant system for influence lines on continuous beams is presented. It consists of a collection of interactive user-friendly applications accessible via Web. It is performed in both Spanish and English languages. Rather than a “black box” system, the procedure involves open interaction with the student, who can simulate and virtually envisage the structural response. Thus, the student is enabled to set the geometric, topologic and mechanic layout of a continuous beam and to change or shift the loading and the support conditions. Simultaneously, the changes in the beam response prompt on the screen, so that the effects of the several issues involved in structural analysis become apparent. The system is performed through a set of web pages which encompasses interactive exercises and problems, written in JavaScript under JQuery and DyGraphs frameworks, given that their efficiency and graphic capabilities are renowned. Students can freely boost their self-study on this subject in order to face their exams more confidently. Besides, this collection is expected to be added to the "Virtual Lab of Continuum Mechanics" of the UPM, launched in 2013 (http://serviciosgate.upm.es/laboratoriosvirtuales/laboratorios/medios-continuos-en-construcci%C3%B3n)

Study of the increase in bending stiffness on timber beams reinforced with composite materials

It is common to find structures that need to be reinforced due to deterioration or because the function of the building changes. The economic cost involved in these forms of interventions is considerable. Therefore, it is interesting to progress in the existing strengthening techniques and the study of new reinforcement systems. This paper analyses the behaviour of timber beams reinforced with carbon and basalt fiber composite materials. The main objective of this study is to test the stiffness increase produced by the carbon and basalt FRP on reinforced beams. The results show the stiffness increase produced by the different types of reinforcement.

Methodology based on FBG sensors for monitoring of FRP-strengthened beams

Advanced composite materials are increasingly used in the strengthening of reinforced concrete (RC) structures. The use of externally bonded strips made of fibre-reinforced plastics (FRP) as strengthening method has gained widespread acceptance in recent years since it has many advantages over the traditional techniques. However, unfortunately, this strengthening method is often associated with a brittle and sudden failure caused by some form of FRP bond failure, originated at the termination of the FRP material or at intermediate areas in the vicinity of flexural cracks in the RC beam. Up to date, little effort in the early prediction of the debonding in its initial instants even though this effect is not noticeable by simple visual observation. An early detection of this phenomenon might help in taking actions to prevent future catastrophes. Fibre-optic Bragg grating (FBG) sensors are able to measure strains locally with high resolution and accuracy. Furthermore, as their physical size is extremely small compared with other strain measuring components, it enables to be embedded at the concrete-FRP interface for determining the strain distribution without influencing the mechanical properties of the host materials. This paper shows the development of a debonding identification methodology based on strains experimentally measured. For, it a simplified model is implemented to simulate the behaviour of FRP-strengthened reinforced concrete beams. This model is taken as a basis to. develop an model updating procedure able to detect minor debonding at the concrete-FRP interface from experimental strains obtained by using FBG sensors embedded at the interface

A simplified spectral approachfor impedance-based damage identification of frp-strengthened rc beams

Hoy en día, el refuerzo y reparación de estructuras de hormigón armado mediante el pegado de bandas de polímeros reforzados con fibras (FRP) se emplea cada vez con más frecuencia a causa de sus numerosas ventajas. Sin embargo, las vigas reforzadas con esta técnica pueden experimentar un modo de fallo frágil a causa del despegue repentino de la banda de FRP a partir de una fisura intermedia. A pesar de su importancia, el número de trabajos que abordan el estudio de este mecanismo de fallo y su monitorización es muy limitado. Por ello, el desarrollo de metodologías capaces de monitorizar a largo plazo la adherencia de este refuerzo a las estructuras de hormigón e identificar cuándo se inicia el despegue de la banda constituyen un importante desafío a abordar. El principal objetivo de esta tesis es la implementación de una metodología fiable y efectiva, capaz de detectar el despegue de una banda de FRP en una viga de hormigón armado a partir de una fisura intermedia. Para alcanzar este objetivo se ha implementado un procedimiento de calibración numérica a partir de ensayos experimentales. Para ello, en primer lugar, se ha desarrollado un modelo numérico unidimensional simple y no costoso representativo del comportamiento de este tipo vigas de hormigón reforzadas con FRP, basado en un modelo de fisura discreta para el hormigón y el método de elementos espectrales. La formación progresiva de fisuras a flexion y el consiguiente despegue en la interface entre el hormigón y el FRP se formulan mediante la introducción de un nuevo elemento capaz de representar ambos fenómenos simultáneamente sin afectar al procedimiento numérico. Además, con el modelo propuesto, se puede obtener de una forma sencilla la respuesta dinámica en altas frecuencias de este tipo de estructuras, lo cual puede hacer muy útil su uso como herramienta de diagnosis y detección del despegue en su fase inicial mediante una monitorización de la variación de las características dinámicas locales de la estructura. Un método de evaluación no destructivo muy prometedor para la monitorización local de las estructuras es el método de la impedancia usando sensores-actuadores piezoeléctricos (PZT). La impedancia eléctrica de los sensores PZT se puede relacionar con la impedancia mecánica de las estructuras donde se encuentran adheridos Ya que la impedancia mecánica de una estructura se verá afectada por su deterioro, se pueden implementar indicadores de daño mediante una comparación del espectro de admitancia (inversa de la impedancia) a lo largo de distintas etapas durante el periodo de servicio de una estructura. Cualquier cambio en el espectro se podría interpretar como una variación en la integridad de la estructura. La impedancia eléctrica se mide a altas frecuencias con lo cual esta metodología debería ser muy sensible a la detección de estados de daño incipiente local, tal como se desea en la aplicación de este trabajo. Se ha implementado un elemento espectral PZT-FRP como extensión del modelo previamente desarrollado, con el objetivo de poder calcular numéricamente la impedancia eléctrica de sensores PZT adheridos a bandas de FRP sobre una viga de hormigón armado. El modelo, combinado con medidas experimentales captadas mediante sensores PZT, se implementa en el marco de una metodología de calibración de modelos para detectar cuantitativamente el despegue en la interfase entre una banda de FRP y una viga de hormigón. El procedimiento de optimización se resuelve empleando el método del enjambre cooperativo con un algoritmo bagging. Los resultados muestran una gran aproximación en la estimación del daño para el problema propuesto. Adicionalmente, se ha desarrollado también un método adaptativo para el mallado de elementos espectrales con el objetivo de localizar las zonas dañadas a partir de los resultados experimentales, el cual contribuye a aumentar la robustez y efectividad del método propuesto a la hora de identificar daños incipientes en su aparición inicial. Finalmente, se ha llevado a cabo un procedimiento de optimización multi-objetivo para detectar el despegue inicial en una viga de hormigón a escala real reforzada con FRP a partir de las impedancias captadas con una red de sensores PZT instrumentada a lo largo de la longitud de la viga. Cada sensor aporta los datos para definir cada una de las funciones objetivo que definen el procedimiento. Combinando el modelo previo de elementos espectrales con un algoritmo PSO multi-objetivo el procedimiento de detección de daño resultante proporciona resultados satisfactorios considerando la escala de la estructura y todas las incertidumbres características ligadas a este proceso. Los resultados obtenidos prueban la viabilidad y capacidad de los métodos antes mencionados y también su potencial en aplicaciones reales. Abstract Nowadays, the external bonding of fibre reinforced polymer (FRP) plates or sheets is increasingly used for the strengthening and retrofitting of reinforced concrete (RC) structures due to its numerous advantages. However, this kind of strengthening often leads to brittle failure modes being the most dominant failure mode the debonding induced by an intermediate crack (IC). In spite of its importance, the number of studies regarding the IC debonding mechanism and bond health monitoring is very limited. Methodologies able to monitor the long-term efficiency of bonding and successfully identify the initiation of FRP debonding constitute a challenge to be met. The main purpose of this thesisis the implementation of a reliable and effective methodology of damage identification able to detect intermediate crack debonding in FRP-strengthened RC beams. To achieve this goal, a model updating procedure based on numerical simulations and experimental tests has been implemented. For it, firstly, a simple and non-expensive one-dimensional model based on the discrete crack approach for concrete and the spectral element method has been developed. The progressive formation of flexural cracks and subsequent concrete-FRP interfacial debonding is formulated by the introduction of a new element able to represent both phenomena simultaneously without perturbing the numerical procedure. Furthermore, with the proposed model, high frequency dynamic response for these kinds of structures can also be obtained in a very simple and non-expensive way, which makes this procedure very useful as a tool for diagnoses and detection of debonding in its initial stage by monitoring the change in local dynamic characteristics. One very promising active non-destructive evaluation method for local monitoring is impedance-based structural health monitoring(SHM)using piezoelectric ceramic (PZT) sensor-actuators. The electrical impedance of the PZT can be directly related to the mechanical impedance of the host structural component where the PZT transducers are attached. Since the structural mechanical impedance will be affected by the presence of structural damage, comparisons of admittance (inverse of impedance) spectra at various times during the service period of the structure can be used as damage indicator. Any change in the spectra might be an indication of a change in the structural integrity. The electrical impedance is measured at high frequencies with which this methodology appears to be very sensitive to incipient damage in structural systems as desired for our application. Abonded-PZT-FRP spectral beam element approach based on an extension of the previous discrete crack approach is implemented in the calculation of the electrical impedance of the PZT transducer bonded to the FRP plates of a RC beam. This approach in conjunction with the experimental measurements of PZT actuator-sensors mounted on the structure is used to present an updating methodology to quantitatively detect interfacial debonding between a FRP strip and the host RC structure. The updating procedure is solved by using an ensemble particle swarm optimization approach with abagging algorithm, and the results demonstrate a big improvement for the performance and accuracy of the damage detection in the proposed problem. Additionally, an adaptive strategy of spectral element mesh has been also developed to detect damage location with experimental results, which shows the robustness and effectiveness of the proposed method to identify initial and incipient damages at its early stage. Lastly, multi-objective optimization has been carried out to detect debonding damage in a real scale FRP-strengthened RC beam by using impedance signatures. A net of PZT sensors is distributed along the beam to construct impedance-based multiple objectives under gradually induced damage scenario. By combining the spectral element model presented previously and an ensemble multi-objective PSO algorithm, the implemented damage detection process yields satisfactory predictions considering the scale and uncertainties of the structure. The obtained results prove the feasibility and capability of the aforementioned methods and also their potentials in real engineering applications.

Application of PZT and FBG impedance sensors for structural health monitoring of reinforced concrete beams strengthened with FRP composite materials

Esta Tesis tiene como objetivo principal el desarrollo de métodos de identificación del daño que sean robustos y fiables, enfocados a sistemas estructurales experimentales, fundamentalmente a las estructuras de hormigón armado reforzadas externamente con bandas fibras de polímeros reforzados (FRP). El modo de fallo de este tipo de sistema estructural es crítico, pues generalmente es debido a un despegue repentino y frágil de la banda del refuerzo FRP originado en grietas intermedias causadas por la flexión. La detección de este despegue en su fase inicial es fundamental para prevenir fallos futuros, que pueden ser catastróficos. Inicialmente, se lleva a cabo una revisión del método de la Impedancia Electro-Mecánica (EMI), de cara a exponer sus capacidades para la detección de daño. Una vez la tecnología apropiada es seleccionada, lo que incluye un analizador de impedancias así como novedosos sensores PZT para monitorización inteligente, se ha diseñado un procedimiento automático basado en los registros de impedancias de distintas estructuras de laboratorio. Basándonos en el hecho de que las mediciones de impedancias son posibles gracias a una colocación adecuada de una red de sensores PZT, la estimación de la presencia de daño se realiza analizando los resultados de distintos indicadores de daño obtenidos de la literatura. Para que este proceso sea automático y que no sean necesarios conocimientos previos sobre el método EMI para realizar un experimento, se ha diseñado e implementado un Interfaz Gráfico de Usuario, transformando la medición de impedancias en un proceso fácil e intuitivo. Se evalúa entonces el daño a través de los correspondientes índices de daño, intentando estimar no sólo su severidad, sino también su localización aproximada. El desarrollo de estos experimentos en cualquier estructura genera grandes cantidades de datos que han de ser procesados, y algunas veces los índices de daño no son suficientes para una evaluación completa de la integridad de una estructura. En la mayoría de los casos se pueden encontrar patrones de daño en los datos, pero no se tiene información a priori del estado de la estructura. En este punto, se ha hecho una importante investigación en técnicas de reconocimiento de patrones particularmente en aprendizaje no supervisado, encontrando aplicaciones interesantes en el campo de la medicina. De ahí surge una idea creativa e innovadora: detectar y seguir la evolución del daño en distintas estructuras como si se tratase de un cáncer propagándose por el cuerpo humano. En ese sentido, las lecturas de impedancias se emplean como información intrínseca de la salud de la propia estructura, de forma que se pueden aplicar las mismas técnicas que las empleadas en la investigación del cáncer. En este caso, se ha aplicado un algoritmo de clasificación jerárquica dado que ilustra además la clasificación de los datos de forma gráfica, incluyendo información cualitativa y cuantitativa sobre el daño. Se ha investigado la efectividad de este procedimiento a través de tres estructuras de laboratorio, como son una viga de aluminio, una unión atornillada de aluminio y un bloque de hormigón reforzado con FRP. La primera ayuda a mostrar la efectividad del método en sencillos escenarios de daño simple y múltiple, de forma que las conclusiones extraídas se aplican sobre los otros dos, diseñados para simular condiciones de despegue en distintas estructuras. Demostrada la efectividad del método de clasificación jerárquica de lecturas de impedancias, se aplica el procedimiento sobre las estructuras de hormigón armado reforzadas con bandas de FRP objeto de esta tesis, detectando y clasificando cada estado de daño. Finalmente, y como alternativa al anterior procedimiento, se propone un método para la monitorización continua de la interfase FRP-Hormigón, a través de una red de sensores FBG permanentemente instalados en dicha interfase. De esta forma, se obtienen medidas de deformación de la interfase en condiciones de carga continua, para ser implementadas en un modelo de optimización multiobjetivo, cuya solución se haya por medio de una expansión multiobjetivo del método Particle Swarm Optimization (PSO). La fiabilidad de este último método de detección se investiga a través de sendos ejemplos tanto numéricos como experimentales. ABSTRACT This thesis aims to develop robust and reliable damage identification methods focused on experimental structural systems, in particular Reinforced Concrete (RC) structures externally strengthened with Fiber Reinforced Polymers (FRP) strips. The failure mode of this type of structural system is critical, since it is usually due to sudden and brittle debonding of the FRP reinforcement originating from intermediate flexural cracks. Detection of the debonding in its initial stage is essential thus to prevent future failure, which might be catastrophic. Initially, a revision of the Electro-Mechanical Impedance (EMI) method is carried out, in order to expose its capabilities for local damage detection. Once the appropriate technology is selected, which includes impedance analyzer as well as novel PZT sensors for smart monitoring, an automated procedure has been design based on the impedance signatures of several lab-scale structures. On the basis that capturing impedance measurements is possible thanks to an adequately deployed PZT sensor network, the estimation of damage presence is done by analyzing the results of different damage indices obtained from the literature. In order to make this process automatic so that it is not necessary a priori knowledge of the EMI method to carry out an experimental test, a Graphical User Interface has been designed, turning the impedance measurements into an easy and intuitive procedure. Damage is then assessed through the analysis of the corresponding damage indices, trying to estimate not only the damage severity, but also its approximate location. The development of these tests on any kind of structure generates large amounts of data to be processed, and sometimes the information provided by damage indices is not enough to achieve a complete analysis of the structural health condition. In most of the cases, some damage patterns can be found in the data, but none a priori knowledge of the health condition is given for any structure. At this point, an important research on pattern recognition techniques has been carried out, particularly on unsupervised learning techniques, finding interesting applications in the medicine field. From this investigation, a creative and innovative idea arose: to detect and track the evolution of damage in different structures, as if it were a cancer propagating through a human body. In that sense, the impedance signatures are used to give intrinsic information of the health condition of the structure, so that the same clustering algorithms applied in the cancer research can be applied to the problem addressed in this dissertation. Hierarchical clustering is then applied since it also provides a graphical display of the clustered data, including quantitative and qualitative information about damage. The performance of this approach is firstly investigated using three lab-scale structures, such as a simple aluminium beam, a bolt-jointed aluminium beam and an FRP-strengthened concrete specimen. The first one shows the performance of the method on simple single and multiple damage scenarios, so that the first conclusions can be extracted and applied to the other two experimental tests, which are designed to simulate a debonding condition on different structures. Once the performance of the impedance-based hierarchical clustering method is proven to be successful, it is then applied to the structural system studied in this dissertation, the RC structures externally strengthened with FRP strips, where the debonding failure in the interface between the FRP and the concrete is successfully detected and classified, proving thus the feasibility of this method. Finally, as an alternative to the previous approach, a continuous monitoring procedure of the FRP-Concrete interface is proposed, based on an FBGsensors Network permanently deployed within that interface. In this way, strain measurements can be obtained under controlled loading conditions, and then they are used in order to implement a multi-objective model updating method solved by a multi-objective expansion of the Particle Swarm Optimization (PSO) method. The feasibility of this last proposal is investigated and successfully proven on both numerical and experimental RC beams strengthened with FRP.

A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage

Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe (Sp), SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. Here we report the identification of a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural, and functional similarity to SpCds1. HuCds1 was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after hydroxyurea treatment. Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins. These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals.

Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2α kinase homolog

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2α kinases. Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2α phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2α kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.

Molecular cloning and characterization of an invertebrate cellular retinoic acid binding protein

We have cloned a cDNA and gene from the tobacco hornworm, Manduca sexta, which is related to the vertebrate cellular retinoic acid binding proteins (CRABPs). CRABPs are members of the superfamily of lipid binding proteins (LBPs) and are thought to mediate the effects of retinoic acid (RA) on morphogenesis, differentiation, and homeostasis. This discovery of a Manduca sexta CRABP (msCRABP) demonstrates the presence of a CRABP in invertebrates. Compared with bovine/murine CRABP I, the deduced amino acid sequence of msCRABP is 71% homologous overall and 88% homologous for the ligand binding pocket. The genomic organization of msCRABP is conserved with other CRABP family members and the larger LBP superfamily. Importantly, the promoter region contains a motif that resembles an RA response element characteristic of the promoter region of most CRABPs analyzed. Three-dimensional molecular modeling based on postulated structural homology with bovine/murine CRABP I shows msCRABP has a ligand binding pocket that can accommodate RA. The existence of an invertebrate CRABP has significant evolutionary implications, suggesting CRABPs appeared during the evolution of the LBP superfamily well before vertebrate/invertebrate divergence, instead of much later in evolution in selected vertebrates.

β3 integrins mediate the cellular entry of hantaviruses that cause respiratory failure

Newly emerged hantaviruses replicate primarily in the pulmonary endothelium, cause acute platelet loss, and result in hantavirus pulmonary syndrome (HPS). We now report that specific integrins expressed on platelets and endothelial cells permit the cellular entry of HPS-associated hantaviruses. Infection with HPS-associated hantaviruses, NY-1 and Sin Nombre virus (SNV), is inhibited by antibodies to β3 integrins and by the β3-integrin ligand, vitronectin. In contrast, infection with the nonpathogenic (no associated human disease) Prospect Hill virus was inhibited by fibronectin and β1-specific antibodies but not by β3-specific antibodies or vitronectin. Transfection with recombinant αIIbβ3 or αvβ3 integrins rendered cells permissive to NY-1 and SNV but not Prospect Hill virus infection, indicating that αIIbβ3 and αvβ3 integrins mediate the entry of NY-1 and SNV hantaviruses. Furthermore, entry is divalent cation independent, not blocked by arginine-glycine-aspartic acid peptides and still mediated by, ligand-binding defective, αIIbβ3-integrin mutants. Hence, NY-1 and SNV entry is independent of β3 integrin binding to physiologic ligands. These findings implicate integrins as cellular receptors for hantaviruses and indicate that hantavirus pathogenicity correlates with integrin usage.

Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation

Studies on the transmission of human (Hu) prions to transgenic (Tg) mice suggested that another molecule provisionally designated protein X participates in the formation of nascent scrapie isoform of prion protein (PrPSc). We report the identification of the site at which protein X binds to the cellular isoform of PrP (PrPC) using scrapie-infected mouse (Mo) neuroblastoma cells transfected with chimeric Hu/MoPrP genes even though protein X has not yet been isolated. Substitution of a Hu residue at position 214 or 218 prevented PrPSc formation. The side chains of these residues protrude from the same surface of the C-terminal α-helix and form a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at positions 167, 171, or 218 also prevented PrPSc formation: at a mechanistic level, these mutant PrPs appear to act as “dominant negatives” by binding protein X and rendering it unavailable for prion propagation. Our findings seem to explain the protective effects of basic polymorphic residues in PrP of humans and sheep and suggest therapeutic and prophylactic approaches to prion diseases.

The small GTP-binding protein Rho links G protein-coupled receptors and Gα12 to the serum response element and to cellular transformation

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Gαs and Gαi2 families were found in human tumors; and members of the Gαq and Gα12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene. One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Gα12 family of heterotrimeric G protein α subunits strongly induced the SRE, while Gβ1γ2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Gα12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.

Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3′ end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.

Developmental disorder associated with increased cellular nucleotidase activity

Four unrelated patients are described with a syndrome that included developmental delay, seizures, ataxia, recurrent infections, severe language deficit, and an unusual behavioral phenotype characterized by hyperactivity, short attention span, and poor social interaction. These manifestations appeared within the first few years of life. Each patient displayed abnormalities on EEG. No unusual metabolites were found in plasma or urine, and metabolic testing was normal except for persistent hypouricosuria. Investigation of purine and pyrimidine metabolism in cultured fibroblasts derived from these patients showed normal incorporation of purine bases into nucleotides but decreased incorporation of uridine. De novo synthesis of purines and cellular phosphoribosyl pyrophosphate content also were moderately decreased. The distribution of incorporated purines and pyrimidines did not reveal a pattern suggestive of a deficient enzyme activity. Assay of individual enzymes in fibroblast lysates showed no deficiencies. However, the activity of cytosolic 5′-nucleotidase was elevated 6- to 10-fold. Based on the possibility that the observed increased catabolic activity and decreased pyrimidine salvage might be causing a deficiency of pyrimidine nucleotides, the patients were treated with oral pyrimidine nucleoside or nucleotide compounds. All patients showed remarkable improvement in speech and behavior as well as decreased seizure activity and frequency of infections. A double-blind placebo trial was undertaken to ascertain the efficacy of this supplementation regimen. Upon replacement of the supplements with placebo, all patients showed rapid regression to their pretreatment states. These observations suggest that increased nucleotide catabolism is related to the symptoms of these patients, and that the effects of this increased catabolism are reversed by administration of uridine.