Comparative study between powers of sigmoid functions, MLP-backpropagation and polynomials in function approximation problems

Function approximation is a very important task in environments where the computation has to be based on extracting information from data samples in real world processes. So, the development of new mathematical model is a very important activity to guarantee the evolution of the function approximation area. In this sense, we will present the Polynomials Powers of Sigmoid (PPS) as a linear neural network. In this paper, we will introduce one series of practical results for the Polynomials Powers of Sigmoid, where we will show some advantages of the use of the powers of sigmiod functions in relationship the traditional MLP-Backpropagation and Polynomials in functions approximation problems.

Development and evaluation of a new instrument to measure visual exploration behavior

Effective visual exploration is required for many activities of daily living and instruments to assess visual exploration are important for the evaluation of the visual and the oculomotor system. In this article, the development of a new instrument to measure central and peripheral target recognition is described. The measurement setup consists of a hemispherical projection which allows presenting images over a large area of ±90° horizontal and vertical angle. In a feasibility study with 14 younger (21–49 years) and 12 older (50–78 years) test persons, 132 targets and 24 distractors were presented within naturalistic color photographs of everyday scenes at 10°, 30°, and 50° eccentricity. After the experiment, both younger and older participants reported in a questionnaire that the task is easy to understand, fun and that it measures a competence that is relevant for activities of daily living. A main result of the pilot study was that younger participants recognized more targets with smaller reaction times than older participants. The group differences were most pronounced for peripheral target detection. This test is feasible and appropriate to assess the functional field of view in younger and older adults.

Dissection of antitumor activity by lymphokine -activated killer cells: Role of FcR+ precursors and obligatory role of tumor necrosis factor-$\alpha$ in antibody -dependent cellular cytotoxicity

Human peripheral blood lymphocytes (PBL) cultured for varying lengths of time in IL-2 are able to mediate antibody independent cellular cytotoxicity (AICC) as well as antibody dependent cellular cytotoxicity (ADCC) against a wide range of tumor targets. The objective of our study is to determine the cytotoxic potential of the subset of LAK cells involved in ADCC, the tumor recognition mechanism in ADCC, the kinetics of ADCC mediated by PBL cultured under various conditions and the role of TNF-$\alpha$ in the development and maturation of ADCC effectors in the LAK population.^ The model system in this study for ADCC used a monoclonal antibody 14G2a (IgG2a), that recognizes the GD2 epitope on human melanoma cell line, SK-Mel-1. The target recognition mechanism operative in AICC (traditionally known as lymphokine activated killing or LAK) is an acquired property of these IL-2 activated cells which confers on them the unique ability to distinguish between tumor and normal cells. This recognition probably involves the presence of a trypsin sensitive N-linked glycoprotein epitope on tumor cells. Proteolytic treatment of the tumor cells with trypsin renders them resistant to AICC by PBL cultured in IL-2. However, ADCC is unaffected. This ADCC, mediated by the relatively small population of cells that are positive for the Fc receptor for IgG (FcR), is an indication that this subset of "LAK" cells does not require the trypsin sensitive epitope on tumor cells to mediate killing. Enriching PBL for FcR+ cells markedly enhanced both AICC and ADCC and also reduced the IL-2 requirement of these cells.^ The stoichiometry of Fc receptor (FcR) expression on the cytotoxic effectors does not correlate with ADCC lytic activity. Although FcRs are necessary to mediate ADCC, other factors, appear to regulate the magnitude of cytolytic activity. In order to investigate these putative factors, the kinetics of ADCC development was studied under various conditions (in IL-2 (10u/ml) and 100u/ml), in IL-2(10u/ml) + TNF$\alpha$ (500u/ml) and in TNF-$\alpha$ (500u/ml) alone). Addition of exogenous TNF-$\alpha$ into the four hour cytotoxicity assay did not increase ADCC, nor did anti-TNF antibodies result in inhibition. On the other hand, addition of anti-TNF antibodies to PBL and IL-2 for 24 hours, resulted in a marked inhibition of the ADCC, suggesting that endogenous TNF-$\alpha$ is obligatory for the maturation and differentiation of ADCC effectors. ^

Evidence that lymphokine -activated killer (LAK) cell function is regulated by both serine and tyrosine kinase pathways

Signal transduction pathways operative in lymphokine activated killer (LAK) cells during execution of cytolytic function have never been characterized. Based on ubiquitous involvement of protein phosphorylation in activation of cytolytic mechanisms used by CTL and NK cells, it was hypothesized that changes in protein phosphorylation should occur when LAK encounter tumor targets. It was further hypothesized that protein kinases would regulate LAK-mediated cytotoxicity. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets consistently led to increased phosphorylation of two 65-kD LAK proteins pp65a and -b, with isoelectric points (pI) of 5.1 and 5.2 respectively. Increased p65 phosphorylation was initiated between 1 and 5 min after tumor coincubation, occurred on Ser residues, required physical contact between LAK and tumors, correlated with target recognition, and also occurred after crosslinking Fc$\gamma$RIIIA in the absence of tumors. Both pp65a and -b were tentatively identified as phosphorylated forms of the actin-bundling protein L-plastin, based on pI, molecular weight, and cross-reactivity with specific antiserum. The known biochemical properties of L-plastin suggest it may be involved in regulating adhesion of LAK to tumor targets. The protein tyrosine kinase-specific inhibitor Herb A did not block p65 phosphorylation, but blocked LAK killing of multiple tumor targets at a post-binding stage. Greater than 50% inhibition of cytotoxicity was observed after a 2.5-h pretreatment with 0.125 $\mu$g/ml Herb A. Inhibition occurred over a period in pretreatment which LAK were not dependent upon IL-2 for maintenance of killing activity, supporting the conclusion that the drug interfered with mobilization of cytotoxic function. Granule exocytosis measured by BLT-esterase release from LAK occurred after coincubation with tumors, and was inhibited by Herb A LAK cytotoxicity was dependent upon extracellular calcium, suggesting that granule exocytosis rather than Fas ligand was the principal pathway leading to target cell death. The data indicate that protein tyrosine kinases play a pivotal role in LAK cytolytic function by regulating granule exocytosis, and that tumor targets can activate an adhesion dependent Ser kinase pathway in LAK resulting in phosphorylation of L-plastin. ^

Modelado y Mitigación de Fenómenos de Multitrayecto en Imágenes Radar mediante Técnicas basadas en Inversión Temporal

Las técnicas SAR (Synthetic Aperture Radar, radar de apertura sintética) e ISAR (Inverse SAR, SAR inverso) son sistemas radar coherentes de alta resolución, capaces de proporcionar un mapa de la sección radar del blanco en el dominio espacial de distancia y acimut. El objetivo de ambas técnicas radica en conseguir una resolución acimutal más fina generando una apertura sintética a partir del movimiento relativo entre radar y blanco. Los radares imagen complementan la labor de los sistemas ópticos e infrarrojos convencionales, especialmente en condiciones meteorológicas adversas. Los sistemas SAR e ISAR convencionales se diseñan para iluminar blancos en situaciones de línea de vista entre sensor y blanco. Por este motivo, presentan un menor rendimiento en escenarios complejos, como por ejemplo en bosques o entornos urbanos, donde los retornos multitrayecto se superponen a los ecos directos procedentes de los blancos. Se conocen como "imágenes fantasma", puesto que enmascaran a los verdaderos blancos y dan lugar a una calidad visual pobre, complicando en gran medida la detección del blanco. El problema de la mitigación del multitrayecto en imágenes radar adquiere una relevancia teórica y práctica. En esta Tesis Doctoral, se hace uso del concepto de inversión temporal (Time Reversal, TR) para mejorar la calidad visual de las imágenes SAR e ISAR eliminando las "imágenes fantasma" originadas por la propagación multitrayecto (algoritmos TR-SAR y TR-ISAR, respectivamente). No obstante, previamente a la aplicación de estas innovadoras técnicas de mitigación del multi-trayecto, es necesario resolver el problema geométrico asociado al multitrayecto. Centrando la atención en la mejora de las prestaciones de TR-ISAR, se implementan una serie de técnicas de procesado de señal avanzadas antes y después de la etapa basada en inversión temporal (el eje central de esta Tesis). Las primeras (técnicas de pre-procesado) están relacionadas con el multilook averaging, las transformadas tiempo-frecuencia y la transformada de Radon, mientras que las segundas (técnicas de post-procesado) se componen de un conjunto de algoritmos de superresolución. En pocas palabras, todas ellas pueden verse como un valor añadido al concepto de TR, en lugar de ser consideradas como técnicas independientes. En resumen, la utilización del algoritmo diseñado basado en inversión temporal, junto con algunas de las técnicas de procesado de señal propuestas, no deben obviarse si se desean obtener imágenes ISAR de gran calidad en escenarios con mucho multitrayecto. De hecho, las imágenes resultantes pueden ser útiles para posteriores esquemas de reconocimiento automático de blancos (Automatic Target Recognition, ATR). Como prueba de concepto, se hace uso tanto de datos simulados como experimentales obtenidos a partir de radares de alta resolución con el fin de verificar los métodos propuestos.

Desarrollo y aplicaciones de radares de alta resolución en bandas milimétricas

Las bandas de las denominadas ondas milimétricas y submilimétricas están situadas en la región del espectro entre las microondas y el infrarrojo. La banda de milimétricas se sitúa entre 30 y 300 GHz, considerada normalmente como la banda EHF (Extremely High Frequency). El margen de frecuencias entre 300 y 3000 GHz es conocido como la banda de ondas submilimétricas o de terahercios (THz). Sin embargo, no toda la comunidad científica está de acuerdo acerca de las frecuencias que limitan la banda de THz. De hecho, 100 GHz y 10 THz son considerados comúnmente como los límites inferior y superior de dicha banda, respectivamente. Hasta hace relativamente pocos años, la banda de THz sólo había sido explotada para aplicaciones en los campos de la espectroscopía y la radioastronomía. Los avances tecnológicos en la electrónica de microondas y la óptica lastraron el desarrollo de la banda de THz. Sin embargo, investigaciones recientes han demostrado las ventajas asociadas a operar en estas longitudes de onda, lo que ha aumentado el interés y los esfuerzos dedicados a la tecnología de THz. A pesar de que han surgido un gran número de aplicaciones, una de las más prometedoras está en el campo de la vigilancia y la seguridad. Esta tesis está dedicada al desarrollo de radares de onda continua y frecuencia modulada (CW-LFM) de alta resolución en la banda de milimétricas, más concretamente, en las ventanas de atenuación situadas en 100 y 300 GHz. Trabajar en estas bandas de frecuencia presenta beneficios tales como la capacidad de las ondas de atravesar ciertos materiales como la ropa o el papel, opacos en el rango visible, y la posibilidad de usar grandes anchos de banda, obteniéndose así elevadas resoluciones en distancia. Los anchos de banda de 9 y 27 GHz seleccionados para los sistemas de 100 y 300 GHz, respectivamente, proporcionan resoluciones en distancia alrededor y por debajo del cm. Por otro lado, las aplicaciones objetivo se centran en la adquisición de imágenes a corto alcance. En el caso del prototipo a 300 GHz, su diseño se ha orientado a aplicaciones de detección a distancia en escenarios de vigilancia y seguridad. La naturaleza no ionizante de esta radiación supone una ventaja frente a las alternativas tradicionalmente usadas tales como los sistemas de rayos X. La presente tesis se centra en el proceso de diseño, implementación y caracterización de ambos sistemas así como de la validación de su funcionamiento. Se ha elegido una solución basada en componentes electrónicos, y no ópticos, debido a su alta fiabilidad, volumen reducido y amplia disponibilidad de componentes comerciales. Durante el proceso de diseño e implementación, se han tenido en cuenta varias directrices tales como la minimización del coste y la versatilidad de los sistemas desarrollados para hacer posible su aplicación para múltiples propósitos. Ambos sistemas se han utilizado en diferentes pruebas experimentales, obteniendo resultados satisfactorios. Aunque son sólo ejemplos dentro del amplio rango de posibles aplicaciones, la adquisición de imágenes ISAR de modelos de blancos a escala para detección automática así como la obtención de datos micro-Range/micro- Doppler para el análisis de patrones humanos han validado el funcionamiento del sistema a 100 GHz. Por otro lado, varios ejemplos de imágenes 3D obtenidas a 300 GHz han demostrado las capacidades del sistema para su uso en tareas de seguridad y detección a distancia. ABSTRACT The millimeter- and submillimeter-wave bands are the regions of the spectrum between the microwaves and the infrared (IR). The millimeter-wave band covers the range of the spectrum from 30 to 300 GHz, which is usually considered as the extremely high frequency (EHF) band. The range of frequencies between 300 and 3000 GHz is known as the submillimeter-wave or terahertz (THz) band. Nevertheless, the boundaries of the THz band are not accepted by the whole research community. In fact, 100 GHz and 10 THz are often considered by some authors as the lower and upper limit of this band, respectively. Until recently, the THz band had not been exploited for practical applications, with the exception of minor uses in the fields of spectroscopy and radio astronomy. The advancements on microwave electronics and optical technology left the well-known THz gap undeveloped. However, recent research has unveiled the advantages of working at these frequencies, which has motivated the increase in research effort devoted to THz technology. Even though the range of upcoming applications is wide, the most promising ones are in the field of security and surveillance. Particularly, this Ph.D. thesis deals with the development of high resolution continuouswave linear-frequency modulated (CW-LFM) radars in the millimeter-wave band, namely, in the attenuation windows located at 100 and 300 GHz. Working at these wavelengths presents several benefits such as the ability of radiation to penetrate certain materials, visibly opaque, and the great availability of bandwidth at these frequencies, which leads to high range resolution. The selected bandwidths of 9 and 27 GHz for these systems at 100 and 300 GHz, respectively, result in cm and sub-cm range resolution. On the other hand, the intended applications are in the field of short-range imaging. In particular, the design of the 300-GHz prototype is oriented to standoff detection for security and surveillance scenarios. The non-ionizing nature of this radiation allows safety concerns to be alleviated, in clear contrast to other traditional alternatives such as X-rays systems. This thesis is focused on the design, implementation and characterization process of both systems as well as the experimental assessment of their performances. An electronic approach has been selected instead of an optical solution so as to take advantage of its high reliability, reduced volume and the availability of commercial components. Through the whole design and implementation process, several guidelines such as low cost and hardware versatility have been also kept in mind. Taking advantage of that versatility, different applications can be carried out with the same hardware concept. Both radar systems have been used in several experimental trials with satisfactory results. Despite being mere examples within the wide range of fields of application, ISAR imaging of scaled model targets for automatic target recognition and micro-Range/micro-Doppler analysis of human patterns have validated the system performance at 100 GHz. In addition, 3D imaging examples at 300 GHz demonstrate the radar system’s capabilities for standoff detection and security tasks.

Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA- or protein-binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.

Identification of eIF2Bγ and eIF2γ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

The 5′-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bγ) and 2 (eIF2γ), respectively. The involvement of eIF2Bγ and eIF2γ in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.

Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA

DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m5C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m5C MTases, including the consensus S-adenosyl-l-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-l-homocysteine (AdoHcy) has been determined at 1.8 Å resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HhaI, a confirmed bacterial m5C MTase, and the smaller target recognition domains of DNMT2 and M.HhaI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HhaI. DNMT2 binds AdoHcy in the same conformation as confirmed m5C MTases and, while DNMT2 shares all sequence and structural features with m5C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.

Redirecting the specificity of ubiquitination by modifying ubiquitin-conjugating enzymes.

Depletion of specific cellular proteins is a powerful tool in biological research and has many medical and agricultural benefits. In contrast to genetic methods currently available to attenuate protein levels, we describe an alternative approach that redirects the ubiquitin-dependent proteolytic pathway to facilitate specific proteolytic removal. Degradation via the ubiquitin pathway requires the prior attachment of multiple ubiquitins to the target protein. This attachment is accomplished, in part, by a family of enzymes designated E2s (or ubiquitin-conjugating enzymes), some of which use domains near their C termini for target recognition. Here, we demonstrate that E2 target recognition can be redefined by engineering E2s to contain appropriate protein-binding peptides fused to their C termini. In five dissimilar examples, chimeric E2s were created that recognized and ubiquitinated their respective binding partners with high specificity. We also show that ubiquitination of one protein targeted by this method led to its ATP-dependent degradation in vitro. Thus, by exploiting interacting domains derived from natural and synthetic ligands, it may be possible to design E2s capable of directing the selective removal of many intracellular proteins.

Cellular and molecular consequences of S100A4-induced motility in rat breast tumour Rama 37 cells

Since the first discovery of S100 members in 1965, their expressions have been affiliated with numerous biological functions in all cells of the body. However, in the recent years, S100A4, a member of this superfamily has emerged as the central target in generating new avenue for cancer therapy as its overexpression has been correlated with cancer patients’ mortality as well as established roles as motility and metastasis promoter. As it has no catalytic activity, S100A4 has to interact with its target proteins to regulate such effects. Up to date, more than 10 S100A4 target proteins have been identified but the mechanical process regulated by S100A4 to induce motility remains vague. In this work, we demonstrated that S100A4 overexpression resulted in actin filaments disorganisation, reduction in focal adhesions, instability of filopodia as well as exhibiting polarised morphology. However, such effects were not observed in truncated versions of S100A4 possibly highlighting the importance of C terminus of S100A4 target recognition. In order to assess some of the intracellular mechanisms that may be involved in promoting migrations, different strategies were used, including active pharmaceutical agents, inhibitors and knockdown experiments. Treatment of S100A4 overexpressing cells with blebbistatin and Y-27632, non muscle myosin IIA (NMMIIA) inhibitors, as well as knockdown of NMMIIA, resulted in motility enhancement and focal adhesions reduction proposing that NMMIIA assisted S100A4 in regulating cell motility but its presence is not essential. Further work done using Cos 7 cell lines, naturally lacking NMMIIA, further demonstrated that S100A4 is capable of regulating cell motility independent of NMMIIA, possibly through poor maturation of focal adhesion. Given that all these experiments highlighted the independency of NMMIIA towards migration, a protein that has been put at the forefront of S100A4-induced motility, we aimed to gather further understanding regarding the other molecular mechanisms that may be at play for motility. Using high throughput imaging (HCI), 3 compounds were identified to be capable of inhibiting S100A4-mediated migration. Although we have yet to investigate the underlying mechanism for their effects, these compounds have been shown to target membrane proteins and the externalisation of S100 proteins, for at least one of the compounds, leading us to speculate that preventing externalisation of S100A4 could potentially regulate cell motility.

A ROS-based Workspace Control and Trajectory Planner for a seven degrees of freedom Robotic arm

In this Bachelor Thesis I want to provide readers with tools and scripts for the control of a 7DOF manipulator, backed up by some theory of Robotics and Computer Science, in order to better contextualize the work done. In practice, we will see most common software, and developing environments, used to cope with our task: these include ROS, along with visual simulation by VREP and RVIZ, and an almost "stand-alone" ROS extension called MoveIt!, a very complete programming interface for trajectory planning and obstacle avoidance. As we will better appreciate and understand in the introduction chapter, the capability of detecting collision objects through a camera sensor, and re-plan to the desired end-effector pose, are not enough. In fact, this work is implemented in a more complex system, where recognition of particular objects is needed. Through a package of ROS and customized scripts, a detailed procedure will be provided on how to distinguish a particular object, retrieve its reference frame with respect to a known one, and then allow navigation to that target. Together with technical details, the aim is also to report working scripts and a specific appendix (A) you can refer to, if desiring to put things together.

LINEUP SIZE AND NUMBER OF CUES: WHEN BIGGER ISN’T BETTER

Larger lineups could protect innocent suspects from being misidentified; however, they can also decrease correct identifications. Bertrand (2006) investigated whether the decrease in correct identifications could be prevented by adding more cues, in the form of additional views of lineup members’ faces, to the lineup. Adding these cues was successful to an extent. The current series of studies attempted to replicate Bertrand’s (2006) findings while addressing some methodological issues—namely, the inconsistency in image size as lineup size increased. First, I investigated whether image size could affect face recognition (Chapter 2) and found it could, but that it also affected previously-seen (“old”) versus previously-unseen (“new”) faces differently. Specifically, smaller image sizes at exposure lowered accuracy for old faces, while these same image sizes at recognition lowered accuracy for new faces. Although these results indicate that target recognition would be unaffected by image size at recognition (i.e., during a lineup), lineups are also comprised of previously-unseen faces, in the form of fillers and innocent suspects. Because image size could affect lineup decisions, as it could become more difficult to realize fillers are previously-unseen, I decided to replicate Bertrand (2006) while keeping image size constant in Chapters 3 (simultaneous lineups) and 4 (simultaneous-presentation, sequential decisions). In both Chapters, the integral findings were the same: correct identification rates decreased as lineup size increased from 6- to 24-person lineups, but adding cues had no effect. The inability to replicate Bertrand (2006) could mean that the original finding was due to chance, but alternate explanations also exist, such as the overall size of the array, the degree to which additional cues overlap, and the length of the target exposure. These alternate explanations, along with directions for future research, are discussed in the following Chapters.

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.

New Technique for Static Target Detection in Dense-Multipath Urban Environments

Synthetic Aperture Radar (SAR) images a target region reflectivity function in the multi-dimensional spatial domain of range and cross-range with a finer azimuth resolution than the one provided by any on-board real antenna. Conventional SAR techniques assume a single reflection of transmitted waveforms from targets. Nevertheless, new uses of Unmanned Aerial Vehicles (UAVs) for civilian-security applications force SAR systems to work in much more complex scenes such as urban environments. Consequently, multiple-bounce returns are additionally superposed to direct-scatter echoes. They are known as ghost images, since they obscure true target image and lead to poor resolution. All this may involve a significant problem in applications related to surveillance and security. In this work, an innovative multipath mitigation technique is presented in which Time Reversal (TR) concept is applied to SAR images when the target is concealed in clutter, leading to TR-SAR technique. This way, the effect of multipath is considerably reduced ?or even removed?, recovering the lost resolution due to multipath propagation. Furthermore, some focusing indicators such as entropy (E), contrast (C) and Rényi entropy (RE) provide us with a good focusing criterion when using TR-SAR.