Temporal segmentation tool for high-quality real time video editing software

The increasing use of video editing software requires faster and more efficient editing tools. As a first step, these tools perform a temporal segmentation in shots that allows a later building of indexes describing the video content. Here, we propose a novel real-time high-quality shot detection strategy, suitable for the last generation of video editing software requiring both low computational cost and high quality results. While abrupt transitions are detected through a very fast pixel-based analysis, gradual transitions are obtained from an efficient edge-based analysis. Both analyses are reinforced with a motion analysis that helps to detect and discard false detections. This motion analysis is carried out exclusively over a reduced set of candidate transitions, thus maintaining the computational requirements demanded by new applications to fulfill user needs.

Volume Haptics and Dendritic Spine Editing

Esta tesis se ha desarrollado en el contexto del proyecto Cajal Blue Brain, una iniciativa europea dedicada al estudio del cerebro. Uno de los objetivos de esta iniciativa es desarrollar nuevos métodos y nuevas tecnologías que simplifiquen el análisis de datos en el campo neurocientífico. El presente trabajo se ha centrado en diseñar herramientas que combinen información proveniente de distintos canales sensoriales con el fin de acelerar la interacción y análisis de imágenes neurocientíficas. En concreto se estudiará la posibilidad de combinar información visual con información háptica. Las espinas dendríticas son pequeñas protuberancias que recubren la superficie dendrítica de muchas neuronas del cerebro. A día de hoy, se cree que tienen un papel clave en la transmisión de señales neuronales. Motivo por el cual, el interés por parte de la comunidad científica por estas estructuras ha ido en aumento a medida que las técnicas de adquisición de imágenes mejoraban hasta alcanzar una calidad suficiente para analizar dichas estructuras. A menudo, los neurocientíficos utilizan técnicas de microscopía con luz para obtener los datos que les permitan analizar estructuras neuronales tales como neuronas, dendritas y espinas dendríticas. A pesar de que estas técnicas ofrezcan ciertas ventajas frente a su equivalente electrónico, las técnicas basadas en luz permiten una menor resolución. En particular, estructuras pequeñas como las espinas dendríticas pueden capturarse de forma incorrecta en las imágenes obtenidas, impidiendo su análisis. En este trabajo, se presenta una nueva técnica, que permite editar imágenes volumétricas, mediante un dispositivo háptico, con el fin de reconstruir de los cuellos de las espinas dendríticas. Con este objetivo, en un primer momento se desarrolló un algoritmo que proporciona retroalimentación háptica en datos volumétricos, completando la información que provine del canal visual. Dicho algoritmo de renderizado háptico permite a los usuarios tocar y percibir una isosuperficie en el volumen de datos. El algoritmo asegura un renderizado robusto y eficiente. Se utiliza un método basado en las técnicas de “marching tetrahedra” para la extracción local de una isosuperficie continua, lineal y definida por intervalos. La robustez deriva tanto de una etapa de detección de colisiones continua de la isosuperficie extraída, como del uso de técnicas eficientes de renderizado basadas en un proxy puntual. El método de “marching tetrahedra” propuesto garantiza que la topología de la isosuperficie extraída coincida con la topología de una isosuperficie equivalente determinada utilizando una interpolación trilineal. Además, con el objetivo de mejorar la coherencia entre la información háptica y la información visual, el algoritmo de renderizado háptico calcula un segundo proxy en la isosuperficie pintada en la pantalla. En este trabajo se demuestra experimentalmente las mejoras en, primero, la etapa de extracción de isosuperficie, segundo, la robustez a la hora de mantener el proxy en la isosuperficie deseada y finalmente la eficiencia del algoritmo. En segundo lugar, a partir del algoritmo de renderizado háptico propuesto, se desarrolló un procedimiento, en cuatro etapas, para la reconstrucción de espinas dendríticas. Este procedimiento, se puede integrar en los cauces de segmentación automática y semiautomática existentes como una etapa de pre-proceso previa. El procedimiento está diseñando para que tanto la navegación como el proceso de edición en sí mismo estén controlados utilizando un dispositivo háptico. Se han diseñado dos experimentos para evaluar esta técnica. El primero evalúa la aportación de la retroalimentación háptica y el segundo se centra en evaluar la idoneidad del uso de un háptico como dispositivo de entrada. En ambos casos, los resultados demuestran que nuestro procedimiento mejora la precisión de la reconstrucción. En este trabajo se describen también dos casos de uso de nuestro procedimiento en el ámbito de la neurociencia: el primero aplicado a neuronas situadas en la corteza cerebral humana y el segundo aplicado a espinas dendríticas situadas a lo largo de neuronas piramidales de la corteza del cerebro de una rata. Por último, presentamos el programa, Neuro Haptic Editor, desarrollado a lo largo de esta tesis junto con los diferentes algoritmos ya mencionados. ABSTRACT This thesis took place within the Cajal Blue Brain project, a European initiative dedicated to the study of the brain. One of the main goals of this project is the development of new methods and technologies simplifying data analysis in neuroscience. This thesis focused on the development of tools combining information originating from distinct sensory channels with the aim of accelerating both the interaction with neuroscience images and their analysis. In concrete terms, the objective is to study the possibility of combining visual information with haptic information. Dendritic spines are thin protrusions that cover the dendritic surface of numerous neurons in the brain and whose function seems to play a key role in neural circuits. The interest of the neuroscience community toward those structures kept increasing as and when acquisition methods improved, eventually to the point that the produced datasets enabled their analysis. Quite often, neuroscientists use light microscopy techniques to produce the dataset that will allow them to analyse neuronal structures such as neurons, dendrites and dendritic spines. While offering some advantages compared to their electronic counterpart, light microscopy techniques achieve lower resolutions. Particularly, small structures such as dendritic spines might suffer from a very low level of fluorescence in the final dataset, preventing further analysis. This thesis introduces a new technique enabling the edition of volumetric datasets in order to recreate dendritic spine necks using a haptic device. In order to fulfil this objective, we first presented an algorithm to provide haptic feedback directly from volumetric datasets, as an aid to regular visualization. The haptic rendering algorithm lets users perceive isosurfaces in volumetric datasets, and it relies on several design features that ensure a robust and efficient rendering. A marching tetrahedra approach enables the dynamic extraction of a piecewise linear continuous isosurface. Robustness is derived using a Continuous Collision Detection step coupled with acknowledged proxy-based rendering methods over the extracted isosurface. The introduced marching tetrahedra approach guarantees that the extracted isosurface will match the topology of an equivalent isosurface computed using trilinear interpolation. The proposed haptic rendering algorithm improves the coherence between haptic and visual cues computing a second proxy on the isosurface displayed on screen. Three experiments demonstrate the improvements on the isosurface extraction stage as well as the robustness and the efficiency of the complete algorithm. We then introduce our four-steps procedure for the complete reconstruction of dendritic spines. Based on our haptic rendering algorithm, this procedure is intended to work as an image processing stage before the automatic segmentation step giving the final representation of the dendritic spines. The procedure is designed to allow both the navigation and the volume image editing to be carried out using a haptic device. We evaluated our procedure through two experiments. The first experiment concerns the benefits of the force feedback and the second checks the suitability of the use of a haptic device as input. In both cases, the results shows that the procedure improves the editing accuracy. We also report two concrete cases where our procedure was employed in the neuroscience field, the first one concerning dendritic spines in the human cortex, the second one referring to an ongoing experiment studying dendritic spines along dendrites of mouse cortical pyramidal neurons. Finally, we present the software program, Neuro Haptic Editor, that was built along the development of the different algorithms implemented during this thesis, and used by neuroscientists to use our procedure.

Engineering precision RNA molecular switches

Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.

Cytoplasmic RNA modulators of an inside-out signal-transduction cascade

A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the β2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathways in vivo could potentially be applied wherever targeted modulation of a signal-transduction cascade is desired.

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA–RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5–200kD and U5–100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5–200kD protein but lack U5–100kD, suggesting that the U5–200kD protein could mediate U4/U6 duplex unwinding. Finally, U5–200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5–200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

DnaA-stimulated transcriptional activation of oriλ: Escherichia coli RNA polymerase β subunit as a transcriptional activator contact site

We present evidence that Escherichia coli RNA polymerase β subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage λ. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of λ plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the β subunit of RNA polymerase during activation of the pR promoter. A new classification of prokaryotic transcriptional activators is proposed.

Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria

In populations that are small and asexual, mutations with slight negative effects on fitness will drift to fixation more often than in large or sexual populations in which they will be eliminated by selection. If such mutations occur in substantial numbers, the combined effects of long-term asexuality and small population size may result in substantial accumulation of mildly deleterious substitutions. Prokaryotic endosymbionts of animals that are transmitted maternally for very long periods are effectively asexual and experience smaller effective population size than their free-living relatives. The contrast between such endosymbionts and related free-living bacteria allows us to test whether a population structure imposing frequent bottlenecks and asexuality does lead to an accumulation of slightly deleterious substitutions. Here we show that several independently derived insect endosymbionts, each with a long history of maternal transmission, have accumulated destabilizing base substitutions in the highly conserved 16S rRNA. Stabilities of Domain I of this subunit are 15–25% lower in endosymbionts than in closely related free-living bacteria. By mapping destabilizing substitutions onto a reconstructed phylogeny, we show that decreased ribosomal stability has evolved separately in each endosymbiont lineage. Our phylogenetic approach allows us to demonstrate statistical significance for this pattern: becoming endosymbiotic predictably results in decreased stability of rRNA secondary structure.

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

The human homologue of Saccharomyces cerevisiae Gle1p is required for poly(A)+ RNA export

The mechanism of mRNA export is a complex issue central to cellular physiology. We characterized previously yeast Gle1p, a protein with a leucine-rich (LR) nuclear export sequence (NES) that is essential for poly(A)+ RNA export in Saccharomyces cerevisiae. To characterize elements of the vertebrate mRNA export pathway, we identified a human homologue of yeast Gle1p and analyzed its function in mammalian cells. hGLE1 encodes a predicted 75-kDa polypeptide with high sequence homology to yeast Gle1p, but hGle1p does not contain a sequence motif matching any of the previously characterized NESs. hGLE1 can complement a yeast gle1 temperature-sensitive export mutant only if a LR-NES is inserted into it. To determine whether hGle1p played a role in nuclear export, anti-hGle1p antibodies were microinjected into HeLa cells. In situ hybridization of injected cells showed that poly(A)+ RNA export was inhibited. In contrast, there was no effect on the nuclear import of a glucocorticoid receptor reporter. We conclude that hGle1p functions in poly(A)+ RNA export, and that human cells facilitate such export with a factor similar to yeast but without a recognizable LR-NES. With hGle1p localized at the nuclear pore complexes, hGle1p is positioned to act at a terminal step in the export of mature RNA messages to the cytoplasm.

Polyadenylation of stable RNA precursors in vivo

Polyadenylation at the 3′ terminus has long been considered a specific feature of mRNA and a few other unstable RNA species. Here we show that stable RNAs in Escherichia coli can be polyadenylated as well. RNA molecules with poly(A) tails are the major products that accumulate for essentially all stable RNA precursors when RNA maturation is slowed because of the absence of processing exoribonucleases; poly(A) tails vary from one to seven residues in length. The polyadenylation process depends on the presence of poly(A) polymerase I. A stochastic competition between the exoribonucleases and poly(A) polymerase is proposed to explain the accumulation of polyadenylated RNAs. These data indicate that polyadenylation is not unique to mRNA, and its widespread occurrence suggests that it serves a more general function in RNA metabolism.

Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-β enhanceosome in vitro

The transcriptional activity of an in vitro assembled human interferon-β gene enhanceosome is highly synergistic. This synergy requires five distinct transcriptional activator proteins (ATF2/c-JUN, interferon regulatory factor 1, and p50/p65 of NF-κB), the high mobility group protein HMG I(Y), and the correct alignment of protein-binding sites on the face of the DNA double helix. Here, we investigate the mechanisms of enhanceosome-dependent transcriptional synergy during preinitiation complex assembly in vitro. We show that the stereospecific assembly of the enhanceosome is critical for the efficient recruitment of TFIIB into a template-committed TFIID-TFIIA-USA (upstream stimulatory activity complex) and for the subsequent recruitment of the RNA polymerase II holoenzyme complex. In addition, we provide evidence that recruitment of the holoenzyme by the enhanceosome is due, at least in part, to interactions between the enhanceosome and the transcriptional coactivator CREB, cAMP responsive element binding protein (CBP). These studies reveal a unique role of enhanceosomes in the cooperative assembly of the transcription machinery on the human interferon-β promoter.

Mammalian capping enzyme binds RNA and uses protein tyrosine phosphatase mechanism

Mammalian capping enzymes are bifunctional proteins with both RNA 5′-triphosphatase and guanylyltransferase activities. The N-terminal 237-aa triphosphatase domain contains (I/V)HCXXGXXR(S/T)G, a sequence corresponding to the conserved active-site motif in protein tyrosine phosphatases (PTPs). Analysis of point mutants of mouse RNA 5′-triphosphatase identified the motif Cys and Arg residues and an upstream Asp as required for activity. Like PTPs, this enzyme was inhibited by iodoacetate and VO43− and independent of Mg2+, providing additional evidence for phosphate removal from RNA 5′ ends by a PTP-like mechanism. The full-length, 597-aa mouse capping enzyme and the C-terminal guanylyltransferase fragment (residues 211–597), unlike the triphosphatase domain, bound poly (U) and were nuclear in transfected cells. RNA binding was increased by GTP, and a guanylylation-defective, active-site mutant was not affected. Ala substitution at positions required for the formation of the enzyme-GMP capping intermediate (R315, R530, K533, or N537) also eliminated poly (U) binding, while proteins with conservative substitutions at these sites retained binding but not guanylyltransferase activity. These results demonstrate that the guanylyltransferase domain of mammalian capping enzyme specifies nuclear localization and RNA binding. Association of capping enzyme with nascent transcripts may act in synergy with RNA polymerase II binding to ensure 5′ cap formation.

Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci

DsrA is an 87-nt untranslated RNA that regulates both the global transcriptional silencer and nucleoid protein H-NS and the stationary phase and stress response sigma factor RpoS (σs). We demonstrate that DsrA acts via specific RNA:RNA base pairing interactions at the hns locus to antagonize H-NS translation. We also give evidence that supports a role for RNA:RNA interactions at the rpoS locus to enhance RpoS translation. Negative regulation of hns by DsrA is achieved by the RNA:RNA interaction blocking translation of hns RNA. In contrast, results suggest that positive regulation of rpoS by DsrA occurs by formation of an RNA structure that activates a cis-acting translational operator. Sequences within DsrA complementary to three additional genes, argR, ilvIH, and rbsD, suggest that DsrA is a riboregulator of gene expression that acts coordinately via RNA:RNA interactions at multiple loci.

DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription

DsrA RNA regulates both transcription, by overcoming transcriptional silencing by the nucleoid-associated H-NS protein, and translation, by promoting efficient translation of the stress σ factor, RpoS. These two activities of DsrA can be separated by mutation: the first of three stem-loops of the 85 nucleotide RNA is necessary for RpoS translation but not for anti-H-NS action, while the second stem-loop is essential for antisilencing and less critical for RpoS translation. The third stem-loop, which behaves as a transcription terminator, can be substituted by the trp transcription terminator without loss of either DsrA function. The sequence of the first stem-loop of DsrA is complementary with the upstream leader portion of rpoS messenger RNA, suggesting that pairing of DsrA with the rpoS message might be important for translational regulation. Mutations in the Rpos leader and compensating mutations in DsrA confirm that this predicted pairing is necessary for DsrA stimulation of RpoS translation. We propose that DsrA pairing stimulates RpoS translation by acting as an anti-antisense RNA, freeing the translation initiation region from the cis-acting antisense RNA and allowing increased translation.

Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains

An assembly of a catalytic unit for aminoacylation of an RNA microhelix is demonstrated here. This assembly may recapitulate a step in the historical development of tRNA synthetases. The class-defining domain of a tRNA synthetase is closely related to the primordial enzyme that catalyzed synthesis of aminoacyl adenylate. RNA binding elements are imagined to have been added so that early RNA substrates could be docked proximal to the activated amino acid. RNA microhelices that recapitulate the acceptor stem of modern tRNAs are potential examples of early substrates. In this work, we examined a fragment of Escherichia coli alanyl-tRNA synthetase, which catalyzes aminoacyl adenylate formation but is virtually inactive for catalysis of RNA microhelix aminoacylation. Fusion to the fragment of either of two unrelated nonspecific RNA binding domains activated microhelix aminoacylation. Although the fusion proteins lacked the RNA sequence specificity of the natural enzyme, their activity was within 1–2 kcal⋅mol−1 of a truncated alanyl-tRNA synthetase that has aminoacylation activity sufficient to sustain cell growth. These results show that, starting with an activity for adenylate synthesis, barriers are relatively low for building catalytic units for aminoacylation of RNA helices.