Using segmented objects in ostensive video shot retrieval

This paper presents a system for video shot retrieval in which shots are retrieved based on matching video objects using a combination of colour, shape and texture. Rather than matching on individual objects, our system supports sets of query objects which in total reflect the user’s object-based information need. Our work also adapts to a shifting user information need by initiating the partitioning of a user’s search into two or more distinct search threads, which can be followed by the user in sequence. This is an automatic process which maps neatly to the ostensive model for information retrieval in that it allows a user to place a virtual checkpoint on their search, explore one thread or aspect of their information need and then return to that checkpoint to then explore an alternative thread. Our system is fully functional and operational and in this paper we illustrate several design decisions we have made in building it

T-cadherin signaling in endothelial cells

The cadherin superfamily comprises transmembrane glycoproteins that mediate calciumdependent homophilic cell-cell adhesion. In addition to their pivotal role in mechanical adhesion between cells, cadherins have multiple functions in tissue morphogenesis, cell recognition and sorting, regulated cell motility and the induction and maintenance of tissue/cell polarity. T-cadherin (T-cad) is unusual member of cadherin superfamily; while possessing the Nterminal tandem cadherin repeat structure (EC domain), it lacks both transmembrane and cytoplasmic domains, and is bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. T-cad has five EC domains and a propeptide in its precursor form (130kDa), which upon cleavage gets converted to mature form (105kDa). A role for T-cad in tissue organization was first demonstrated in the avian embryonic nervous system where the protein influenced the pattern of neural crest cell migration and maintained somite polarity. Many cancer cell lines (e.g. breast, colon, lung, inter alia) display allelic loss of T-cad which is correlated with tumor progression, and hence T-cad has been described as tumor suppressor gene. T-cad is widely expressed in the vasculature and is upregulated in proliferative vascular disorders such as atherosclerosis and restenosis. GPI-anchored T-cad is not localized at adherent junctions but rather distributed globally over the cell surface. T-cad is localized within lipid rafts. In vitro data supports participation of T-cad in many cellular processes such as vascular differentiation, migration and proliferation of smooth muscle cells (SMC) and endothelial cells (EC) and angiogenesis. Adenoviral mediated overexpression of T-cad in EC and SMC results in cell cycle progression and a concomitant promotion of proliferation. T-cad exhibits deadhesive functions upon homophilic ligation with antibody against T-cad or with immobilized recombinant protein. Homophilic ligation of T-cad induces polarization and migration of endothelial cells in a RhoA/ROCK and Rac dependent fashion. T-cad stimulates in-gel outgrowth of endothelial sprouts in 3-dimensional EC-spheroid and heart tissue models of angiogenesis. In vivo, myoblast-mediated delivery of recombinant soluble T-cad to mouse skeletal muscle facilitates VEGF-induced angiogenesis, supporting a physiological role for Tcad as a proangiogenic protein. The present thesis is focused on the regulation of T-cad expression and the signaling mechanisms whereby T-cad affects vascular cell behavior. The conditions of proliferative vascular cell disorders in which T-cad is upregulated are associated with oxidative stress and cell survival/cell death. We observed an elevation in T-cad levels under condition of oxidative stress induced by serum-deprivation and H2O2; this response was normalized upon inclusion of an antioxidant, N-acetyl cysteine or NADPH oxidase inhibitor diphenyleneiodonium, suggesting T- cad induction by reactive oxygen species is NADPH oxidase dependent. Adenoviral mediated overexpression of T-cad in EC facilitated EC survival upon induction of apoptosis by serumdeprivation and various apoptosis-inducing pharmacologicals. Western blot analysis of lysates infected with Empty –adenovirus (E-EC) and T-cad adenovirus (T-cad+-EC) resulted in hyperactivity of anti-apoptotic proteins (Akt and mTOR target p70S6 kinase) and diminished activity of pro-apoptotic proteins (p38MAPK and active caspase3). PI3 kinase inhibitor, wortmannin, and mTOR inhibitor, rapamycin, normalized anti-apoptotic effects of T-cad; these data suggest that upregulation of T-cad in response to oxidative stress functions to protect EC by concomitant induction of PI3K/Akt/mTOR pathway and suppression of p38/caspase3 pathways. Subsequently we focused on identifying downstream targets of Akt and candidate proximal molecular mediators for T-cad. T-cad+-EC exhibited hyperphosphorylation of glycogen synthase kinase β (GSK3β) and concomitant nuclear accumulation of active β-catenin, a transcription factor regulating cell cycle proteins. Using various GSK3β-carrying adenovectors (kinase mutant, dominant negative or wild type) we demonstrated that T-cad induced nuclear accumulation of β-catenin is GSK3β-dependent. siRNA mediated knockdown of T-cad resulted in decreased phosphorylation of Akt and GSK3β and also in reduced nuclear accumulation of β- catenin. T cell factor (TCF) and Leukocyte enhancer factor (LEF) are co-factors for β-catenin; we found that luciferase (reporter) activity of TCF/LEF elements in T-cad+-EC was markedly increased as compared to E-EC. Cyclin D1, one of the important regulators of the cell cycle is a target of β-catenin/TCF/LEF transcription machinery; T-cad+-EC showed increased mRNA and protein levels of cyclin D1 and increased cell proliferation. In searching for molecular mediators of T-cad we considered Integrin linked kinase (ILK) as a putative candidate because both the proteins are located in rafts and ILK acts upstream of Akt and GSK3β in a PI3K-dependent fashion. T-cad+-EC exhibited increased ILK “kinase” activity in a pull-down assay. RNAimediated knockdown of ILK abrogated effects of T-cad on both phosphorylation of Akt and GSK3β and the nuclear accumulation β-catenin, suggesting involvement of ILK in T-cad signaling. Confocal microscopy studies revealed colocalisation of T-cad and ILK in EC which was most prominent within leading edges of migratory cells and at focal adhesions. Anti-ILK immunoprecipitates contained T-cad indicating the existence of T-cad/ILK complexes, and supporting our hypothesis that ILK can function as a proximal molecular mediator for T-cadelicited PI3K/Akt/GSK3β signaling. Transcriptional regulation of T-cad in endothelial cells is poorly understood. To characterize the minimal promoter region of T-cad, we cloned serially deleted fragments of Tcad promoter stretches into luciferase reporter vector (pGL3). Reporter gene analysis exhibited basal levels of luciferase activity within -285bps suggesting existence of minimal promoter region within -285bps from translational start site. Oxidative stress elevated reporter activity of -285 bps construct, suggesting the minimal promoter region might be responsible for the redox sensitivity of T-cad expression. To identify regulatory elements (transcription factors) responsible for T-cad regulation gel shift assays were performed using nuclear extracts of EC and various oligos designed from T-cad promoter region from -1 to -284 bps We identified specific binding of regulatory protein(s) between -156 to -203 bps. Nuclear extracts from serumdeprived EC exhibited increased binding to -156 to -203 bps oligo, suggesting that the identified nucleoprotein complex could function to induce T-cad expression under conditions of oxidative stress. To identify transcription factor(s) within the identified nucleoprotein complex we performed pull-down assay using nuclear extracts of EC, biotinylated -156 to -203 bps and streptavidin agarose beads. Proteins pulled down were subjected to microsequencing by mass spectrometry. Interestingly thioredoxin (TRX1) was found to be present. TRX1 is a 12kDa protein induced by NADPH oxidase under stress and it acts as an antioxidant by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange. Following its translocation to the nucleus TRX1 reduces transcription factors, enabling their binding to regulatory elements. Preliminary data using RNAi-mediated knockdown of TRX1 abrogates oxidative stress-induced upregulation of T-cad in EC, suggesting that NADPH dependent-induction of T-cad involves nuclear translocation of TRX1. These data may explain the observations of upregulation of Tcad on vascular cells in atherosclerotic lesions where oxidative stress plays a key pathogenic role