Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Background: One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape. Methodology/Principal Findings: We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally. Conclusions/Significance: The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development

Non-Invasive Measurement of Frog Skin Reflectivity in High Spatial Resolution Using a Dual Hyperspectral Approach

Background:Most spectral data for the amphibian integument are limited to the visible spectrum of light and have been collected using point measurements with low spatial resolution. In the present study a dual camera setup consisting of two push broom hyperspectral imaging systems was employed, which produces reflectance images between 400 and 2500 nm with high spectral and spatial resolution and a high dynamic range.Methodology/Principal Findings:We briefly introduce the system and document the high efficiency of this technique analyzing exemplarily the spectral reflectivity of the integument of three arboreal anuran species (Litoria caerulea, Agalychnis callidryas and Hyla arborea), all of which appear green to the human eye. The imaging setup generates a high number of spectral bands within seconds and allows non-invasive characterization of spectral characteristics with relatively high working distance. Despite the comparatively uniform coloration, spectral reflectivity between 700 and 1100 nm differed markedly among the species. In contrast to H. arborea, L. caerulea and A. callidryas showed reflection in this range. For all three species, reflectivity above 1100 nm is primarily defined by water absorption. Furthermore, the high resolution allowed examining even small structures such as fingers and toes, which in A. callidryas showed an increased reflectivity in the near infrared part of the spectrum.Conclusion/Significance:Hyperspectral imaging was found to be a very useful alternative technique combining the spectral resolution of spectrometric measurements with a higher spatial resolution. In addition, we used Digital Infrared/Red-Edge Photography as new simple method to roughly determine the near infrared reflectivity of frog specimens in field, where hyperspectral imaging is typically difficult. © 2013 Pinto et al

Exploring Virtual Reality and Doppelganger Avatars for the Treatment of Chronic Back Pain

Cognitive-behavioral models of chronic pain assume that fear of pain and subsequent avoidance behavior contribute to pain chronicity and the maintenance of chronic pain. In chronic back pain (CBP), avoidance of movements often plays a major role in pain perseverance and interference with daily life activities. In treatment, avoidance is often addressed by teaching patients to reduce pain behaviors and increase healthy behaviors. The current project explored the use of personalized virtual characters (doppelganger avatars) in virtual reality (VR), to influence motor imitation and avoidance, fear of pain and experienced pain in CBP. We developed a method to create virtual doppelgangers, to animate them with movements captured from real-world models, and to present them to participants in an immersive cave virtual environment (CAVE) as autonomous movement models for imitation. Study 1 investigated interactions between model and observer characteristics in imitation behavior of healthy participants. We tested the hypothesis that perceived affiliative characteristics of a virtual model, such as similarity to the observer and likeability, would facilitate observers’ engagement in voluntary motor imitation. In a within-subject design (N=33), participants were exposed to four virtual characters of different degrees of realism and observer similarity, ranging from an abstract stickperson to a personalized doppelganger avatar designed from 3d scans of the observer. The characters performed different trunk movements and participants were asked to imitate these. We defined functional ranges of motion (ROM) for spinal extension (bending backward, BB), lateral flexion (bending sideward, BS) and rotation in the horizontal plane (RH) based on shoulder marker trajectories as behavioral indicators of imitation. Participants’ ratings on perceived avatar appearance were recorded in an Autonomous Avatar Questionnaire (AAQ), based on an explorative factor analysis. Linear mixed effects models revealed that for lateral flexion (BS), a facilitating influence of avatar type on ROM was mediated by perceived identification with the avatar including avatar likeability, avatar-observer-similarity and other affiliative characteristics. These findings suggest that maximizing model-observer similarity may indeed be useful to stimulate observational modeling. Study 2 employed the techniques developed in study 1 with participants who suffered from CBP and extended the setup with real-world elements, creating an immersive mixed reality. The research question was whether virtual doppelgangers could modify motor behaviors, pain expectancy and pain. In a randomized controlled between-subject design, participants observed and imitated an avatar (AVA, N=17) or a videotaped model (VID, N=16) over three sessions, during which the movements BS and RH as well as a new movement (moving a beverage crate) were shown. Again, self-reports and ROMs were used as measures. The AVA group reported reduced avoidance with no significant group differences in ROM. Pain expectancy increased in AVA but not VID over the sessions. Pain and limitations did not significantly differ. We observed a moderation effect of group, with prior pain expectancy predicting pain and avoidance in the VID but not in the AVA group. This can be interpreted as an effect of personalized movement models decoupling pain behavior from movement-related fear and pain expectancy by increasing pain tolerance and task persistence. Our findings suggest that personalized virtual movement models can stimulate observational modeling in general, and that they can increase pain tolerance and persistence in chronic pain conditions. Thus, they may provide a tool for exposure and exercise treatments in cognitive behavioral treatment approaches to CBP

Evolutionary diversification and historical biogeography of orchidaceae in Central America with emphasis on Costa Rica and Panama

In this thesis, I targeted the orchid genus Lepanthes, one of the six genera of angiosperms that surpasses 1,000 species in the Neotropics, as a study model to investigate the evolutionary processes that promoted species diversifications. To investigate this, we improved the taxonomy of the group integrating a solid phylogenetic framework with morphological evolution, assessing inter-specific relationships in species complexes with hundreds of DNA markers using anchored hybrid enrichment approach, and describing new species. In addition, we addressed the pollination of Trichosalpinx through the study of floral anatomy, pollinator behavior, and floral traits. Trichosalpinx flowers are pollinated exclusively by female biting midges that are attracted by the small quantities of proteins secreted on the flowers. Finally, we inferred the biogeographical history and diversification dynamics of the two largest Neotropical orchid groups (Cymbidieae and Pleurothallidinae), using densely sampled phylogenies coupled with geological datasets and discussed the impact of biogeographical events and orogeny on the species richness of Lepanthes. Species diversification is correlated with Andean orogeny, and multiple migrations and recolonizations across the Andes indicate that mountains do not constrain orchid dispersal over long timescales. This thesis provides new insights into the complex evolution of one of the most species-rich angiosperm.Leiden University/[]//Países BajosCentro de Biodiversidad Naturalis/[]//Países BajosUniversidad de Costa Rica/[]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL

Hieroglyph: Hierarchical Glia Graph Skeletonization and Matching

Automatic 3D reconstruction of glia morphology is a powerful tool necessary for investigating the role of microglia in neurological disorders in the central nervous system. Current glia skeleton reconstruction techniques fail to capture an accurate tracing of the processes over time, useful for the study of the microglia motility and morphology in the brain during healthy and diseased states. We propose Hieroglyph, a fully automatic temporal 3D skeleton reconstruction algorithm for glia imaged via 3D multiphoton microscopy. Hieroglyph yielded a 21% performance increase compared to state of the art automatic skeleton reconstruction methods and outperforms the state of the art in different measures of consistency on datasets of 3D images of microglia. The results from this method provide a 3D graph and digital reconstruction of glia useful for a myriad of morphological analyses that could impact studies in brain immunology and disease.Comment: submitted to IEEE International Conference on Image Processing, 201

The Paradox behind the Pattern of Rapid Adaptive Radiation: How Can the Speciation Process Sustain Itself Through an Early Burst?

Rapid adaptive radiation poses two distinct questions apart from speciation and adaptation: What happens after one speciation event and how do some lineages continue speciating through a rapid burst? We review major features of rapid radiations and their mismatch with theoretical models and speciation mechanisms. The paradox is that the hallmark rapid burst pattern of adaptive radiation is contradicted by most speciation models, which predict continuously decelerating diversification and niche subdivision. Furthermore, it is unclear if and how speciation-promoting mechanisms such as magic traits, phenotype matching, and physical linkage of coadapted alleles promote rapid bursts of speciation. We review additional mechanisms beyond ecological opportunity to explain rapid radiations: (a) ancient adaptive alleles and the transporter hypothesis, (b) sexual signal complexity, (c) fitness landscape connectivity, (d) diversity begets diversity, and (e) plasticity first. We propose new questions and predictions connecting microevolutionary processes to macroevolutionary patterns through the study of rapid radiations

Computational visualization for critical thinking

This paper looks back at historical precedents for how computational systems and ideas have been visualized as a means of access to and engagement with a broader audience, and to develop a new more tangible language to address abstraction. These precedents share a subversive ground in using a visual language to provoke new ways of engaging with about complex ideas. Two new approaches to visualizing algorithmic systems are proposed for the emerging context of algorithmic ethics in society, looking at prototypical algorithms in computer vision and machine learning systems, to think through the meaning created by algorithmic structure and process. The aim is to use visual design to provoke new kinds of thinking and criticality that can offer opportunities to address algorithms in their increasingly more politicized role today. These new approaches are developed from an arts research perspective to support critical thinking and arts knowledge through creative coding and interactive design

Unravelling the Conformational Dynamics of a-Synuclein's Folding Pathway By Hydrogen-Deuterium Exchange Mass Spectrometry in the Presence of Phospholipid Nanodiscs

Conformational changes of the -synuclein protein during docking into a phospholipid membrane is suspected to play a central role in neuronal cell death during Parkinsons disease. This intrinsically disordered protein obtains its secondary helical structure through a pathway of transition states. These states, when improperly folded, are prone to stack into hydrophobic -sheets, which later lead to the formation of fibrils and Lewy bodies the hallmarks of Parkinsons. The ability to elucidate key transition intermediates of -synuclein will clarify the mechanism used in the development of this disease and help facilitate the screening of drugs for the treatment of all -synucleinopathies. In this research, Time-Resolved Hydrogen/Deuterium Exchange Mass Spectrometry coupled with the nanodiscs technology as artificial membrane was employed to unravel structural behaviour of this protein in the presence of phospholipid bilayer. As a result, the conformational behaviour of SN indicated rather a transient interaction with phospholipid bilayer than binding to the nanodisc. The key regions of the most structural rearrangements during the interaction with the lipid membrane were identified and were consistent with the published literature. Based on the results of the kinetic HDX studies, the data proposed an evidence for the structural intermediate of SN which was proposed in different theories on SNs conformational behaviour in vivo