The haemocytes of the colonial aplousobranch ascidian Diplosoma listerianum: Structural, cytochemical and functional analyses

Diplosoma listerianum is a colonial aplousobranch ascidian of the family Didemnidae that is native to the northeast Atlantic and exhibits a cosmopolitan distribution in temperate waters. It lacks a shared colonial circulation crossing the tunic, and the zooids are connected only by the common tunic. In the present study, the haemocytes of this ascidian were analysed via light and electron microscopy. Their phagocytic and enzymatic activities, staining and immunostaining properties, and lectin affinity were examined with various classical methods reconsidered and modified for small marine invertebrates. Eight morphotypes were identified in reference to corresponding cell types described in other ascidians: undifferentiated cells (haemoblasts), storage cells for nitrogenous catabolites (nephrocytes) and immunocytes. The immunocytes are involved in immune responses, acting as (1) phagocytes, rich in hydrolases and involved in the clearance of both foreign particles and effete cells (hyaline amoebocytes and macrophage-like cells); (2) cytotoxic cells, able to degranulate and induce cytotoxicity through the release of the enzyme phenoloxidase after an immune stimulus (granular amoebocytes and morula cells); and (3) basophilic cells with an affinity for ConA and NPA that contain heparin and histamine and that show sensitivity to the compound 48/80, promoting their degranulation (mast cell-like granulocytes). In addition, a particular cell type showing exceptional development of the Golgi apparatus and large vacuoles containing a filamentous material has been recognised (spherule cell), for which a role in tunic repair and fibrogenesis has been hypothesised

BsTLR: a new member of the TLR family of recognition proteins from the colonial ascidian Botryllus schlosseri.

Toll-like receptors (TLRs) represent a well-known family of conserved pattern recognition receptors the importance of which, in non-self recognition, was demonstrated in both vertebrates and invertebrates. Tunicates represent the vertebrate sister group and, as invertebrates, they rely only on innate immunity for their defense. As regards TLRs, two transcripts have been described and characterized in the solitary species Ciona robusta, referred to as CiTLR1 and CiTLR2. Using the Ciona TLR nucleotide sequences, we examined the available transcriptomes of Botryllus schlosseri looking for similar sequences. We were able to identify a sequence, with similarity to CiTLR2 and, through in silico transduction and subsequent sequence analysis, we studied the domain content of the putative protein. The sequence, called BsTLR, has a TIR and a transmembrane domain, four LLR and two LRR-CT domains. In addition, we analised bstlr transcription in vivo and in vitro, under various experimental conditions and in different phases of the Botryllus blastogenetic cycle. Our data show that, in different phases, there is a change in gene transcription and mRNA location, according to the blastogenetic phase

Urban planning support based on the photovoltaic potential of buildings: a multi-scenario ranking system

An increased use of renewable energy and of energy efficiency measures in buildings is needed to face the urgency of climate change. Buildings are in fact among the highest worldwide consumers of primary energy, mostly of fossil fuel origin, while still making insufficient use of in-situ renewable energy sources. To find a solution to this situation, many municipalities have promoted the use of solar cadastres mapping the solar energy potential of the existing building stock. However, their implementation has limits from different points of view including assessment accuracy, representation methods, and decision-support. To overcome these limits, this thesis proposes a planning-support system based on the photovoltaic (PV) potential of buildings. The goal is to provide decision-makers and stakeholders with a robust method to assess the potential of photovoltaic electricity generation of existing buildings under uncertain environmental conditions. The developed methodology is based on an urban-scale modeling workflow that includes the simulation of the photovoltaic electricity production and a simplified estimation of the building energy retrofit potential. Existing state-of-the-art models for solar radiation, building energy and PV performance are coupled in the workflow, which relies on a vector 3D city model featuring an accurate representation of buildings, terrain, and vegetation. The proposed modeling workflow also includes an innovative approach for simulating the arrangement of PV modules on the building envelope, which influences both the energy yield and the acceptability of the system. The modeling workflow is in turn integrated into a planning-support system that provides a robust assessment of the photovoltaic potential through risk-averse scenarios. We consider here two crucial yet underestimated uncertainty factors: weather and vegetation. The results are aggregated at different scales and, for each scale, the spatial locations are ranked through pairwise comparisons according to relevant energy indicators. The results are finally displayed in a 3D-mapping tool featuring false-color overlays at the considered aggregation scales to address different objectives and inform decision-makers. We conducted sensitivity analyses towards different input data resolutions and modeling scenarios so as to achieve a good trade-off between accuracy and computational cost and define confidence intervals for the calculated values. The simulated PV yield was also compared against measured data from an existing PV installation. The proposed modeling workflow and planning-support system were tested in an urban district within the city of Neuchâtel (Switzerland). The analysis highlighted areas with the highest potential and provided a priority list of interventions. It also showed the impact of vegetation on absolute results and especially on the ranking of the spatial locations evaluated by their energy potential

Complement-mediated cooperation between immunocytes in the compound ascidian Botryllus schlosseri

Two main kinds of innate immune responses are present in ascidians: phagocytosis and cytotoxicity. They are mediated by two different types of circulating immunocytes: phagocytes and cytotoxic morula cells (MCs). MCs, once activated by non-self-recognition, can stimulate phagocytosis by the release of soluble factors able to act as opsonins. BsC3, the complement C3 homologue, like mammalian C3, contains the thioester bond required to split the molecule into BsC3a and BsC3b. BsC3b likely represents the MC opsonin as it can enhances phagocytosis. The tenet is supported by the observed reduction in phagocytosing cells after exposure of hemocytes to compstatin, a drug preventing C3 activation, or after the bsc3 knockdown by iRNA injection. In addition, the transcript for BsCR1, homologous to mammalian CR1, is present in Botryllus phagocytes and the transcription is modulated during the blastogenetic cycle. MCs also release cytokines (chemokines) able to recruit immunocytes to the infection site. The activity is inhibited by antibodies raised against human TNFa. Since no genes for TNFa are present in the Botryllus genome, the observed activity is probably related to a TNF-domain containing protein, member of the Botryllus complement system. Conversely, activated phagocytes release a rhamnose-binding lectin able to interact with microbial surfaces and act as opsonin. It can also activate MCs by inducing the release of the reported cytokine and stimulate their degranulation. Overall, the results obtained so far indicate the presence of a well-defined cross-talk between the two types of immunocytes during the immune responses of B. schlosseri

Expression study of molecular markers involved in staminality and differentiation in the colonial ascidians Botryllus schlosseri

Ascidians are invertebrate chordates, members of the subphylum Tunicata that represents the sister group of vertebrates. They offer the opportunity to investigate and compare the behaviour of both embryonic and adult stem cells. Morphological data suggest the presence of undifferentiated haemocytes (haemoblasts) able to proliferate and give rise to terminally differentiated cells. Relevant studies were also carried out in the neural lineage, in which neural progenitor cells regenerate the brain after extirpation. In B. schlosseri, during the cyclical generation change, bud primordial cells, probably deriving from a pool of long-living stem cells, are able to give rise to the neural complex. We screened the B. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic and neural stem cells. Four sequences, orthologous to mammalian transcripts considered markers of haematopoietic progenitor cells, were identified in B. schlosseri. They are: bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. In situ hybridization on haemocyte monolayers and colony sections, resulted in labelling of cells in the sub-endostylar haemolymph lacunae. This results matches previously morphological data that identified the endostyle as a stem cell niche. Quantitative real time PCR (qRT-PCR) highlighted the over-expression of the considered genes in the mid-cycle phase of the blastogenetic cycle. During this phase, there is the formation of new secondary buds emerging from the primary buds. The high expression levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for giving rise to the new blastogenetic generation. For the neural lineage, we identified and characterised two transcripts orthologues of vertebrate neural stem cell markers (BsSox2 and BsMsi2). We also studied the expression, during the blastogenetic cycle, of a panel of genes already known to be involved in ascidian larvae neurogenesis, i.e., orthologues of Pax2/5/8, Hox1 and Hox3. ISH with riboprobes for BsSox2, BsMsi2, BsPax2/5/8, BsHox1 and BsHox3 revealed a common labelling in the endostyle niche. The presence of bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 transcripts in the cells of the region known to be a stem cell niche, led us to conclude, not only that our probes identified undifferentiated cells but even that in B. schlosseri are probably present a single population of pluripotent stem cells that could differentiate into haematopoietic or neural cells. The qRT-PCR, showed an high expression level in the mid-cycle phase of all the putative neural markers considered. In this phase new secondary buds are produced from primary buds. Each new bud needs its own neural complex and this requires the proliferation of undifferentiated cells to originate neural gland rudiment and cerebral ganglion. Bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 increased their expression associated with these neurogenesis events and this support their involvement in neural stem cell differentiation

Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3

The compound ascidian Botryllus schlosseri is a reliable model organism for the study of immunobiology. As an invertebrate, it relies only on innate immunity for its defense. We already demonstrated the presence, in Botryllus, of homologues of mammalian C3, Bf, MBL and MASP1, referred to as BsC3, BsBf, BsMBL and BsMASP, respectively. All the complement components identified so far, are expressed by morula cells, the most abundant circulating hemocytes. In mammals, once the complement system is activated, a cascade of reactions occurs resulting in the cleavage of the third complement component (C3) to C3a and C3b, the former exerting a chemotactic activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. The best-known receptor for C3a in mammals is C3aR, whereas CR1 is the receptor able to recognize and bind C3b on the microbial surfaces. Here, we describe, in B. schlosseri, new genes showing homology with vertebrate C3aR and CR1, respectively, and studied their transcription in the course of the colonial blastogenetic cycle. In addition, we continued our analysis of the role of C3 in Botryllus immunity by studying the modulation of BsC3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. Results indicate that only morula cells, and no other immunocytes type, are labelled by the antisense probe for BsC3aR, whereas phagocytes and young, undifferentiated cells, known as hemoblasts, are the cells stained by the probe for BsCR1. Both the bsc3ar and bscr1 genes are constitutively transcribed. However, a modulation in the extent of transcription occurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at TO, whereas no differences were observed when EC and MC were compared. This is probably related to the renewing of circulating cells at TO, that are replaced by new, differentiating cells entering the circulation in the same period

Characterization of the complement system in a colonial tunicate: C3 complement receptors and opsonic role of C3

The complement system is one of the most ancient immune modulator mechanism of bilaterian metazoans. In vertebrates, three complement-activation pathways are known: the classical, the alternative and the lectin pathways: all of them converge on the cleavage of C3. The compound ascidian Botryllus schlosseri is a reliable model organism for the study of immunobiology. As an invertebrate, B. schlosseri relies only on innate immunity for its defense and immunocytes. We already demonstrated the presence, in Botryllus, of homologues of mammalian C3, Bf, MBL and MASP1, referred to as BsC3, BsBf, BsMBL and BsMASP, respectively. All the complement components identified so far, are expressed by morula cells, the most abundant circulatingemocytes. In mammals, once the complement system isactivated, a cascade of reactions that involves proteolysis and polymerization occurs resulting in the cleavage of the third complement component (C3) to C3a and C3b, the former exerting a chemotactic activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. The best-known receptor for C3a in mammals is C3aR, whereas CR1 is the receptor able to recognize and bind C3b on the microbial surfaces. Here, we describe, in B. schlosseri, two newgenes showing homology with vertebrate C3aR and CR1, respectively, and studied their transcription in the course of the colonial blastogeneticcycle. In addition, we continued our analysis ofthe role of C3 in Botryllus immunity by studying the modulation of BsC3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. Results indicate that only morula cells, and no other immunocytes type, are labelled by the antisense probe for BsC3aR, whereas phagocytes and young, undifferentiated cells, known as hemoblasts, are the cells stained by the probe for BsCR1. This suggests the presence of an important cross-talk between these two immunocytes types. Both the bsc3ar and bscr1 genes are constitutively transcribed as almost all morula cells and phagocytes, respectively, resulted labelled by the antisense probe in the ISH assay, independently of their previous challenge with zymosan, a known activator of B. schlosseri hemocytes. However, a modulation in the extent of transcriptionoccurs during the colonial blastogenetic cycle as the amount of BsC3aR mRNA abruptly decreased at TO, whereas no differences were observed when EC and MC were compared. This is probably related to the renewing of circulating cells at TO, when 20-30% of hemocytes undergo cell death by apoptosis and are replaced by new, differentiating cells entering the circulation in the same period

A parametric design-based methodology to visualize building performance at the neighborhood scale

This paper focuses on parametric design-based visualization methods to represent building performance at the neighborhood scale in the perspective of an integrated design-support system. The goal of the developed methodology is to convey the relative effectiveness of different design alternatives according to a wide range of building performance indicators, including the potential for active solar applications, the energy need for space heating/cooling and (spatial) daylight autonomy. The proposed methodology is applied to a case study of a typical urban renewal project in Switzerland for which 768 design variants were analyzed using validated climate-based simulation engines. For each design variant, simulation results are represented qualitatively using multiple false-color maps and quantitatively through comprehensive plots. We conclude by showing the applicability of this methodology to a large number of neighborhood-scale design variants as well as the complementarity of the proposed visualization methods. On the basis of the case study application, a possible implementation as a design-support tool is finally discussed

Assessing the photovoltaic potential of flat roofs: insights from the analysis of optimised array arrangements

PV installations on flat roofs offer a wide range of design options, which are usually neglected in urban-scale assessments as these typically assume horizontal or other fixed arrangements. In this study, we analyse the influence of common design parameters (tilt and inter-row distance) in evaluating the potential of PV arrays installed on flat roofs, using three different performance indicators. By comparing optimised arrangements to horizontal ones, we show that the latter could be misleading, unless building- and indicator-specific correction coefficients are applied.Postprint (published version

3D-modeling of vegetation from LiDAR point clouds and assessment of its impact on façade solar irradiation

The presence of vegetation can significantly affect the solar irradiation received on building surfaces. Due to the complex shape and seasonal variability of vegetation geometry, this topic has gained much attention from researchers. However, existing methods are limited to rooftops as they are based on 2.5D geometry and use simplified radiation algorithms based on view-sheds. This work contributes to overcoming some of these limitations, providing support for 3D geometry to include facades. Thanks to the use of ray-tracing-based simulations and detailed characterization of the 3D surfaces, we can also account for inter-reflections, which might have a significant impact on fac¸ade irradiation. In order to construct confidence intervals on our results, we modeled vegetation from LiDAR point clouds as 3D convex hulls, which provide the biggest volume and hence the most conservative obstruction scenario. The limits of the confidence intervals were characterized with some extreme scenarios (e.g. opaque trees and absence of trees). Results show that uncertainty can vary significantly depending on the characteristics of the urban area and the granularity of the analysis (sensor, building and group of buildings). We argue that this method can give us a better understanding of the uncertainties due to vegetation in the assessment of solar irradiation in urban environments, and therefore, the potential for the installation of solar energy systems