Screening of algal cells using microdroplet technology

The increased interest in algae as feedstock for biofuel and high value chemicals, has accentuated the need for a more in-depth study of algal cells at the single cell level. Droplet microfluidics has been established as a multifunctional platform, with many advantages for the study of biological systems in the microscale. In this thesis, droplet microfluidic technology was used to optimise and expand the application of a platform designed for the study and screening of single algal cells. Single Phaeodactylum tricornutum (Pt) cells were encapsulated in microdroplets and their growth was monitored over 11 days and was found to be heterogeneous. To limit cell sedimentation during encapsulation and to increase the time over which the encapsulation procedure could be run, 10% v/v OptiPrep density matching medium was added to the cell suspension prior to the encapsulation. A fluorescence-activated droplet sorting platform was used to detect the fluorescence of encapsulated algal cells. Following modifications to the optics of a previously used platform, the fluorescence measurement uncertainty was reduced from 87% to 8%. This improved platform was used to detect the chlorophyll fluorescence of encapsulated Pt and Ng (N. gaditana) cells, confirming that the screening of cells of different sizes and shapes is possible. Chlorophyll fluorescence sorting was used to isolate droplets containing cells from empty droplets to overcome the issue of the random encapsulation of the cells in droplets. There were no false negatives during the sorting procedure, minimising the loss of cells. GFP fluorescence detection was used to sort GFP-expressing Pt cells from a mixture with wild type cells. Furthermore, droplet sorting was combined with droplet dispensing to collect single GFP-expressing cells and obtain monoclonal cell cultures. Single droplet dispensing enables the screening of mutagenized libraries of cells and the selection of potentially rare cell clones, which is much harder to achieve using other selection methods. To screen cells based on their lipid content, Pt cells encapsulated in microdroplets were stained with BODIPY 505/515. The diffusion of the dye to and from the droplets was studied by fluorescence imaging. Furthermore, the BODIPY 505/515 fluorescence of the droplets was detected with the laser-induced fluorescence detection platform. Several experimental parameters were modified to reduce the high fluorescence background caused by the dye dissolved in the oil surrounding the droplets. A new cell staining method was developed using micelle/hydrogel composite beads loaded with BODIPY 505/515 for dye delivery. The success of this method was assessed with fluorescence microscopy and flow cytometry. The flow cytometry results showed that Pt cells could be stained by these beads within 30 minutes and that the beads could also stain other types of cells, such as Ng cells. The cells could still grow after the staining. The beads were co-encapsulated with Pt cells in microdroplets to test whether this was a viable solution to the issue of leakage of BODIPY 505/515 from the droplets over time. With the microfluidic platform established and optimised, it is now possible to transition from mainly proof-of-concept experiments on algal cell fluorescence detection, to targeted experiments aimed towards answering specific biological questions

Persisting digital society territorial divides

The ‘Europe 2020 Strategy’ was issued in 2010 by the European Commission. This document constitutes a growth scheme for the decade 2010-2020 that aims to help the European Union to emerge from the current crisis through the so-called smart, sustainable and inclusive dimensions of growth. In this context, the basic aim of the SIESTA (“Spatial Indicators for a ‘Europe 2020 Strategy’ Territorial Analysis”) Project has been to illustrate the territorial dimension of the ‘Europe 2020 Strategy’. In other words, to show how this document acts territorially, particularly at the regional scale, but, when possible, also at the urban level. The SIESTA Project has been funded by ESPON (“European Observation Network for Territorial Development and Cohesion”), a European Commission Programme whose mission is to support policy development in relation to the aim of territorial cohesion and a harmonious development of the European territory. This book includes most of the main findings and conclusions obtained through research of the SIESTA Project. The contents were presented and discussed as keynote addresses or communications at the SIESTA Final Conference held in Santiago de Compostela, Galicia, on 4-5 April 2013

Scale of hospitality firms and local economic development: evidence from Crete

The hospitality industry generates benefits for many host communities including employment generation and foreign exchange earnings. However, the hospitality industry often leads to external dependency contributing to a loss of local control over resources, migrant workforce and leakages outside the local economy, seriously reducing industry's potential for generating net financial advantages and growth for the local economy. Despite the variation of size of hospitality firms, there is still limited research on how well different size hospitality firms contribute to local economic development, something which this paper addresses, taking as a case the island of Crete. The findings suggest that the smaller the size of hospitality firm the larger the benefits to the local economy

Towards reviving post-Olympic Athens as a cultural destination

This paper examines the effects of global change on the status and qualities of the Greek national capital, Athens, focusing on how they affect the development of cultural tourism in the city. Although Athens constituted one of the most significant destinations for Greek tourism in the past, in recent years it started to weaken. Athens is characterised by a series of problems, among them are the degradation of its environment and quality of life and traffic congestion. However, in terms of tourism development, the Olympic Games helped in re-imaging the city and upgrading its infrastructure. This study based on semi-structured interviews with top officials reveals how global change has affected Athens’ socio-cultural/economic status, identity and image. Despite the tourism policy/planning responses to global changes, Athens’ tourism continues to decline leaving unexplored potential such as its rich cultural heritage, new multicultural identity and the New Acropolis Museum. The paper suggests that cultural elements of capital cities must be multidimensional including a variety of attractions and amenities. The use of cultural heritage assets needs to be in line with global developments in order for cities to effectively leverage heritage for cultural tourism

Droplet-based microfluidic screening and sorting of microalgal populations for strain engineering applications

The application of microfluidic technologies to microalgal research is particularly appealing since these approaches allow the precise control of the extracellular environment and offer a high-throughput approach to studying dynamic cellular processes. To expand the portfolio of applications, here we present a droplet-based microfluidic method for analysis and screening of Phaeodactylum tricornutum and Nannochloropsis gaditana, which can be integrated into a genetic transformation workflow. Following encapsulation of single cells in picolitre-sized droplets, fluorescence signals arising from each cell can be used to assess its phenotypic state. In this work, the chlorophyll fluorescence intensity of each cell was quantified and used to identify populations of P. tricornutum cells grown in different light conditions. Further, individual P. tricornutum or N. gaditana cells engineered to express green fluorescent protein were distinguished and sorted from wild-type cells. This has been exploited as a rapid screen for transformed cells within a population, bypassing a major bottleneck in algal transformation workflows and offering an alternative strategy for the identification of genetically modified strains

Plant biodiversity assessment through pollen DNA metabarcoding in Natura 2000 habitats (Italian Alps)

11openInternationalInternational coauthor/editorMonitoring biodiversity is of increasing importance in natural ecosystems. Metabarcoding can be used as a powerful molecular tool to complement traditional biodiversity monitoring, as total environmental DNA can be analyzed from complex samples containing DNA of different origin. The aim of this research was to demonstrate the potential of pollen DNA metabarcoding using the chloroplast trnL partial gene sequencing to characterize plant biodiversity. Collecting airborne biological particles with gravimetric Tauber traps in four Natura 2000 habitats within the Natural Park of Paneveggio Pale di San Martino (Italian Alps), at three-time intervals in 1 year, metabarcoding identified 68 taxa belonging to 32 local plant families. Metabarcoding could identify with finer taxonomic resolution almost all non-rare families found by conventional light microscopy concurrently applied. However, compared to microscopy quantitative results, Poaceae, Betulaceae, and Oleaceae were found to contribute to a lesser extent to the plant biodiversity and Pinaceae were more represented. Temporal changes detected by metabarcoding matched the features of each pollen season, as defined by aerobiological studies running in parallel, and spatial heterogeneity was revealed between sites. Our results showcase that pollen metabarcoding is a promising approach in detecting plant species composition which could provide support to continuous monitoring required in Natura 2000 habitats for biodiversity conservation.openLeontidou, Kleopatra; Vokou, Despoina; Sandionigi, Anna; Bruno, Antonia; Lazarina, Maria; De Groeve, Johannes; Li, Mingai; Varotto, Claudio; Girardi, Matteo; Casiraghi, Maurizio; Cristofori, AntonellaLeontidou, K.; Vokou, D.; Sandionigi, A.; Bruno, A.; Lazarina, M.; De Groeve, J.; Li, M.; Varotto, C.; Girardi, M.; Casiraghi, M.; Cristofori, A

Incorporating density in spatiotemporal land use/cover change patterns: The case of Attica, Greece

This paper looks at the periodic land use/cover (LUC) changes that occurred in Attica, Greece from 1991 to 2016. During this period, land transformations were mostly related to the artificial LUC categories; therefore, the aim was to map LUC with a high thematic resolution aimed at these specific categories, according to their density and continuity. The classification was implemented using the Random Forests (RF) machine learning algorithm and the presented methodological framework involved a high degree of automation. The results revealed that the majority of the expansion of the built-up areas took place at the expense of agricultural land. Moreover, mapping and quantifying the LUC changes revealed three uneven phases of development, which reflect the socioeconomic circumstances of each period. The discontinuous low-density urban fabric started to increase rapidly around 2003, reaching 7% (from 2.5% in 1991), and this trend continued, reaching 12% in 2016. The continuous as well as the discontinuous dense urban fabric, almost doubled throughout the study period. Agricultural areas were dramatically reduced to almost half of what they were in 1991, while forests, scrubs, and other natural areas remained relatively stable, decreasing only by 3% in 25 years

Staging equality in Greek squares: hybrid spaces of political subjectification

This article stages a dialogue between Jacques Rancière's political writings and the squares movement in Greece. From May to July 2011, a heterogeneous multitude of protesters reclaimed the squares of the country from their allocation in the police order and articulated a multiplicity of divergent discursive, organizational and spatial repertoires. This was an urban political event that reasserted the importance of urban spaces in expressing political dissent and experimented with new ways of being and acting in common. This article draws on Rancière's conceptualization of politics to read the squares movement as an opening of spaces of political subjectification. At the same time, through a close ethnography of the squares, it highlights the tensions that marked this process and focuses on two of these: the coexistence of nationalist and equalibertarian discursive and performative repertoires and the co-implication of horizontal and vertical organizational practices. The article builds on this analysis to argue that the squares movement opened hybrid spaces of political subjectification and to explore some of the tensions in Rancière's political writings. This reading, in turn, informs a discussion of the legacies of the squares movement