Research

Research context / Theoretical framework Controlling and understanding adhesion of cells on artificial surfaces remains as a critical topic in materials and life sciences. In this regard, combination of top-down (contact printing) and bottom-up approaches (ATRP polymerization + layer-by-layer adsorption of polyelectrolytes and proteins) appears as a promising strategy for the design and fabrication of cell-appealing interfaces. Interestingly, this methodology allows going from 2D to 3D-like hierarchical structures of hybrid content (niches) that influence a subsequent cell attachment on top, by better exposing the specific binding sites (RGD, IKVAV moieties) towards target membrane receptors (i.e. integrins, CD44). Complementary use of Atomic Force Microscopy (AFM), with a living cell as probe, together with Quartz Crystal Microbalance with Dissipation (QCM-D), will enable an early-stage analysis and quantification of these cell-substrate interactions on the nanoscale. Hypotheses/ Research questions / Objectives The main hypotheses of the project are the following: i) Combination of substrate-anchored polymer brushes and layer-by-layer deposited polyelectrolyte chains give rise to soft 3D niches for the enhanced adsorption of ECM proteins. The transformation of 2D interfaces into 3D-like architectures will, in turn, enhance cell attachment and proliferation of cells, with particular impact on both cell morphology and the number of cell-substrate connections formed; ii) The use of Contact-Printing techniques before the grafting-from of the brushes allows the fabrication of localized individual 3D attachment points. The localized presence of specific molecules will influence the cell-substrate affinity with final impact on cell morphology and the establishment of a different number of cell-surface contacts; iii) Single-Cell Probe Force Spectroscopy (SCPFS) technique is sensitive enough to identify early stage attachment events in cell-substrate contacts. The use of a living cell acting as indenting probe will determine events taking place on the nano- and microscale. Approach / Methods The following methods will be used to study substrate preparation and cell adhesive behaviour: Atomic force microscopy (AFM) in SCPFS mode, (confocal) fluorescence microscopy, quartz crystal microbalance with dissipation (QCMD), scanning electron microscopy (SEM), and cell culture protocols.

The impact of heat on plants is intrinsically tied to plant water use. Heatwaves have more than tripled, warmed by 2.3K and usually combine with drought. Under heat, stomata act at the leeway between cooling and critical water loss. Once they close, cooling ceases, leaf overheating boosts transpiration and water loss across cuticles becomes decisive. Unfortunately, permeance of cuticles rises exponentially with heat. Little is known about the structural basis of changes, whether increased cuticular transpiration is reversible and acclimation is possible. Also the tolerance to various heat doses and effect on cuticular transpiration is largely unknown. To close these knowledge gaps plants in temperate alpine vs. tropical habitats are compared, experiencing different heat doses (intensity x duration). Thereby the following questions will be addressed: What are the habitat-specific heat doses, leaf to air temperature differences and vapor pressure deficits? Does heat dose affect heat survival? Which heat dose, vapor pressure deficits and plant water potential closes stomata? How does cuticular transpiration respond to various heat doses and vapor pressure deficits? Are heat-induced changes of cuticular transpiration reversible? Does heat exposure alter cuticle structure? Does excess water loss explain heat damage? So the project explores the reversibility and acclimation potential of heat treshold of cuticular transpirationt by different approaches. To get insights into the cuticle of plants from cold vs. hot habitats RAMAN microscopy and transmission electron microscopy will be applied. Reaching from laboratory to the field and from molecules to individuals, the study promises comprehensively new insights into heat survival of plants. Results will be important to assess the future heat risk to plants in a globally warmer world.

Freezing events significantly impair plant life. Biophysical aspects of freezing are less studied than molecular responses but are fundamental to the understanding of freezing resistance. During harmless freezing of plant tissues ice accumulates extracellularly and cells usually freeze dehydrate. How ice growth is controlled and how cellular water is segregated to the ice is not understood. As plants consist in large part of water, the amount of ice formed must be considerable. While reduction of water content is part of cold acclimation, spring and alpine plants survive freezing with high water content. Hardly anything is known about how the growing ice masses are managed. There is, however, recent evidence that ice accumulates in predetermined spaces. Some of them seem to pre-exist, others are formed by tissue rupture. We hypothesize that 1) The spatial confinement of ice masses must be managed by targeted ice segregation at specific loci. 2) Ice masses show a temperature dependent growth and 3) ice mass growth is regulated by ice affecting molecules that locally promote or inhibit ice mass formation, facilitate targeted ice segregation and affect ice crystal morphology. With a new set of new and innovative methods we tackle the biophysical and chemical aspects of ice growth in plant tissue: (1) A new cryo-microscope using reflected-polarised-light (CMrpl) allows an unambiguous and rapid visualisation of ice masses and the analysis of ice crystal shape and their attachment to cell walls. (2) Recent results obtained with a new calorimeter (µDSC 7 Evo) uncover till now invisible very slight freezing processes in leaves. 3) By GC-MS molecular components of ice crystals and by RAMAN microscopy also molecular components of cell walls and cell lumina close to ice will be revealed.