Uncovering the molecular basis of compartmentalization as a principle of neuronal organization

Cells are faced with coordinating countless, simultaneous, partly antagonistic biochemical reactions. This is especially true for neurons that must orchestrate the complex task of neurotransmission. One solution to this problem is the formation of specialized compartments. To understand the molecular mechanisms of such compartments this thesis investigates two system in neuronal cells: i. the plasma membrane and its underlying cytoskeleton and ii. synaptic vesicle clusters inside synaptic boutons. Towards this end, a combinatorial approach of computational modeling, single particle tracking and super-resolution microscopy is employed. A periodic array of actin rings in the neuronal axon initial segment has been known to confine membrane protein motion. Still, a local enrichment of ion channels offers an alternative explanation. Using computational modeling this thesis now shows that ion channels, in contrast to actin rings, cannot mediate confinement. Furthermore, by employing single particle tracking and super-resolution microscopy, this work shows that actin rings are close to the plasma membrane and that actin rings confine membrane proteins in several neuronal cell types. Further, it is shown that actin ring disruption leads to a reduction of membrane compartmentalization. Synaptic boutons in the axon of neurons are the location of synaptic vesicle release. Synaptic vesicles form dense clusters inside boutons, that are essential for pre-synaptic function. In vitro experiments have suggested that the soluble phosphoprotein synapsin 1 controls synaptic clustering via liquid liquid phase separation. However, the in vivo mechanism remains elusive. This work now shows via two-color single molecule tracking in live neurons that synapsin 1 drives synaptic vesicle clustering. Furthermore, using a synapsin knock-out model it is shown that synapsin 1 controls the mobility of synaptic vesicles through its intrinsically disordered region, which is responsible for phase separation. By studying the dynamics of compartmentalized systems in neuronal cells this work uncovers two molecular mechanisms: actin rings form membrane diffusion barriers and synapsin 1 controls synaptic vesicle clustering and mobility through liquid liquid phase separation. Thus, this thesis makes important strides towards deepening the understanding of neuronal function by uncovering how compartmentalization operates in both the plasma membrane and the cytosol of neuronal cells.Zellen müssen unzählige, gleichzeitige, teilweise gegensätzliche biochemische Reaktionen koordinieren. Dies gilt insbesondere für Neuronen, die die komplexe Aufgabe der Neurotransmission koordinieren. Eine Lösung für dieses Problem ist die Bildung spezialisierter Kompartimente. Um die molekulare Funktionsweise solcher Kompartimente zu verstehen, werden in dieser Arbeit zwei Systeme in neuronalen Zellen untersucht: i. die Plasmamembran und das darunterliegende Zytoskelett und ii. synaptische Vesikel-Cluster in Boutons. Zu diesem Zweck, wurde ein kombinatorischer Ansatz aus Computermodellierung, Einzelpartikelverfolgung und superauflösender Mikroskopie verwendet. Periodische Aktinringe im neuronalen Axoninitialsegment schränken die Bewegung von Membranproteinen ein. Jedoch liefert eine lokale Anreicherung von Ionenkanälen eine alternative Erklärung. Durch Computermodellierung wird in dieser Arbeit nun gezeigt, dass Ionenkanäle keine Einschränkung der Membranmolekülbewegung bewirken. Darüber hinaus wird durch Einzelpartikelverfolgung und superauflösende Mikroskopie gezeigt, dass Aktinringe nahe der Plasmamembran sind und dass Aktinringe Membranproteine in verschiedenen neuronalen Zelltypen in ihrer Bewegung einschränken. Weiterhin wird gezeigt, dass die Zerstörung der Aktinringe Membrankompartmentalisierung reduziert. Synaptische Boutons im Axon sind der Ort der Freisetzung synaptischer Vesikel. Synaptische Vesikel bilden dichte Cluster in Boutons, welche für die Funktion der Präsynapse essenziell sind. In vitro Experimente haben gezeigt, dass das lösliche Phosphoprotein Synapsin 1 das Clustern durch Flüssig-Flüssig-Phasentrennung steuert, der Mechanismus in vivo ist jedoch unklar. Diese Arbeit zeigt nun mittels Zweifarben-Einzelmolekülverfolgung in lebenden Neuronen, dass Synapsin 1 das Clustern synaptischer Vesikel steuert. Anhand eines Synapsin-Knock-out-Modells wird gezeigt, dass Synapsin 1 die Mobilität synaptischer Vesikel durch seine intrinsisch ungeordnete Region kontrolliert, die für die Phasentrennung verantwortlich ist. Durch Untersuchungen der Dynamik kompartmentalisierter Systeme in neuronalen Zellen deckt diese Arbeit zwei molekulare Mechanismen auf: Aktinringe bilden Membrandiffusionsbarrieren und Synapsin 1 steuert Clustern und Mobilität synaptischer Vesikel durch Flüssig-Flüssig-Phasentrennung. Somit macht diese Arbeit wichtige Fortschritte zum Verständnis der Funktionsweise neuronaler Zellen, indem sie aufdeckt, wie die Kompartmentalisierung der Plasmamembran und des Zytosols gesteuert wird

Automated Correlative Light and Electron Microscopy using FIB-SEM as a tool to screen for ultrastructural phenotypes

In Correlative Light and Electron Microscopy (CLEM), two imaging modalities are combined to take advantage of the localization capabilities of light microscopy (LM) to guide the capture of high-resolution details in the electron microscope (EM). However, traditional approaches have proven to be very laborious, thus yielding a too low throughput for quantitative or exploratory studies of populations. Recently, in the electron microscopy field, FIB-SEM (Focused Ion Beam -Scanning Electron Microscope) tomography has emerged as a flexible method that enables semi-automated 3D volume acquisitions. During my thesis, I developed CLEMSite, a tool that takes advantage of the semi-automation and scanning capabilities of the FIB-SEM to automatically acquire volumes of adherent cultured cells. CLEMSite is a combination of computer vision and machine learning applications with a library for controlling the microscope ( product from a collaboration with Carl Zeiss GmbH and Fibics Inc.). Thanks to this, the microscope was able to automatically track, find and acquire cell regions previously identified in the light microscope. More specifically, two main modules were implemented. First, a correlation module was designed to detect and record reference points from a grid pattern present on the culture substrate in both modalities (LM and EM). Second, I designed a module that retrieves the regions of interest in the FIB-SEM and that drives the acquisition of image stacks between different targets in an unattended fashion. The automated CLEM approach is demonstrated on a project where 3D EM volumes are examined upon multiple siRNA treatments for knocking down genes involved in the morphogenesis of the Golgi apparatus. Additionally, the power of CLEM approaches using FIB-SEM is demonstrated with the detailed structural analysis of two events: the breakage of the nuclear envelope within constricted cells and an intriguing catastrophic DNA Damage Response in binucleated cells. Our results demonstrate that executing high throughput volume acquisition in electron microscopy is possible and that EM can provide incredible insights to guide new biological discoveries

Data Acquisition Applications

Data acquisition systems have numerous applications. This book has a total of 13 chapters and is divided into three sections: Industrial applications, Medical applications and Scientific experiments. The chapters are written by experts from around the world, while the targeted audience for this book includes professionals who are designers or researchers in the field of data acquisition systems. Faculty members and graduate students could also benefit from the book

Glaucoma

This book addresses the basic and clinical science of glaucomas, a group of diseases that affect the optic nerve and visual fields and is usually accompanied by increased intraocular pressure. The book incorporates the latest development as well as future perspectives in glaucoma, since it has expedited publication. It is aimed for specialists in glaucoma, researchers, general ophthalmologists and trainees to increase knowledge and encourage further progress in understanding and managing these complicated diseases

Numerical modelling of additive manufacturing process for stainless steel tension testing samples

Nowadays additive manufacturing (AM) technologies including 3D printing grow rapidly and they are expected to replace conventional subtractive manufacturing technologies to some extents. During a selective laser melting (SLM) process as one of popular AM technologies for metals, large amount of heats is required to melt metal powders, and this leads to distortions and/or shrinkages of additively manufactured parts. It is useful to predict the 3D printed parts to control unwanted distortions and shrinkages before their 3D printing. This study develops a two-phase numerical modelling and simulation process of AM process for 17-4PH stainless steel and it considers the importance of post-processing and the need for calibration to achieve a high-quality printing at the end. By using this proposed AM modelling and simulation process, optimal process parameters, material properties, and topology can be obtained to ensure a part 3D printed successfully

Applications and Experiences of Quality Control

The rich palette of topics set out in this book provides a sufficiently broad overview of the developments in the field of quality control. By providing detailed information on various aspects of quality control, this book can serve as a basis for starting interdisciplinary cooperation, which has increasingly become an integral part of scientific and applied research

Book of Abstracts 15th International Symposium on Computer Methods in Biomechanics and Biomedical Engineering and 3rd Conference on Imaging and Visualization

In this edition, the two events will run together as a single conference, highlighting the strong connection with the Taylor & Francis journals: Computer Methods in Biomechanics and Biomedical Engineering (John Middleton and Christopher Jacobs, Eds.) and Computer Methods in Biomechanics and Biomedical Engineering: Imaging and Visualization (JoãoManuel R.S. Tavares, Ed.). The conference has become a major international meeting on computational biomechanics, imaging andvisualization. In this edition, the main program includes 212 presentations. In addition, sixteen renowned researchers will give plenary keynotes, addressing current challenges in computational biomechanics and biomedical imaging. In Lisbon, for the first time, a session dedicated to award the winner of the Best Paper in CMBBE Journal will take place. We believe that CMBBE2018 will have a strong impact on the development of computational biomechanics and biomedical imaging and visualization, identifying emerging areas of research and promoting the collaboration and networking between participants. This impact is evidenced through the well-known research groups, commercial companies and scientific organizations, who continue to support and sponsor the CMBBE meeting series. In fact, the conference is enriched with five workshops on specific scientific topics and commercial software.info:eu-repo/semantics/draf

Overhearing: An Attuning Approach to Noise in Danish Hospitals

Denmark is building new and improved super hospitals, based on a vision of improving overall quality by switching the focus from hospitals for treatment to hospitals for healing, guided by research in the field of evidence-based design and healing architecture. Users mention noise as one of the main stressors and research has discovered that noise levels in hospitals continue to rise. Noise has therefore become a central point of concern, recommending strategies to reduce measurable and perceived noise levels.However, these strategies do not support the need to feel like an integral part of the shared hospital environment, which is also a key element in creating healing environments linked to a reductionist framework underlying the field. This framework regards broad concepts such as noise and silence as objects with quantifiable properties, and assumes that these properties can be understood independently of the perceiver as a bodily and situated subject. The aim of this dissertation is accordingly to develop an alternative framework capable of accommodating the multi-sensory, affective and atmospheric conditions that influence the experience of noise, with a view to complementing the existing approaches in the field.  Consequently, the dissertation develops an ecological framework capable of accommodating these issues, established by viewing sound and listening through the lens of atmospheres. The attuning approach highlights the reciprocal relationship between the way in which atmospheres condition shared rhythms that shape us, but also the way in which we can tune them in different ways. In the context of sound and listening, this creates the potential of ecological overhearing as an atmospheric mode of listening capable of reconfiguring habitual background and foregrounding relationships. Attuning strategies should thus provide opportunities for diverse acoustic situations and possibilities for active choice-making to meet different and shifting needs through an enactive approach in order to enhance empowerment and ecological overhearing. Embedding diverse enactive sound installations and interactive sound technology in hospitals can facilitate such zones of overhearing. These zones become places for ruptures that strengthen the possibilities for engaging in counter-attunements of existing negative atmospheres. In this way, zones of overhearing not only provide continual sense of presence without demanding full attention, but also create ample opportunities for the restoration of  attention.The dissertation takes an experimental practice-based approach through artistic- and constructive design-research and comprises six peer-reviewed papers (Part IV), framed by a general overview article (Parts I-III) that develops the theoretical and methodological foundation for the papers, and provides a synthesis and discussion of their main findings. The practice-based work is founded on a range of experiments, but focuses on two main experiments: Light, Landscape & Voices and KidKit, and the way in which they elicit sensitivities within the topic of investigation. This contribution also concerns the concrete development of installations through the experiments. These installations are in themselves manifestations of and challenges to hypotheses about the topic I aim to address.