Heat-induced changes in the material properties of cytoplasm

Organisms are frequently exposed to fluctuating environmental conditions and might consequently experience stress. Environmental stress can damage cellular components, which can threaten especially single-celled organisms, such as yeast, as they cannot escape. To survive, cells mount protective stress responses, which serve to preserve cellular components and architecture. Recent findings in yeast show that the stress response upon energy depletion stress involves a gelation of the cytoplasm due to macromolecular protein assembly, characterized by drastic changes in cytoplasmic material properties. Remarkably, the stress-induced cytoplasmic gelation is protective, raising the question whether this could be a common strategy of cells to cope with severe stress. I hypothesized that protein aggregation induced by another common stress, severe heat shock, might cause a similar cytoplasmic gelation in yeast. Furthermore, I hypothesized that the reversibility of cytoplasmic gelation is provided by molecular chaperones, which are known regulators of protein aggregation. In this thesis, I therefore aimed to characterize the changes in the material properties of the cytoplasm upon severe heat shock as well as their underlying causes and how molecular chaperones affect these changes. To characterize heat-induced changes in the material properties of the cytoplasm, I monitored Schizosaccharomyces pombe cells during recovery from severe heat shock using a combination of cell mechanical assays, time-lapse microscopy and single-particle tracking. I found that the cells entered a prolonged growth arrested state upon stress, which coincided with significant cell stiffening and a long-range motion arrest of lipid droplets in the cytoplasm, while smaller cytoplasmic nanoparticles remained mostly mobile. At the same time, a significant fraction of proteins aggregated in the cytoplasm, forming insoluble inclusions such as heat shock granules. After stress cessation, the observed changes were reversed as stiffened cells softened and lipid droplets resumed long-range motion. Cell softening and lipid droplet motion recovery coincided with protein disaggregation. These processes could be delayed by impairing protein disaggregation through genetic perturbation of the molecular chaperone Hsp104, which functions as a protein disaggregase. In contrast, no influence on protein disaggregation or heat-induced cytoplasmic material property changes was detected for the small heat shock protein Hsp16. These results suggest that the cytoplasm gels upon severe heat shock due to protein aggregation and is refluidized during recovery with the help of Hsp104. Remarkably, cells resumed growth only after refluidization of the cytoplasm, suggesting that reversible cytoplasmic gelation may contribute to regulation of the heat-induced growth arrest. In addition, cytoplasmic gelation could potentially preserve cellular architecture during heat shock. Overall, the results from my thesis work indicate that reversible cytoplasmic gelation due to macromolecular protein assembly may be a universal cellular response to severe stress which is associated with a stress-protective growth arrest. A likely stress-specific part of this response is the chaperone-dependent refluidization of the cytoplasm, which might explain the prolonged growth arrest seen upon severe heat shock as compared to other stresses and might allow more time for the repair of heat-induced damage.:Abstract Zusammenfassung Table of contents Figure index List of abbreviations 1 Introduction 1.1 Heat shock affects cellular function and fitness 1.1.1 Cells respond to stress in phases 1.1.2 Heat shock threatens cellular homeostasis and structural integrity 1.1.3 Stress severity determines detrimental effects of heat shock 1.1.4 Heat stress causes protein aggregation 1.1.5 Heat shock granules are functional aggregates in yeast 1.2 The heat shock response protects cellular fitness 1.2.1 Cells change transcription to adapt to stress 1.2.2 Molecular chaperones are important in stress protection 1.2.3 Hsp104 is a protein disaggregase chaperone 1.2.4 Small heat shock proteins modulate protein aggregation 1.2.5 Stress severity determines modules of the heat shock response 1.3 Cytoplasmic material properties change during stress 1.3.1 Cells homeostatically adapt cytoplasmic material properties during stress 1.3.2 The cytoplasm is viscoelastic 1.3.3 Is the cytoplasm a gel? 1.3.4 Stress can induce cytoplasmic gelation 1.4 Research aims 2 Materials and Methods 2.1 S. pombe strains and growth conditions 2.1.1 Growth conditions 2.1.2 Construction of S. pombe strains 2.1.3 S. pombe transformation 2.1.4 S. pombe colony PCR 2.1.5 S. pombe strains used in this thesis 2.2 Plasmids and cloning 2.2.1 Plasmids used in this thesis 2.2.2 Construction of plasmid for fluorescent GEM nanoparticle expression 2.2.3 E. coli transformation 2.2.4 Plasmid purification from E. coli 2.3 S. pombe stress treatments 2.3.1 Heat shock treatment 2.3.2 Osmoadaptation 2.4 Cell biological methods 2.4.1 Viability assay 2.4.2 Growth assay 2.5 Cell bulk mechanical assays 2.5.1 Spheroplasting assay 2.5.2 Atomic force microscopy 2.5.3 Real-time deformability cytometry 2.5.4 RT-DC sample preparation 2.5.5 RT-DC setup and measurements 2.5.6 RT-DC data evaluation 2.6 Microscopy 2.6.1 Microscopy of GEM particles 2.6.2 Fluorescence microscopy of endogenously labeled Pabp-mCherry 2.6.3 Microscopy of µNS particles 2.7 Image analysis 2.7.1 Image analysis of Pabp-mCherry in vivo fluorescence microscopy 2.7.2 Differenced brightfield image analysis 2.7.3 Kymographs 2.8 Single-particle tracking analysis 2.8.1 Particle tracking 2.8.2 Mean squared displacement analysis 2.9 Optical diffraction tomography (ODT) 2.9.1 ODT sample preparation 2.9.2 ODT optical setup and measurements 2.9.3 ODT tomogram reconstruction and quantitative analysis 2.10 Lysis and sedimentation assay 2.10.1 Lysis buffer 2.10.2 S. pombe heat shock treatment and lysis 2.10.3 Sedimentation assay 2.10.4 Protein concentration measurement 2.10.5 SDS-PAGE 2.10.6 Coomassie staining 2.10.7 Western Blot 3 Results 3.1 Physical and chemical conditions affect heat shock survival and heat-induced growth arrest of S. pombe 3.1.1 S. pombe arrests growth during severe heat shock 3.1.2 Heat-induced growth arrest is dose-responsive 3.1.3 Heat-induced growth arrest depends on experimental conditions 3.1.4 Buffer pH and energy source have a strong impact on heat shock survival 3.1.5 Osmoadaptation protects cells during heat shock 3.2 Severe heat shock induces reversible cellular stiffening 3.2.1 Cellular rounding upon cell wall removal is delayed after heat shock 3.2.2 Elastic modulus of S. pombe cells is increased after heat shock 3.2.3 Recovery from heat-induced growth arrest is preceded by cell softening 3.3 Long-range particle dynamics in cytoplasm are abolished after heat shock 3.3.1 Small particle dynamics are largely independent of heat shock treatment 3.3.2 Lipid droplets are confined in space after heat shock 3.4 Cytoplasmic crowding increases during heat shock 3.5 Heat shock induces reversible protein aggregation 3.5.1 Insoluble protein fraction is increased after heat shock 3.5.2 Heat shock granules form reversibly during heat shock 3.5.3 HSG formation and dissolution are correlated with changes in cytoplasmic long-range dynamics 3.6 Molecular chaperones modulate cytoplasmic material property changes during heat stress recovery 3.6.1 Hsp104 but not Hsp16 is required for disaggregation of heat shock granules 3.6.2 Hsp104 but not Hsp16 is required for recovery from heat-induced growth arrest 3.6.3 Hsp104 but not Hsp16 is required for recovery of cytoplasmic long-range dynamics 3.6.4 Hsp104 but not Hsp16 is required for rapid reversal of cellular stiffening which coincides with growth recovery 4 Discussion 4.1 Summary and model 4.2 Which mechanism underlies cell stiffening upon heat shock? 4.2.1 Heat-induced protein aggregation might cause cell stiffening 4.2.2 Heat-induced protein aggregation might lead to cytoplasmic gelation 4.2.3 Many factors could contribute to protein aggregation and cytoplasmic gelation 4.3 The heat-induced growth arrest state is associated with reversible cytoplasmic gelation 4.3.1 Cytoplasmic material property changes mark the severe heat-induced growth arrest state 4.3.2 Is cytoplasmic gelation a common response to severe stress? 4.4 What are the biological consequences of cytoplasmic gelation? 4.4.1 Cytoplasmic gelation might obstruct processes that require motion of large structures 4.4.2 Is cytoplasmic gelation upon heat shock protective? 4.5 Heat shock recovery involves the chaperone-mediated refluidization of the cytoplasm 4.5.1 Cytoplasmic refluidization is required for growth recovery 4.5.2 Stress tolerance is marked by enhanced reversibility of cytoplasmic gelation 4.5.3 The protein disaggregase chaperone Hsp104 regulates the reversal of heat-induced cytoplasmic material property changes 4.6 Conclusion References Acknowledgements Publications and Contributions 5 Erklärung entsprechend §5.5 der Promotionsordnung

PROFINET– Zukünftige OT-Security- Anforderungen : Was fordern NIS2, CER, CRA und IEC 62443

Hersteller, Integratoren und Betreiber von Produktionsanlagen müssen sich zunehmend regulatorischen Anforderungen in Bezug auf die OT-Security stellen. Darüber hinaus sind Hersteller gut beraten, wenn sie einschlägigen OT-Security-Normen wie der IEC 62443 folgen. Aus den beschriebenen Anforderungen folgt, dass die „Security“ der industriellen Kommunikation über entsprechende Schutzmaßnahmen sicherzustellen ist. Dieser Beitrag zeigt, wie das Kommunikationsprotokoll PROFINET zurzeit ertüchtigt wird, um die Anforderungen zu erfüllen

Additive manufacturing of β-tricalcium phosphate components via Fused Deposition of Ceramics (FDC)

Das Paper beschreibt die Compoundierung bioaktiver Keramik (Beta-Tricalciumphosphat) in einer organischen Matrix, die anschließende Extrusion zu Filamenten, die für den FDM-3D-Druck geeignet sind, sowie die Formgebund zu Scaffolds mittels additiver Fertigung. Weiterhin werden fertigungsprozessbegleitende Untersuchungen zur Entbinderung und Sinterung durchgeführt

Exploring adaptive behavior of non-linear hexagonal frameworks

© Copyright © 2020 O'Donnell, Towes, Groh and Chenchiah. Non-linear structural responses offer a rich design space that can be integrated into the development of novel (meta)-materials to enable transformative capabilities. By exploiting robust, repeatable, non-linear elasticity the (meta)-material's performance can be tailored to significantly exceed what has been demonstrated through traditional linear design paradigms. Embracing geometric non-linearity offers the designer the potential for truly bespoke large-range elastic behavior. Herein we explore the behavior of bistable elements that can be arranged into a space-filling triangular lattice, constructed in a hierarchical manner. We develop an analysis of the system that can be readily extended to more complex hierarchies, such as the hexagonal arrangement considered herein, whilst retaining physical insight. Specifically, we present the geometric restrictions of lattice elements that govern the existence of stable stress-free states and subsequently characterize the transitions between such states by searching for the minimum energetic transition pathway. Through our approach, we explore how hierarchical multi-stable systems can be analyzed, thus contributing to the development of truly bespoke adaptive (meta-)materials

Intracellular Mass Density Increase Is Accompanying but Not Sufficient for Stiffening and Growth Arrest of Yeast Cells

Many organisms, including yeast cells, bacteria, nematodes, and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography (ODT) and atomic force microscopy (AFM). We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of a solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms

Happy Catastrophe: Recent Progress in Analysis and Exploitation of Elastic Instability

A synthesis of recent progress is presented on a topic that lies at the heart of both structural engineering and nonlinear science. The emphasis is on thin elastic structures that lose stability subcritically — without a nearby stable post-buckled state — a canonical example being a uniformly axially-loaded cylindrical shell. Such structures are hard to design and certify because imperfections or shocks trigger buckling at loads well below the threshold of linear stability. A resurgence of interest in structural instability phenomena suggests practical stability assessment require stochastic approaches and imperfection maps. This article surveys a different philosophy; the buckling process and ultimate post-buckled state are well described by the perfect problem. The significance of the Maxwell load is emphasised, where energy of the unbuckled and fully developed buckle patterns are equal, as is the energetic preference of localised states, stable and unstable versions of which connect in a snaking load-deflection path. The state of the art is presented on analytical, numerical and experimental methods. Pseudo15 arclength continuation (path-following) of a finite-element approximation computes families of complex localised states. Numerical implementation of a mountain-pass energy method then predicts the energy barrier through which the buckling process occurs. Recent developments also indicate how such procedures can be replicated experimentally; unstable states being accessed by careful control of constraints, and stability margins assessed by shock sensitivity experiments. Finally, the fact that subcritical instabilities can be robust, not being undone by reversal of the loading path, opens up potential for technological exploitation. Several examples at different length scales are discussed; a cable-stayed prestressed column, two examples of adaptive structures inspired by morphing aeroelastic surfaces, and a model for a functional auxetic material

Influence of tiapride versus lormetazepam and placebo on subjective sleep perception, polysomnographic parameters and cognitive functions in sleep-disturbed elderly individuals

Ein gesunder Schlaf ist für die volle körperliche und psychische Leistungsfähigkeit des Menschen notwendig. Auch bei älteren Menschen treten jedoch häufig Störungen des Schlafs auf. Diese können in Form von primären oder organischen Insomnien oder als Symptome bei psychischen oder körperlichen Erkrankungen vorkommen. Zur Behandlung von Schlafstörungen stehen neben der Therapie der Grunderkrankung, die bei sekundären Schlafstörungen primär erfolgen sollte, und verhaltenstherapeutischen Methoden verschiedene Medikamente zur Verfügung. Die Einsatzmöglichkeiten dieser Medikamente sind jedoch gerade bei älteren Patienten durch ihre unerwünschten Wirkungen, vor allem auf vegetative und kognitive Funktionen, eingeschränkt. Als alternatives Schlafmedikament für ältere Menschen wurde wegen der fehlenden anticholinergen Wirkung und da keine negativen Effekte auf die kognitive Leistungsfähigkeit bekannt sind, das Benzamid-Derivat Tiaprid in einer Pilotstudie untersucht. Es zeigte dabei eine mit Lormetazepam vergleichbare Wirkung auf das subjektive Schlafempfinden. Ziel der vorliegenden Studie war es daher, diese Ergebnisse polysomnographisch abzusichern und mögliche unerwünschte Wirkungen zu erfassen. Als Vergleichssubstanz diente erneut Lormetazepam, ein seit langem gegen Schlafstörungen eingesetztes Benzodiazepin. Es wurden 29 gerontopsychiatrische Patienten der Klinik für Psychiatrie und Psychotherapie der Universitätsklinik Gießen im Schlaflabor der Klinik in einer Doppelblindstudie untersucht. Die Patienten erhielten in randomisierter Reihenfolge Tiaprid (150 oder 300 mg), Lormetazepam (0,5 oder 1 mg) und Plazebo. Für jede Bedingung wurden zu je einer Nacht polysomnographische und subjektive Schlafdaten erhoben, sowie Befragungen bezüglich der subjektiven Befindlichkeit durchgeführt. Die kognitive Leistungsfähigkeit wurde mittels Reaktionszeitaufgaben und einer Labyrinth-Lernaufgabe untersucht. Weder in den subjektiven noch in den objektiven, polysomnographisch gemessenen Schlafparametern zeigte sich ein positiver Effekt des Tiaprid als Schlafmedikament, während sich die erwarteten Effekte des Lormetazepam mit einer Verbesserung der Schlafzeit, Schlafeffizienz, dem Anteil and Stadium zwei an der Schlafperiode und einer Verringerung der Wachzeit, Vermehrung der Bewegungszeit und Reduktion der REM-Dichte nachweisen ließen. Es fanden sich keine Auswirkungen beider Medikamente auf die allgemeine Befindlichkeit oder basale Kreislaufparameter. Die kognitive Leistungsfähigkeit war unter Lormetazepam in Bereich des Arbeitsgedächtnisses vermindert. Unerwarteterweise fand sich auch für Tiaprid eine Störung kognitiver Funktionen, die wahrscheinlich auf eine reduzierte Konsolidierung von Gedächtnisinhalten im Schlaf zurückzuführen war.Da die Ergebnisse der Schlafuntersuchungen in deutlichem Gegensatz zu den in der Pilotstudie gefundenen Tiaprid-Wirkungen standen, wurden verschiedene methodische Faktoren der vorliegenden Studie als mögliche Einflussfaktoren untersucht: weder durch geringe Störungen in der Ausbalancierung der Messreihenfolge des Cross-over-Designs, noch durch die Inhomogenität der Stichprobe konnte die fehlende Wirkung des Tiaprid erklärt werden. Eine Wirksamkeit in einer höheren Dosis von mindestens 300 mg konnte durch die Ergebnisse der Studie jedoch nicht ausgeschlossen werden. Als Erklärung für die Diskrepanz zu den Ergebnissen der Pilotstudie muss schließlich auch eine Abweichung vom strengen Cross-over-Design in der Pilotstudie in Betracht gezogen werden. Einen unerwarteten Befund erbrachte die Untersuchung der Korrelation subjektiver und objektiver Schlafparameter, die unter Tiaprid deutlich besser war, was für eine genauere Wahrnehmung des eigenen Schlafs unter diesem Medikament spricht. Ob sich hieraus eine klinische Anwendbarkeit, zum Beispiel zur Behandlung der Fehlwahrnehmung des Schlafzustandes, ableiten ließe, könnte Gegenstand weiterer Studien sein.Healthy sleep is essential for maintaining full mental and physical performance. Sleep disorders are, however, quite frequent also in the elderly population. They occur as primary insomnia, organic sleep disorders or as a symptom of psychiatric or somatic diseases. Possible therapeutic interventions include the treatment of the underlying disorder in the case of secondary insomnias, behavioural therapy and various drugs. The applicability of drugs is limited especially in the elderly population due to unwanted side effects on vegetative and cognitive functioning. Because of its lack of anticholinergic properties and because no negative side effects on cognitive functioning have been shown, tiapride, a benzamide derivative, was tested in a pilot study for sleep inducing properties. It could be shown that tiapride had positive effects on subjective sleep quality similar to lormetazepam. The aim of the present study was to add polysomnographic evidence to these findings and to detect possible side effects. Lormetazepam, a benzodiazepine that has long been used to treat insomnia, was again utilised as a comparison substance. Twenty-nine gerontopsychiatric patients of the Psychiatric Clinic of the University Hospital of Giessen were enrolled in a double-blind study in the sleep laboratory of the clinic. They were given tiapride (150 300 mg), lormetazepam (0.5 1 mg) or placebo in randomized sequence. Under each condition, polysomnographic and subjective sleep parameters were measured, and questionnaires on subjective well-being were completed. Cognitive functioning was measured using reaction time tasks and a maze learning task.There was no positive effect of tiapride on subjective and on objective, polysomnographic sleep parameters of the subjects. Lormetazepam, however, showed the expected effects: an increase in total sleep time, sleep efficiency, percentage of stage 2 sleep and movement time and a decrease of wake time and REM density. Both drugs had no effects on subjective well-being and basal cardiovascular parameters. Cognitive functioning in the domain of working memory was impaired under lormetazepam. Unexpectedly, there was also an impairment of cognitive functioning under tiapride, most likely in the area of memory consolidation during sleep. Because the results concerning sleep parameters were contrary to the effects of tiapride found in the pilot study, several methodological factors of the present study were examined as possible confounds: neither the slight imbalance of the sequence in which the substances were administered in the cross-over design, nor the inhomogeneity of the subject sample could explain the missing effects of tiapride as a sleep medication. An efficacy of higher doses of at least 300 mg, however, could not be ruled out by the results of the study. Finally, a methodological deviation from a strict cross-over-design in the pilot study must be taken into account as a possible explanation for the discrepancy in findings. There was an unexpected finding concerning the correlation between subjective and objective sleep parameters, which was notably stronger under tiapride indicating a more accurate perception of one s sleep under this medication. It could be the subject of future studies to examine the clinical relevance of this finding, for example as a treatment for sleep state misperception