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Etude des altérations morphologiques et biochimiques des érythrocytes au cours du sepsis / Studies of the alterations of shape and biochemistry of erythrocytes during sepsis.Piagnerelli, Michaël 05 November 2009 (has links)
La microcirculation est rapidement altérée dans le sepsis et la persistance de ces altérations est associée à un mauvais pronostic. La microcirculation est composée de vaisseaux invisibles à l’œil (< 100 µm), de l’endothélium, du glycocalyx, des cellules musculaires lisses et des éléments sanguins dont les GR. <p>De nombreuses études animales et humaines ont rapporté des altérations rhéologiques des GR dans le sepsis. Ces modifications comprennent une diminution de la déformabilité, une augmentation de l’agrégation et de l’adhérence globulaire. <p>De plus, l’altération de la déformabilité peut induire des altérations du flux microcirculatoire dans des modèles expérimentaux animaux. Ces mêmes altérations rhéologiques sont rapportées dans le diabète. Dans cette pathologie, les GR présentent une diminution du contenu membranaire en AS, comme dans les processus de sénescence. <p>La déformabilité des GR dépend des caractéristiques cellulaires incluant surtout les propriétés de la membrane, la géométrie cellulaire et dans une moindre mesure la viscosité cellulaire. Malgré la connaissance des altérations de la rhéologie dans le sepsis, peu de travaux, au contraire du diabète, s’interessent aux modifications de la membrane.<p>Nous avons étudié, par analogie aux altérations globulaires rapportées dans le diabète, la membrane des GR de patients admis en soins intensifs pour un sepsis, et comparé à des GR de patients non septiques et de volontaires sains. Le contenu membranaire en AS était significativement diminué chez les patients septiques par rapport aux patients non-septiques et aux volontaires sains. De plus, les GR des patients septiques, analysés par une technique de cytométrie en flux indépendante de la température de l’échantillon, étaient rapidement plus sphériques (dans les 24 heures du sepsis) et incapables de modifier leurs formes en hypoosmolalité. Cette technique de cytométrie a par ailleurs aussi été utilisée pour l’analyse de GR de patients diabétiques et en insuffisance rénale terminale. <p> La diminution du contenu en AS est aussi rapidement observée sur la transferrine, suggérant une augmentation de la concentration et/ou de l’activité de la neuraminidase, enzyme clivant l’AS. Dans un modèle de choc septique induit chez l’ovin, nous avons confirmé la rapidité de ce phénomène. En effet, la concentration en AS libre augmente dès la 15ième heure après induction du sepsis.<p>In-vitro, nous avons pu reproduire les modifications de forme des GR observés chez les patients septiques par incubation de GR de volontaire avec de la neuraminidase, et ce en 10 heures, quelles que soient les concentrations utilisées. Ces modifications de forme et de membrane s’accompagnent d’une augmentation significative du contenu en lactate, suggérant une stimulation de la glycolyse érythrocytaire et en 2,3-DPG, facilitant la libération de l’O2 de l’Hb vers les tissus. <p>Toutes ces modifications touchant la membrane des GR des patients de soins intensifs, surtout septiques, peuvent être responsables des altérations de rhéologie que nous avons observé grâce au LORCA sur une large population admis aux soins intensifs.<p>Une meilleure compréhension des mécanismes conduisant aux altérations rhéologiques des GR dans le sepsis, et ses effets potentiellement déletères sur la microcirculation, sont nécessaires avant d’envisager les GR comme cible thérapeutique.<p><p><p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished
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Heat-induced changes in the material properties of cytoplasmEßlinger, Anne Hilke 26 June 2023 (has links)
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
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