O trabalho tem como objetivo correlacionar as propriedades mecânicas de blocos vazados com as do concreto que o constitui. Moldam-se blocos vazados e corpos-de-prova de dimensões distintas com concreto plástico em três níveis de resistência (10, 20 e 30 MPa) e caracterizam-se as propriedades mecânicas por meio de ensaios à compressão axial e à tração. Ensaiam-se à compressão axial prismas constituídos por dois e três blocos de concreto sem junta de argamassa, unidos por adesivo à base de epóxi. Observa-se que a relação entre as resistências do bloco e prisma e a resistência do concreto diminui com o aumento da altura desses elementos. Obtém-se o módulo de elasticidade longitudinal do concreto, a partir de ensaios com corpos-de-prova. Analisam-se as deformações em diversos pontos do bloco quando submetidos à compressão axial. Devido à distribuição não uniforme de tensões nos blocos e prismas, obtêm-se diferentes valores de deformação ao longo das paredes dos elementos. Os blocos, por possuírem menor altura que os prismas, apresentam maior diferença entre os valores de deformação. Por meio de simulações numéricas, no regime linear, observa-se que a placa de ensaio não se desloca uniformemente, acarretando os distintos valores de deformação ao longo do bloco. Uma forma peculiar de cálculo, baseada nessas deformações, permite a previsão da capacidade resistente do bloco e conduz a valores próximos da força máxima de ensaio. Apresenta-se ainda, uma tentativa de prever a deformabilidade do bloco vazado de concreto a partir das propriedades mecânicas do concreto. / This research aims to correlate the mechanical properties of hollow blocks to those of the concrete used to build them. Concrete hollow blocks and test samples of different shapes and sizes (cylinder 50 x 100, 100 x 200 and 150 x 300 mm) were molded with plastic consistency concrete in three different levels of strength (nominally 10, 20 and 30 MPa). Their mechanical properties and structural behavior in axial compression and tension tests were determined. Also two and three block prisms were built without mortar joints, pilled up with epoxy glue. It could be observed that the relationship between block strength or prism strength and concrete strength (measured in test samples) decreases with increasing of the element\'s height. Stress and strain analyses were made based on the concrete\'s modulus of elasticity obtained in sample tests and on the measured strain in the blocks\' faces and webs. By linear numerical simulations it was observed that loading plate does not show uniform displacement values, causing significant differences between theoretical and experimental behavior. A peculiar stress-strain analysis permitted to calculate the block load capacity based in its deformations. Also a tentative method to preview the block deformability from the concrete mechanical properties is described and tested.

Nous avons étudié les marqueurs hémorhéologiques, hématologiques et biochimiques chez des sujets drépanocytaires homozygotes SS (HbS/HbS) et hétérozygotes composites SC (HbS/HbC) dans deux cohortes, pédiatriques et adultes, de patients drépanocytaires, et ce, à travers 7 complications récurrentes de la drépanocytose : 2 appartenant au profil hémolytique (l’ulcère de jambes et la glomérulopathie) et 5 appartenant au phénotype visqueux/vaso-occlusif (l’hypertension artérielle, le syndrome thoracique aigu (STA), la crise vaso-occlusive (CVO), la rétinopathie et l’ostéonécrose). Nous avons montré que : 1) une viscosité sanguine et une déformabilité érythrocytaire élevées sont des facteurs de risques de CVO chez les enfants homozygotes ; 2) Une viscosité sanguine élevée est associée à une hypertension artérielle systémique relative chez des adultes SS ; 3) les enfants SC présente une fonction vasculaire mieux préservée que les enfants SS pour faire face à une augmentation de la viscosité sanguine ; 4) les patients adultes SS avec une ostéonécrose présentent une déformabilité érythrocytaire plus élevée que les patients sans ostéonécrose ; 5) une viscosité sanguine élevée est associée à la présence d’une rétinopathie chez les adultes SC mais pas chez les SS ; 6) les patients adultes SS présentant une glomérulopathie ont un taux d’hémolyse élevé, une déformabilité érythrocytaire réduite et des agrégats érythrocytaires très robustes ; 7) les patients adultes SS avec des ulcères de jambes récurrents ont un taux d’hémolyse accru et une déformabilité érythrocytaire réduite. De plus, nos travaux confirment que l’-thalassémie module les propriétés de déformabilité érythrocytaire, mais montrent pour la première fois qu’elle module aussi les propriétés d’agrégation érythrocytaire, et notamment la force des agrégats érythrocytaires. En conclusion, ces travaux permettent de préciser le rôle de la rhéologie sanguine dans un certain nombre de complications de la drépanocytose et d’enrichir le modèle préexistant divisant les complications de la drépanocytose selon 2 phénotypes : hémolytique versus visqueux/vaso-occlusif. Nous montrons pour la première fois que le phénotype hémolytique est caractérisé aussi par des anomalies de la rhéologie du globule rouge : rigidité accrue et agrégats érythrocytaire robustes. / Hemorheological, hemathological, and biochemical marquers of patients with sickle cell anemia (SS) and patients with sickle cell SC disease (SC) were studied in 2 cohorts: children and adults. We focused on 7 recurrent complications: 5 belonging to the viscosity/vaso-occlusion phenotype (systemic hypertension, acute chest syndrome (ACS), vaso-occlusive crisis (VOC), retinopathy and osteonecrosis) and 2 belonging to the hemolytic phenotype (leg ulcer and glomerulopathy). Our results show that 1) high viscosity is associated with increased risk for VOC in SS children; 2) blood viscosity is increased in SS adults with systemic relative hypertension; 3) SC children have preserved vascular function compared to SS children; 4) SS adults with osteonecrosis are characterized by higher red blood cell (RBC) deformability than SS adults without osteonecrosis; 5) high blood viscosity is associated with retinopathy in SC adults but not in SS adults; 6) SS adults affected by glomerulopathy have high hemolytic rate, low RBC deformability and increased RBC aggregates strenght; 7) SS adults with recurrent leg ulcers have high hemolytic rate and reduced RBC deformability. Moreover, our studies shows that alpha-thalassemia modulate RBC deformability and RBC aggregation properties. In conclusion, this work shows for the first time that the hemolytic phenotype is characterized by an abnormal RBC rheology which may play a role in several sickle cell complications.

Mechanical properties of cells and their environment have an undeniable impact on physiological and pathological processes such as tissue development or cancer metastasis. Hence, there is a pressing need for establishing and validating methodologies for measuring the mechanical properties of cells, as well as for deciphering the molecular underpinnings that govern the mechanical phenotype. During my doctoral research, I addressed these needs by pushing the boundaries of the field of single-cell mechanics in four projects, two of which were method-oriented and two explored important biological questions. First, I consolidated real-time deformability cytometry as a method for high-throughput single-cell mechanical phenotyping and contributed to its transformation into a versatile image-based cell characterization and sorting platform. Importantly, this platform can be used not only to sort cells based on image-derived parameters, but also to train neural networks to recognize and sort cells of interest based on raw images. Second, I performed a cross-laboratory study comparing three microfluidics-based deformability cytometry approaches operating at different timescales in two standardized assays of osmotic shock and actin disassembly. This study revealed that while all three methods are sensitive to osmotic shock-induced changes in cell deformability, the method operating at the shortest timescale is not suited for detection of actin cytoskeleton changes. Third, I demonstrated changes in cell mechanical phenotype associated with cell fate specification on the example of differentiation and de-differentiation along the neural lineage. In the process of reprogramming to pluripotency, neural precursor cells acquired progressively stiffer phenotype, that was reversed in the process of neural differentiation. The stiff phenotype of induced pluripotent stem cells was equivalent to that of embryonic stem cells, suggesting that mechanical properties of cells are inherent to their developmental stage. Finally, I identified and validated novel target genes involved in the regulation of mechanical properties of cells. The targets were identified using machine learning-based network analysis of transcriptomic profiles associated with mechanical phenotype change, and validated computationally as well as in genetic perturbation experiments. In particular, I showed that the gene with the best in silico performance, CAV1, changes the mechanical properties of cells when silenced or overexpressed. Identification of novel targets for mechanical phenotype modification is crucial for future explorations of physiological and pathological roles of cell mechanics. Together, this thesis encompasses a collection of contributions at the frontier of single-cell mechanical characterization across timescales and cell state transitions, and lays ground for turning cell mechanics from a correlative phenomenological parameter to a controllable property.:Abstract Kurzfassung List of Publications Contents Introduction Chapter 1 — Background 1.1. Mechanical properties as a marker of cell state in health and disease 1.2. Functional relevance of single-cell mechanical properties 1.3. Internal structures determining mechanical properties of cells 1.4. Cell as a viscoelastic material 1.5. Methods to measure single-cell mechanical properties Aims and scope of this thesis Chapter 2 — RT-DC as a versatile method for image-based cell characterization and sorting 2.1. RT-DC for mechanical characterization of cells 2.1.1. Operation of the RT-DC setup 2.1.2. Extracting Young’s modulus from RT-DC data 2.2. Additional functionalities implemented to the RT-DC setup 2.2.1. 1D fluorescence readout in three spectral channels 2.2.2. SSAW-based active cell sorting 2.3. Beyond assessment of cell mechanics — emerging applications 2.3.1. Deformation-assisted population separation and sorting 2.3.2. Brightness-based identification and sorting of blood cells 2.3.3. Transferring molecular specificity into label-free cell sorting 2.4. Discussion 2.5. Key conclusions 2.6. Materials and experimental procedures 2.7. Data analysis Chapter 3 — A comparison of three deformability cytometry classes operating at different timescales 3.1. Results 3.1.1. Representatives of the three deformability cytometry classes 3.1.2. Osmotic shock-induced deformability changes are detectable in all three methods 3.1.3. Ability to detect actin disassembly is method-dependent 3.1.4. Strain rate increase decreases the range of deformability response to actin disassembly in sDC 3.2. Discussion 3.3. Key conclusions 3.4. Materials and methods Chapter 4 — Mechanical journey of neural progenitor cells to pluripotency and back 4.1. Results 4.1.1. fNPCs become progressively stiffer during reprogramming to pluripotency 4.1.2. Transgene-dependent F-class cells are more compliant than ESC-like iPSCs 4.1.3. Surface markers unravel mechanical subpopulations at intermediate reprogramming stages 4.1.4. Neural differentiation of iPSCs mechanically mirrors reprogramming of fNPCs 4.1.5. The closer to the pluripotency, the higher the cell stiffness 4.2. Discussion 4.3. Key conclusions 4.4. Materials and methods Chapter 5 — Data-driven approach for de novo identification of cell mechanics regulators 5.1. Results 5.1.1. An overview of the mechanomics approach 5.1.2. Model systems characterized by mechanical phenotype changes 5.1.3. Discriminative network analysis on discovery datasets 5.1.4. Conserved functional network module comprises five genes 5.1.5. CAV1 performs best at classifying soft and stiff cell states in validation datasets 5.1.6. Perturbing expression levels of CAV1 changes cells stiffness 5.2. Discussion 5.3. Key conclusions 5.4. Materials and methods Conclusions and Outlook Appendix A Appendix B Supplementary Tables B.1 – B.2 Supplementary Figures B.1 – B.9 Appendix C Supplementary Tables C.1 – C.2. Supplementary Figures C.1 – C.5 Appendix D Supplementary Tables D.1 – D.6 Supplementary Figures D.1 – D.7 List of Figures List of Tables List of Abbreviations. List of Symbols References Acknowledgements

A resistência à compressão uniaxial e o módulo de deformabilidade são parâmetros que descrevem o comportamento de materiais rochosos frente a solicitações por esforços. É prática corriqueira em investigações geológico-geotécnicas para projetos de engenharia estimar a resistência de maneira preliminar com base em propriedades de tenacidade, dureza e friabilidade, aferindo à rocha graus de coerência. Dados laboratoriais referentes a aspectos físicos, texturais e mineralógicos de arenitos da Formação Piramboia, Bacia do Paraná, amostrados via sondagens mistas na região de Anhembi e Santa Maria da Serra, Estado de São Paulo, foram correlacionados entre si e a parâmetros geomecânicos como resistência à compressão uniaxial, módulo de deformabilidade e graus de coerência. As fácies reconhecidas nos arenitos foram agrupadas em associações de dunas e interdunas e suas rochas classificadas como quartzo arenitos e arenitos feldspáticos ou subarcóseos, com cimentação principalmente de esmectita ou material argilo- ferruginoso. Encontraram-se correlações significativas da resistência à compressão uniaxial com tamanho médio dos grãos e densidade, inversa no primeiro caso e direta no segundo. Já o módulo de deformabilidade apresentou correlação significativa inversa com proporção de pelíticos, e direta com tamanho médio dos grãos, desvio padrão da distribuição granulométrica e grau de empacotamento. Arenitos característicos de inundações efêmeras em domínio de interdunas úmidas, com maiores teores de pelíticos, apresentaram módulos de deformabilidade baixos e resistências altas em comparação aos arenitos de demais fácies. Variações da composição mineralógica do arcabouço não se mostraram suficientes para influenciar significativamente o comportamento geomecânico dos arenitos. / The uniaxial compressive strength and the deformability modulus are parameters that describe the behavior of rock materials under stress. It is an everyday practice in geological and geotechnical investigations for engineering projects to estimate strength in a preliminary fashion, based on properties such as tenacity, toughness and friability, attributing to the rock a qualitative term describing its strength. Laboratorial data on physical, textural and mineralogical aspects of sandstones of the Piramboia Formation, Paraná Basin, obtained by core drillings in the region of Anhembi and Santa Maria da Serra, state of São Paulo, were correlated to each other and to geomechanical parameters such as uniaxial compressive strength, deformability modulus and qualitative terms describing their strength. The recognized sandstone facies were grouped in dune and interdune associations and their rocks were classified as quartz arenites and feldspathic arenites or subarkoses, whose cementation is mainly composed of smectite or ferruginous clay material. Significant correlations of uniaxial compressive strength with mean grain size and density were found, inverse in the first case and direct in the second. On the other hand, the deformability modulus showed a significant inverse correlation with the amount of pelitic material, and direct with mean grain size, standard deviation of grain size distribution and packing density. Sandstones characteristic of ephemeral floods in the domain of wet interdunes, with greater amounts of pelitic material, presented low values of deformability modulus and high strength values compared to the other facies. Variations in the mineral composition of the framework were not sufficient to significantly influence the geomechanical behavior of the sandstones.

Este trabalho caracterizou os siltitos do Grupo Itararé, rocha branda proveniente da amostragem de pedreira localizada nas proximidades de futuro empreendimento brasileiro, o Trem de Alta Velocidade. Objetivou entender as causas da pronunciada e rápida alteração da rocha intacta, e simular, em condições controladas de laboratório, a degradação hidromecânica causada pela simples variação da umidade relativa do ar. Para tal, estabeleceu-se técnica de amostragem de testemunhos por meio da extração via seca com ar-comprimido, e processo de intemperismo artificial, composto por 4 ciclos de 2 etapas (secagem e umedecimento). O processo se deu em dessecadores, sendo a variação da umidade ocasionada pela técnica do vapor e sílica gel. O acompanhamento da degradação dos corpos de prova efetuou-se com duas técnicas não-destrutivas de propagação de ondas: o pulso ultrassônico e a excitação por impulso (sistema Sonelastic). Em paralelo, realizou-se a caracterização geotécnica dos siltitos em umidade natural, com ensaios mineralógicos, físicos, físico-mecânicos, físico-químicos e de alteração laboratorial. Por meio desta metodologia, foi possível estimar as minorações do módulo de deformabilidade e da resistência, além das causas físico-químicas da degradação. / This work characterized the siltstones of the Itararé Group, a soft rock sampled in a quarry located near a future Brazilian venture, the High Speed Train. It aims at understanding the causes of the pronounced and fast changes of the intact rock, and simulate in controlled laboratory conditions the hydro degradation process caused by simple variation of relative air humidity. Dry sampling technique with compressed air was used to extract samples from blocks, and the process of artificial weathering consisted on 4 cycles of 2 steps (drying and wetting). The process occurred in desiccators, where moisture variation was caused by the vapor technique and silica gel. The monitoring of the specimens degradation was performed with two non-destructive wave propagation techniques: the ultrasonic pulse and the impulse excitation (Sonelastic system). A geotechnical characterization of the siltstones in natural moisture condition was made in parallel, consisting on mineralogical, physical, physical-mechanical, physical-chemical and durability tests. By this methodology, it was possible to estimate the mitigation of the deformability modulus and strength besides the physical- chemical causes of degradation.

A resistência à compressão uniaxial e o módulo de deformabilidade são parâmetros que descrevem o comportamento de materiais rochosos frente a solicitações por esforços. É prática corriqueira em investigações geológico-geotécnicas para projetos de engenharia estimar a resistência de maneira preliminar com base em propriedades de tenacidade, dureza e friabilidade, aferindo à rocha graus de coerência. Dados laboratoriais referentes a aspectos físicos, texturais e mineralógicos de arenitos da Formação Piramboia, Bacia do Paraná, amostrados via sondagens mistas na região de Anhembi e Santa Maria da Serra, Estado de São Paulo, foram correlacionados entre si e a parâmetros geomecânicos como resistência à compressão uniaxial, módulo de deformabilidade e graus de coerência. As fácies reconhecidas nos arenitos foram agrupadas em associações de dunas e interdunas e suas rochas classificadas como quartzo arenitos e arenitos feldspáticos ou subarcóseos, com cimentação principalmente de esmectita ou material argilo- ferruginoso. Encontraram-se correlações significativas da resistência à compressão uniaxial com tamanho médio dos grãos e densidade, inversa no primeiro caso e direta no segundo. Já o módulo de deformabilidade apresentou correlação significativa inversa com proporção de pelíticos, e direta com tamanho médio dos grãos, desvio padrão da distribuição granulométrica e grau de empacotamento. Arenitos característicos de inundações efêmeras em domínio de interdunas úmidas, com maiores teores de pelíticos, apresentaram módulos de deformabilidade baixos e resistências altas em comparação aos arenitos de demais fácies. Variações da composição mineralógica do arcabouço não se mostraram suficientes para influenciar significativamente o comportamento geomecânico dos arenitos. / The uniaxial compressive strength and the deformability modulus are parameters that describe the behavior of rock materials under stress. It is an everyday practice in geological and geotechnical investigations for engineering projects to estimate strength in a preliminary fashion, based on properties such as tenacity, toughness and friability, attributing to the rock a qualitative term describing its strength. Laboratorial data on physical, textural and mineralogical aspects of sandstones of the Piramboia Formation, Paraná Basin, obtained by core drillings in the region of Anhembi and Santa Maria da Serra, state of São Paulo, were correlated to each other and to geomechanical parameters such as uniaxial compressive strength, deformability modulus and qualitative terms describing their strength. The recognized sandstone facies were grouped in dune and interdune associations and their rocks were classified as quartz arenites and feldspathic arenites or subarkoses, whose cementation is mainly composed of smectite or ferruginous clay material. Significant correlations of uniaxial compressive strength with mean grain size and density were found, inverse in the first case and direct in the second. On the other hand, the deformability modulus showed a significant inverse correlation with the amount of pelitic material, and direct with mean grain size, standard deviation of grain size distribution and packing density. Sandstones characteristic of ephemeral floods in the domain of wet interdunes, with greater amounts of pelitic material, presented low values of deformability modulus and high strength values compared to the other facies. Variations in the mineral composition of the framework were not sufficient to significantly influence the geomechanical behavior of the sandstones.

The flexural performance of composite systems made of reinforced concrete, Fibre Reinforced Polymers (FRPs) and adhesives was studied during the current research. The experimental investigation was principally concentrated on the potential use of Kevlar® 49 (aramid fibre) for RC beam strengthening. The main aims of research have been; (a) to investigate the relative merits of using Aramids in comparison to other FRPs, (b) strength optimisation of systems to prevent excessive losses of ductility, (c) to examine the failure mode and crack patterns, together with salient strength factors at ultimate limit state and (d) to carry out analytical modelling using a commercial FE package. The experimental investigation comprised of testing 55 simply supported RC beams of either 1.5m or 2.6m length. In addition to the parametric studies included in points (a)-(d) above (to assess the section characteristics), further experimentation was conducted to investigate the beam performance by varying the factors of; (e) beam shear span, (f) FRP anchorage length, (g) concrete surface preparation, (h) FRP end-anchoring, (i) beam precracking, (j) introduction of air-voids within the bond line of FRP/concrete, (k) influence of cyclic loading and, (1) exposure to aggressive environment. The results from current tests confirm elements of reports from other researchers (by thorough review of literature) that all FRPs have great potential for flexural strengthening of RC members. This is valid even in cases where additional environmental degradation and/or cracking (due to serviceability loads), had taken place. Aramid fibres were found to result in favourable outcomes concerning both strength and ductility enhancements. It was determined, both from experiments and non-linear modelling, that the amount of FRP fibre content is an important factor in every strengthening application. Experimentation showed that depending on the existing condition of the structure (concrete strength, internal reinforcement ratio, section dimensions, degradation level and load configuration), there seems to be a unique level of optimum fibre content. The FRP levels in excess of the optimum were seen to lead to premature brittle tearing-off failure modes. It was also found that to prevent premature beam failure (due to incompatibility of stress at concrete and FRP interface), a maximum possible anchorage length should be considered in order to deliver an optimum section performance. The results from the analytical modelling indicated a most satisfactory agreement with the experimental data after the initial mechanical properties were calibrated. It was found that actual representation of material properties (e.g. steel constitutive law) are of great significance, for an accurate modelling of RC element loaded behaviour. The bond developed between the FRP and concrete is one of the key parameters for achieving good performance of the systems. It was determined that concrete surface preparation and priming is beneficial, while the introduction of air-voids due to poor workmanship can reduce the section load bearing capabilities. Cyclic loading on FRP strengthened sections was found to curtail the full rotational capacity utilisation of the beam. However, even the above mentioned curtailed behaviour was more advantageous than cyclically loaded beam performance without FRP strengthening.

Este trabalho caracterizou os siltitos do Grupo Itararé, rocha branda proveniente da amostragem de pedreira localizada nas proximidades de futuro empreendimento brasileiro, o Trem de Alta Velocidade. Objetivou entender as causas da pronunciada e rápida alteração da rocha intacta, e simular, em condições controladas de laboratório, a degradação hidromecânica causada pela simples variação da umidade relativa do ar. Para tal, estabeleceu-se técnica de amostragem de testemunhos por meio da extração via seca com ar-comprimido, e processo de intemperismo artificial, composto por 4 ciclos de 2 etapas (secagem e umedecimento). O processo se deu em dessecadores, sendo a variação da umidade ocasionada pela técnica do vapor e sílica gel. O acompanhamento da degradação dos corpos de prova efetuou-se com duas técnicas não-destrutivas de propagação de ondas: o pulso ultrassônico e a excitação por impulso (sistema Sonelastic). Em paralelo, realizou-se a caracterização geotécnica dos siltitos em umidade natural, com ensaios mineralógicos, físicos, físico-mecânicos, físico-químicos e de alteração laboratorial. Por meio desta metodologia, foi possível estimar as minorações do módulo de deformabilidade e da resistência, além das causas físico-químicas da degradação. / This work characterized the siltstones of the Itararé Group, a soft rock sampled in a quarry located near a future Brazilian venture, the High Speed Train. It aims at understanding the causes of the pronounced and fast changes of the intact rock, and simulate in controlled laboratory conditions the hydro degradation process caused by simple variation of relative air humidity. Dry sampling technique with compressed air was used to extract samples from blocks, and the process of artificial weathering consisted on 4 cycles of 2 steps (drying and wetting). The process occurred in desiccators, where moisture variation was caused by the vapor technique and silica gel. The monitoring of the specimens degradation was performed with two non-destructive wave propagation techniques: the ultrasonic pulse and the impulse excitation (Sonelastic system). A geotechnical characterization of the siltstones in natural moisture condition was made in parallel, consisting on mineralogical, physical, physical-mechanical, physical-chemical and durability tests. By this methodology, it was possible to estimate the mitigation of the deformability modulus and strength besides the physical- chemical causes of degradation.

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

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