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The relationship between intracellular forces and cellular stiffness investigated by atomic force microscopyMandriota, Nicola January 2016 (has links)
The characterization of the mechanical behavior of cells has always captured the interest of scientists and, in the last decades, has been facilitated by the development of techniques capable of measuring a cell’s deformability. However, if on one hand, cells are active, living materials that regulate their physiology by generating and transmitting forces throughout their volume, common mechanical characterizations of cells involve material science approaches, which mostly address them as inert materials. As a consequence, although mechanical characterizations of cells have so far provided a wealth of correlations between stiffness and physio-pathological states, they have rarely provided insights into biological function and regulation.
In this thesis, a cell nanomechanical platform is presented, whose resolution allows the isolation of the mechanical contribution of load-bearing cellular components. We first demonstrated that tensional forces - rather than the passive viscoelastic properties of the cytoplasm - govern the stiffness of cells at the nanoscale. We then quantitatively characterized the relationship between intracellular forces and the µm-scale patterns of stiffness across the cell surface. This analysis allowed us to calculate multiple physiologically-relevant quantities, such as membrane tension, cortex tension, actin bundle tension, tension-free elastic modulus, and mechanical coupling distances, all from single high-resolution cell stiffness images, providing an unprecedented connection between distinct mechanobiology fields.
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Non-canonical aspects in cell and nuclear mechanicsChan, Chii Jou January 2015 (has links)
No description available.
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Actin-based propulsion and entropic forces generated by single filamentHu, Bin, 胡斌 January 2011 (has links)
published_or_final_version / Mechanical Engineering / Master / Master of Philosophy
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A Mechanism of Mechanotransduction Mediated by the Primary CiliumLee, Kristen Lauren January 2014 (has links)
Mechanotransduction is a process by which cells sense and convert mechanical loads into biochemical signals and transcriptional changes. This process is particularly critical in bone, a metabolically active tissue that continously remodels and adapts to mechanical loads in its local environment. Osteocytes are the most prevalent bone cell type and are responsible for coordinating skeletal adaptation. Recently, the loss of primary cilia, nonmotile antenna-like cellular structures, has been attributed to causing defects in skeletal development and loading-induced bone formation. While primary cilia have been implicated in osteocyte mechanotransduction, the molecular mechanism associated with this process is not understood. In this thesis, we demonstrate that the osteocyte primary cilium forms a microdomain that mediates osteogenic responses to mechanical loads. In the first study, we build a genetically encoded primary cilium-localized calcium biosensor and characterize ciliary calcium mobilization in response to mechanical loading with unprecedented sensitivity. Next, we apply similar techniques to monitor levels of another second messenger, cyclic AMP (cAMP), and are the first to demonstrate that the primary cilium segregates ciliary cAMP from the cytosol. In the third study, we link loading-induced bone formation in vivo to adenylyl cyclase 6 enzyme function, a component of the primary cilium-mediated mechanotransduction mechanism. Collectively, this thesis elucidates how osteocyte primary cilia convert mechanical stimuli into osteogenic responses at the molecular and tissue levels and characterizes the primary cilium as a microdomain that serves as a biochemical and mechanical signaling nexus. Improvements in our understanding of primary cilia-regulated mechanotransduction will advance research efforts in the bone, tissue engineering, and mechanobiology communities.
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Cell fate restriction in Caenorhabditis elegans is orchestrated by precise chromatin organization and transcription factor activityPatel, Tulsi January 2016 (has links)
The plasticity of cells in a multicellular organism is progressively lost during differentiation. This loss is reflected in studies involving the ectopic misexpression of fate-specifying or terminal selector transcription factors (TFs). These TFs can efficiently activate target genes in undifferentiated cells, but lose this ability as cells differentiate. While this phenomenon of cell fate restriction is widely observed, the mechanisms orchestrating it are poorly understood. In this thesis, I have used the ubiquitous overexpression of Zn-finger-TF CHE-1 as a tool to understand the mechanisms that restrict cell fate in Caenorhabditis elegans. When CHE-1 is ubiquitously expressed at embryonic stages, it activates target gene expression in many cell types, while in adults it can only act in a few neurons. To uncover factors that inhibit plasticity of all other adult cells, I first performed an RNAi screen against chromatin-associated factors. Using this approach I found that the removal of either the PRC2 complex, which deposits the H3K27me3 mark, or loss of proteins that indirectly regulate domains of H3K27me3, allows CHE-1 and two other terminal selector TFs to activate target genes in the germline. These data show that the correct distribution of H3K27me3 is crucial for the restriction of germ cell fate. I next took a candidate approach to identify genes that regulate fate restriction in other cell types. We hypothesized that terminal selector TFs themselves, in addition to specifying cellular identity by controlling large gene sets, may also act to inhibit plasticity. To test this, I first assayed the activity of CHE-1 in mutants of COE-TF unc-3, the terminal selector for a subset of cholinergic motor neurons (MNs). I found that in contrast to wildtype MNs, unc-3 mutant MNs remain plastic as CHE-1 can induce expression of target genes in these cells even at the adult stage. This phenotype is also observed in four of six additional terminal selector mutants tested. I further found that the removal of met-2, a protein required for H3K9 methylation, or mes-2, a PRC2 component, also makes differentiated cholinergic MNs amenable to the activity of CHE-1. Preliminary evidence suggests that met-2 may act in the same pathway as unc-3. These results raise the exciting possibility that selector TFs play a role in restricting cell fate by organizing the heterochromatin domains in differentiated cells. Overall, in this work I provide functional evidence to show that specific chromatin-modifying enzymes restrict the fate of germ cells and that both fate-specifying TFs and chromatin-modifying enzymes are required for the fate restriction in neurons.
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Mathematically Modeling the Mechanics of Cell DivisionWang, Shuyuan January 2018 (has links)
The final stage of the cell cycle is cell division by cytokinesis, when the cell physically separates into two daughter cells. Improper timing or location of the division site results in incorrect segregation of chromosomes and thus genetically unstable aneuploid cells, which is associated with tumorigenesis. Cytokinesis in animal, fungal and amoeboid cells occurs through the assembly and constriction of an actomyosin contractile ring, a mechanism that dates back about one billion years in the common ancestor of these organisms. However, it is not well understood how the ring generates tension or how the rate of ring constriction is set. Long ago a sliding filament mechanism similar to skeletal muscle was proposed, but definitive evidence for muscle-like sarcomeric order in the ring is lacking.
Here we build mathematical models of cytokinesis in the fission yeast Schizosaccharomyces pombe, where the most complete inventory of more than 150 cytokinesis genes have been documented. The models explicitly represent proteins in the contractile ring such as formin, myosin, actin, α-actinin, etc. and implements their quantities, biomechanical properties and organizations from the best available experimental information. At the same time, the models adopt coarse-grain approaches that are able to describe the collective behaviors of thousands of ring components, which include tension production, constriction, and disassembly of the ring.
In the first part of this thesis, we modeled the extraordinarily rapid constriction of the partially unanchored ring in fission yeast cell ghosts. Experiments on isolated fission yeast rings showed sections of ring unanchoring from the membrane and shortening ~30-fold faster than normal (1). We demonstrated that anchoring of actin to the plasma membrane generates tension in the fission yeast cytokinetic ring by showing (1) unanchored segments in these experiments were tensionless, and (2) only a barbed-end anchoring of actin can generate tension in the normally anchored ring, and can explain the extraordinary behavior of unanchored segments. Molecularly explicit simulations accurately reproduced experimental constriction rates, and showed a novel non-contractile reeling-in mechanism by which the unanchored segment shortens, despite being tensionless.
In the second part of this thesis, we built a highly coarse-grained model to study how ring tension is generated and how structural stability is maintained. Recently, a super-resolution microscopy study of the fission yeast ring revealed that myosins and formins that nucleate actin filaments colocalize in plasma membrane-anchored complexes called nodes in the constricting ring (2). The nodes move bidirectionally around the ring. Here we construct and analyze a coarse-grained mathematical model of the fission yeast ring to explore essential consequences of the recently discovered ring ultrastructure. The model reproduces experimentally measured values of ring tension, explains why nodes move bidirectionally and shows that tension is generated by myosin pulling on barbed-end-anchored actin filaments in a stochastic sliding-filament mechanism. This mechanism is not based on an ordered sarcomeric organization. We show that the ring is vulnerable to intrinsic contractile instabilities, and protection from these instabilities and organizational homeostasis require both component turnover and anchoring of components to the plasma membrane.
In the third part of this thesis, we measured ring tension in fission yeast protoplasts. We found ~650 pN tension in wild type cells, ~65% the normal tension in myp2 deletion mutants and ~40% normal tension in myo2-E1 mutant cells with negligible ATPase activity and reduced actin binding. To understand the relation between organization and tension, we developed a molecularly explicit simulation of the fission yeast ring with the above organization. Our simulations revealed a clear division of labor between the 2 myosin-II isoforms, which maintains organization and maximal tension. (1) Myo2 anchors the ring to the plasma membrane, and transmits ring tension to the membrane. (2) Myo2, extending ~100 nm away from the membrane, bundles half (~25) of the actin filaments in the cross-section due to filament packing constraints, as only ~25 filaments are within reach. (3) To increase tension requires that the ring be thickened, as tensions in the ~25 membrane-proximal filaments are close to fracture. (4) Unanchored Myp2 indeed enables thickening, by bundling an additional ~25 filaments and doubling tension. Anchoring of these filaments to the membrane is indirect, via filaments shared with the anchored Myo2. (5) In simulated myo2-E1 rings ~20% of the actin filaments peeled away from the ring and formed Myp2-dressed bridges, as observed experimentally in myo2-E1 cells. (6) The organization in simulated Δmyp2 rings was highly disrupted, with ~ 50% of the actin filaments unbundled. Therefore, beyond their widely recognized job to pull actin and generate tension, myosin-II isoforms are vital crosslinking organizational elements of the ring. Two isoforms in the ring cooperate to organize the ring for maximal actomyosin interaction and tension.
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The MicroPIVOT : an Integrated Particle Image Velocimeter and Optical Tweezers Instrument for Microscale InvestigationsNeve de Mevergnies, Nathalie 01 January 2010 (has links)
This dissertation describes the development of a device capable of suspending a microscale object in a controlled flow. The uPIVOT is a system integrating two laser-based techniques: micron particle image velocimetry (uPIV) and optical tweezers (OT). The OT allows the suspension and manipulation of micron-sized objects such as microspheres or biological cells. uPIV provides imaging of the suspended object and velocity measurements from which fluid induced stresses can be determined. Using this device, we measured fluid velocities around an optically suspended polystyrene microsphere (an experimental first) and studied the interaction between two particles suspended in a uniform flow. The results were consistent with theoretical low Reynolds number, Newtonian flow predictions. Additionally, we analyzed a single cell's mechanical response to a controlled and measurable multiaxial external force (fluid flow) without the cell being physically attached to a surface. The cell's mechanical response was monitored by observing its morphology and measuring its deformation. The results show significant deformations of optically suspended cells at substantially smaller stresses than previously reported and demonstrate the opportunity to optically distinguish a cell by its trapping efficiency. These initial applications of the uPIVOT demonstrate the potential of this unique device as a research tool for novel studies in the fields of fluid/particle(s) interactions, non-Newtonian fluid mechanics, and single cell biomechanics.
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Characterization of flow within a polymer scaffold inside a compression-perfusion bioreactorMoreau, Damien 12 1900 (has links)
No description available.
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Quantitative measurements of flow within a polymer scaffold inside a compression-perfusion bioreactorJouan, Gurvan 05 1900 (has links)
No description available.
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Cell disruption mechanics / by Andrew Royce Kleinig.Kleinig, Andrew Royce January 1997 (has links)
Bibliography: leaves 213-223. / xv, 223 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis examines the cell-fluid interactions that occur during homogenization and combines them with an investigation of the mechanical properties of the cell. This results in a predictive model for cell-disruption efficiency during high-pressure homogenization. The mechanical properties of individual cells are characterised using a micro-manipulation technique. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 1997
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