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Trapping and Manipulating Single Molecules of DNAShon, Min Ju 25 February 2014 (has links)
This thesis presents the development and application of nanoscale techniques to trap and / Chemistry and Chemical Biology
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Environmentally controlled magnetic nano-tweezer for living cells and extracellular matricesAermes, Christian, Hayn, Alexander, Fischer, Tony, Mierke, Claudia Tanja 11 February 2022 (has links)
The magnetic tweezer technique has become a versatile tool for unfolding or folding of individual molecules, mainly DNA. In addition to single molecule analysis, the magnetic tweezer can be used to analyze the mechanical properties of cells and extracellular matrices. We have established a magnetic tweezer that is capable of measuring the linear and non-linear viscoelastic behavior of a wide range of soft matter in precisely controlled environmental conditions, such as temperature, CO2 and humidity. The magnetic tweezer presented in this study is suitable to detect specific differences in the mechanical properties of different cell lines, such as human breast cancer cells and mouse embryonic fibroblasts, as well as collagen matrices of distinct concentrations in the presence and absence of fibronectin crosslinks. The precise calibration and control mechanism employed in the presented magnetic tweezer setup provides the ability to apply physiological force up to 5 nN on 4.5 µm superparamagnetic beads coated with fibronectin and coupled to the cells or collagen matrices. These measurements reveal specific local linear and non-linear viscoelastic behavior of the investigated samples. The viscoelastic response of cells and collagen matrices to the force application is best described by a weak power law behavior. Our results demonstrate that the stress stiffening response and the fluidization of cells is cell type specific and varies largely between differently invasive and aggressive cancer cells. Finally, we showed that the viscoelastic behavior of collagen matrices with and without fibronectin crosslinks measured by the magnetic tweezer can be related to the microstructure of these matrices.
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Effect of PAK Inhibition on Cell Mechanics Depends on Rac1Mierke, Claudia Tanja, Puder, Stefanie, Aermes, Christian, Fischer, Tony, Kunschmann, Tom 03 April 2023 (has links)
Besides biochemical and molecular regulation, the migration and invasion of cells
is controlled by the environmental mechanics and cellular mechanics. Hence, the
mechanical phenotype of cells, such as fibroblasts, seems to be crucial for the
migratory capacity in confined 3D extracellular matrices. Recently, we have shown
that the migratory and invasive capacity of mouse embryonic fibroblasts depends on
the expression of the Rho-GTPase Rac1, similarly it has been demonstrated that the
Rho-GTPase Cdc42 affects cell motility. The p21-activated kinase (PAK) is an effector
down-stream target of both Rho-GTPases Rac1 and Cdc42, and it can activate via the
LIM kinase-1 its down-stream target cofilin and subsequently support the cell migration
and invasion through the polymerization of actin filaments. Since Rac1 deficient cells
become mechanically softer than controls, we investigated the effect of group I PAKs
and PAK1 inhibition on cell mechanics in the presence and absence of Rac1. Therefore,
we determined whether mouse embryonic fibroblasts, in which Rac1 was knockedout,
and control cells, displayed cell mechanical alterations after treatment with group I
PAKs or PAK1 inhibitors using a magnetic tweezer (adhesive cell state) and an optical
cell stretcher (non-adhesive cell state). In fact, we found that group I PAKs and Pak1
inhibition decreased the stiffness and the Young’s modulus of fibroblasts in the presence
of Rac1 independent of their adhesive state. However, in the absence of Rac1 the
effect was abolished in the adhesive cell state for both inhibitors and in their nonadhesive
state, the effect was abolished for the FRAX597 inhibitor, but not for the IPA3
inhibitor. The migration and invasion were additionally reduced by both PAK inhibitors
in the presence of Rac1. In the absence of Rac1, only FRAX597 inhibitor reduced their
invasiveness, whereas IPA3 had no effect. These findings indicate that group I PAKs
and PAK1 inhibition is solely possible in the presence of Rac1 highlighting Rac1/PAK I
(PAK1, 2, and 3) as major players in cell mechanics.
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