Magnetogenetic Control of Intracellular Signaling Pathways
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Controlling the spatio-temporal organization of biomolecules inside living cells is a major rerequisite for deciphering mechanisms governing cell signaling and its regulation. In this thesis project, a new method to induce a highly specific and local perturbation of signaling pathways inside living cells is presented: magnetogenetics. It is based on the use of biofunctionalized magnetic nanoparticles, to induce and maintain protein gradients inside living cells. We tailored the size and surface properties of both synthetic silica core shell nanoparticles and superparamagnetic GFP-ferritin-based nanoparticles in order to ensure unhindered mobility in the cytosol. These nanoparticles can be rapidly localized in living cells by exploiting biased diffusion at weak magnetic forces in the fN range. In combination with nanoparticles' surface functionalization for specific in situ capturing of target proteins as well as efficient delivery into the cytosplasm, this work presents a novel technology for controlling intracellular protein gradients with a spatial resolution of micrometers and a temporal resolution of a few tens of seconds. In this work we showed the possibility to precisely control the perturbation of the signaling pathways associated to the small Rho GTPases proteins with relative quantification on signal propagation in terms of effector recruitment and morphological changes.Keywords:
Biomolecule
Surface Modification
Signaling proteins
We developed nanoparticles with tailored magnetic properties for direct and sensitive detection of biomolecules in biological samples in a single step. Thermally blocked nanoparticles obtained by thermal hydrolysis, functionalized with specific ligands, are mixed with sample solutions, and the variation of the magnetic relaxation due to surface binding is used to detect the presence of biomolecules. The binding significantly increases the hydrodynamic volume of nanoparticles, thus changing their Brownian relaxation frequency which is measured by a specifically developed AC susceptometer. The system was tested for the presence of Brucella antibodies, a dangerous pathogen causing brucellosis with severe effects both on humans and animals, in serum samples from infected cows and the surface of the nanoparticles was functionalized with lipopolysaccarides (LPS) from Brucella abortus. The hydrodynamic volume of LPS-functionalized particles increased by 25−35% as a result of the binding of the antibodies, measured by changes in the susceptibility in an alternating magnetic field. The method has shown high sensitivity, with detection limit of 0.05 μg·mL−1 of antibody in the biological samples without any pretreatment. This magnetic-based assay is very sensitive, cost-efficient, and versatile, giving a direct indication whether the animal is infected or not, making it suitable for point-of-care applications. The functionalization of tailored magnetic nanoparticles can be modified to suit numerous homogeneous assays for a wide range of applications.
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Surface Modification
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In addition to synthesizing biofunctionalized magnetic nanopaticles for the purpose of magnetically labeling biomolecules, a system to measure the ac magnetic susceptibility of the labeled sample was developed. When a targeted biomolecule was mixed with magnetic fluid possessing biofunctionalized magnetic nanoparticles, portions of magnetic nanoparticles agglomerated to form clusters due to the association with the targeted biomolecule. Due to the formation of magnetic clusters, the measured ac magnetic susceptibility reduced. The relationship between the mixed-frequency ac magnetic susceptibility reduction and the amount of the detected biomolecule was established.
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Magnetic separation
Biomagnetism
Molecular biophysics
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Subthreshold conduction
Subthreshold slope
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This review summarizes several photochemical methods of immobilization biomolecules on polymer materials in order to enhance its biomedical performance. Proteins such as albumin, polysaccharides such as heparin, other biomolecules such as enzyme, antibody, peptides, DNA fragments and so on are all available by those methods. It can retain the reactivity of immobilized biomolecules and do district designed modification without undermining the substrate performance. Polymer materials immobilized with biomolecules can obtain favorable biocompatibility and biomedical performance.
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Polymer substrate
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Horticultural crops suffer from bacterial, fungal, and oomycete pathogens. Effectors are one of the main weapons deployed by those pathogens, especially in the early stages of infection. Pathogens secrete effectors with diverse functions to avoid recognition by plants, inhibit or manipulate plant immunity, and induce programmed cell death. Most identified effectors are proteinaceous, such as the well-studied type-III secretion system effectors (T3SEs) in bacteria, RXLR and CRN (crinkling and necrosis) motif effectors in oomycetes, and LysM (lysin motifs) domain effectors in fungi. In addition, some non-proteinaceous effectors such as toxins and sRNA also play crucial roles in infection. To cope with effectors, plants have evolved specific mechanisms to recognize them and activate effector-triggered immunity (ETI). This review summarizes the functions and mechanisms of action of typical proteinaceous and non-proteinaceous effectors secreted by important horticultural crop pathogens. The defense responses of plant hosts are also briefly introduced. Moreover, potential application of effector biology in disease management and the breeding of resistant varieties is discussed.
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Plant Immunity
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Antibody-functionalized magnetic nanoparticles dispersed in phosphate-buffered saline solution were used as reagents in immunomagnetic reduction assays. Biomolecules are detected in bioliquid samples when they associate with magnetic nanoparticles and reduce the AC magnetic susceptibility χac of the reagent at a given frequency. In this study, the chemical kinetics for the real-time χac during the association was investigated. The association kinetics between biomolecules and nanoparticles consists of diffusion and binding steps. It was found that the diffusion speeds up in samples with higher concentrations of molecules. Furthermore, the period of association was longer for samples having higher concentrations of molecules. The association rates were proportional to the T-Tau concentration. The results showed that one biomolecule was associated with one magnetic nanoparticle.
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Magnetic nanoparticles biofunctionalized with antibodies are useful for specifically labeling target biomolecules. By measuring magnetic signals after the association between biofunctionalized magnetic nanoparticles and target biomolecules, the concentration of target biomolecules can be quantitatively detected. One of measuring methodologies is so-called immunomagnetic reduction (IMR), in which the reduction in ac magnetic susceptibility of magnetic reagent is a function of the concentration of target biomolecules. In this letter, the relationship between the magnetic reduction signal of reagent and the concentration of target biomolecules is explored. According to the experimental results on various kinds of target biomolecules, such as proteins and chemicals, the magnetic reduction signal as a function of concentration of various target biomolecules can be scaled to a universal curve. This universal curve is a logistic function. This implies that there exists a unique mechanism for the association between the target biomolecules and biofunctionalized magnetic nanoparticles in an IMR assay.
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Immunomagnetic separation
Magnetic separation
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