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1.
Biophys J ; 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600698

RESUMO

The micropipette-aspiration technique is commonly used in the field of mechanobiology, offering a variety of measurement types. To extract biophysical parameters from the experiments, numerical analysis is required. Although previous works have developed techniques for the partial automation of these analyses, these approaches are relatively time consuming for the researchers. In this article, we describe the development and application of an artificial-intelligence tool for the completely automatic analysis of micropipette-aspiration experiments. The use of this tool is compared with previous methods and the impressive reduction in the time required for these analyses is discussed. The new tool opens new possibilities for the micropipette-aspiration technique by enabling dealing with large numbers of experiments and real-time measurements.

2.
Immunology ; 167(4): 622-639, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36054660

RESUMO

Age-associated changes in T-cell function play a central role in immunosenescence. The role of aging in the decreased T-cell repertoire, primarily because of thymic involution, has been extensively studied. However, increasing evidence indicates that aging also modulates the mechanical properties of cells and the internal ordering of diverse cell components. Cellular functions are generally dictated by the biophysical phenotype of cells, which itself is also tightly regulated at the molecular level. Based on previous evidence suggesting that the relative nuclear size contributes to variations of T-cell stiffness, here we examined whether age-associated changes in T-cell migration are dictated by biophysical parameters, in part through nuclear cytoskeleton organization and cell deformability. In this study, we first performed longitudinal analyses of a repertoire of 111 functional, biophysical and biomolecular features of the nucleus and cytoskeleton of mice CD4+ and CD8+ T cells, in both naive and memory state. Focusing on the pairwise correlations, we found that age-related changes in nuclear architecture and internal ordering were correlated with T-cell stiffening and declined interstitial migration. A similarity analysis confirmed that cell-to-cell variation was a direct result of the aging process and we applied regression models to identify biomarkers that can accurately estimate individuals' age. Finally, we propose a biophysical model for a comprehensive understanding of the results: aging involves an evolution of the relative nuclear size, in part through DNA-hypomethylation and nuclear lamin B1, which implies an increased cell stiffness, thus inducing a decline in cell migration.


Assuntos
Linfócitos T CD8-Positivos , Imunossenescência , Camundongos , Animais , Timo/fisiologia , Linfócitos T CD4-Positivos , Envelhecimento
3.
Expert Rev Mol Med ; 24: e35, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36111609

RESUMO

Ageing is interrelated with the development of immunosenescence. This article focuses on one of the cell sets of the adaptive immune system, T cells, and provides a review of the known changes in T cells associated with ageing. Such fundamental changes affect both cell molecular content and internal ordering. However, acquiring a complete description of the changes at these levels would require extensive measurements of parameters and, furthermore, important fine details of the internal ordering that may be difficult to detect. Therefore, an alternative approach for the characterisation of cells consists of the performance of physical measurements of the whole cell, such as deformability measurements or migration measurements: the physical parameters, complementing the commonly used chemical biomarkers, may contribute to a better understanding of the evolution of T-cell states during ageing. Mechanical measurements, among other biophysical measurements, have the advantage of their relative simplicity: one single parameter agglutinates the complex effects of the variety of changes that gradually appear in cells during ageing.


Assuntos
Imunossenescência , Linfócitos T , Envelhecimento , Biomarcadores , Humanos
4.
Mech Ageing Dev ; 207: 111722, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35961414

RESUMO

Mitochondrial DNA sequences were found inserted in the nuclear genome of mouse peritoneal T lymphocytes that increased progressively with aging. These insertions were preferentially located at the pericentromeric heterochromatin. In the same individuals, binucleated T-cells with micronuclei showed a significantly increased frequency associated with age. Most of them were positive for centromere sequences, reflecting the loss of chromatids or whole chromosomes. The proliferative capacity of T lymphocytes decreased with age as well as the glutathione reductase activity, whereas the oxidized glutathione and malondialdehyde concentrations exhibited a significant increase. These results may point to a common process that provides insights for a new approach to understanding immunosenescence. We propose a novel mechanism in which mitochondrial fragments, originated by the increased oxidative stress status during aging, accumulate inside the nuclear genome of T lymphocytes in a time-dependent way. The primary entrance of mitochondrial fragments at the pericentromeric regions may compromise chromosome segregation, causing genetic loss that leads to micronuclei formation, rendering aneuploid cells with reduced proliferation capacity, one of the hallmark of immunosenescence. Future experiments deciphering the mechanistic basis of this phenomenon are needed.


Assuntos
DNA Mitocondrial , Imunossenescência , Animais , Segregação de Cromossomos , DNA Mitocondrial/genética , Dissulfeto de Glutationa/genética , Glutationa Redutase/genética , Heterocromatina , Malondialdeído , Camundongos
5.
Soft Matter ; 16(24): 5669-5678, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32519732

RESUMO

Deformability and internal ordering are key features related to cell function, particularly critical for cells that routinely undergo large deformations, like T cells during extravasation and migration. In the measurement of cell deformability, a considerable variability is typically obtained, masking the identification of possible interrelationships between deformability, internal ordering and cell function. We report the development of a single-cell methodology that combines measurements of living-cell deformability, using micropipette aspiration, and three-dimensional confocal analysis of the nucleus and cytoskeleton. We show that this single-cell approach can serve as a powerful tool to identify appropriate parameters that characterize deformability within a population of cells, not readably discernable in population-averaged data. By applying this single-cell methodology to mouse CD4+ T cells, our results demonstrate that the relative size of the nucleus, better than other geometrical or cytoskeletal features, effectively determines the overall deformability of the cells within the population.


Assuntos
Linfócitos T CD4-Positivos/citologia , Modelos Biológicos , Animais , Fenômenos Biofísicos , Núcleo Celular , Dimetilpolisiloxanos , Módulo de Elasticidade , Feminino , Fluorescência , Camundongos Endogâmicos ICR , Microscopia Confocal , Análise de Célula Única , Viscosidade
6.
J Mech Behav Biomed Mater ; 95: 103-115, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30986755

RESUMO

A comparative analysis of T-lymphocyte mechanical data obtained from Micropipette Aspiration (MPA) and Atomic Force Microscopy (AFM) is presented. Results obtained by fitting the experimental data to simple Hertz and Theret models led to non-Gaussian distributions and significantly different values of the elastic moduli obtained by both techniques. The use of more refined models, taking into account the finite size of cells (simplified double contact and Zhou models) reduces the differences in the values calculated for the elastic moduli. Several possible sources for the discrepancy between the techniques are considered. The analysis suggests that the local nature of AFM measurements compared with the more general character of MPA measurements probably contributed to the differences observed.


Assuntos
Teste de Materiais/métodos , Fenômenos Mecânicos , Microscopia de Força Atômica , Microtecnologia/instrumentação , Linfócitos T/citologia , Animais , Adesão Celular , Citoesqueleto/metabolismo , Módulo de Elasticidade , Feminino , Teste de Materiais/instrumentação , Camundongos
7.
Biophys J ; 116(4): 587-594, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30683304

RESUMO

With five decades of sustained application, micropipette aspiration has enabled a wide range of biomechanical studies in the field of cell mechanics. Here, we provide an update on the use of the technique, with a focus on recent developments in the analysis of the experiments, innovative microaspiration-based approaches, and applications in a broad variety of cell types. We first recapitulate experimental variations of the technique. We then discuss analysis models focusing on important limitations of widely used biomechanical models, which underpin the urge to adopt the appropriate ones to avoid misleading conclusions. The possibilities of performing different studies on the same cell are also considered.


Assuntos
Células/citologia , Fenômenos Mecânicos , Micromanipulação/métodos , Modelos Biológicos , Fenômenos Biomecânicos , Forma Celular , Humanos , Micromanipulação/instrumentação
8.
Phys Rev E ; 96(2-1): 022402, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28950493

RESUMO

The study of fungal cells is of great interest due to their importance as pathogens and as fermenting fungi and for their appropriateness as model organisms. The differential pressure between the hyphal cytoplasm and the bordering medium is essential for the growth process, because the pressure is correlated with the growth rate. Notably, during the invasion of tissues, the external pressure at the tip of the hypha may be different from the pressure in the surrounding medium. We report the use of a method, based on the micropipette-aspiration technique, to study the influence of this external pressure at the hyphal tip. Moreover, this technique makes it possible to study hyphal growth mechanics in the case of very thin hyphae, not accessible to turgor pressure probes. We found a correlation between the local pressure at the tip and the growth rate for the species Arpergillus nidulans. Importantly, the proposed method allows one to measure the pressure at the tip required to arrest the hyphal growth. Determining that pressure could be useful to develop new medical treatments for fungal infections. Finally, we provide a mechanical model for these experiments, taking into account the cytoplasm flow and the wall deformation.


Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/fisiologia , Resinas Acrílicas , Fenômenos Biomecânicos , Parede Celular/fisiologia , Citoplasma/fisiologia , Corrente Citoplasmática , Desenho de Equipamento , Microscopia , Modelos Biológicos , Pressão , Água
9.
Ann Biomed Eng ; 45(5): 1375-1385, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28097526

RESUMO

Mechanical deformability of cells is an important property for their function and development, as well as a useful marker of cell state. The classical technique of micropipette aspiration allows single-cell studies and we provide here a method to measure the two basic mechanical parameters, elastic modulus and Poisson's ratio. The proposed method, developed from finite-element analysis of micropipette aspiration experiments, may be implemented in future technologies for the automated measurement of mechanical properties of cells, based on the micropipette aspiration technique or on the cell transit through flow constrictions. We applied this method to measure the elastic parameters of lymphocytes, in which the mechanical properties depend on their activation state. Additionally, we discuss in this work the accuracy of previous models to estimate the elastic modulus of cells, in particular the analytical model by Theret et al., widely used in the field. We show the necessity of using an improved model, taking into account the finite size of the cells, to obtain new insights that may remain hidden otherwise.


Assuntos
Módulo de Elasticidade , Análise de Elementos Finitos , Ativação Linfocitária , Linfócitos/química , Animais , Feminino , Camundongos , Camundongos Endogâmicos ICR
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