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- Mojica Benavides, Martin, 1983, et al.
(författare)
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An Optical Tweezers, Epi-Fluorescence/Spinning disk confocal- and microfluidic-setup for synchronization studies of glycolytic oscillations in living yeast cells
- 2016
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Ingår i: Proceedings SPIE 9922, Optical Trapping and Optical Micromanipulation XIII. San Diego; USA. 28 August -1 September 2016. - : SPIE. - 9781510602359
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Konferensbidrag (refereegranskat)abstract
- Due to the significant importance of glycolytic oscillations studies and the recent breakthroughs on single cell analysis, a further interest arrives with intracellular and intercellular responses. Understanding cell-cell communication can give insight to oscillatory behaviors in biological systems, such as insulin secretion from pancreatic beta-cells. The aim of this work consists on the manipulation of living yeast cells to study propagation and synchronization of induced glycolytic oscillations. A setup, consisting of an optical tweezers system and microfluidic devices coupled with fluorescence imaging was designed to perform a time dependent observation during artificially induced glycolytic oscillations. Multi-channel flow devices and diffusion chambers were fabricated using soft lithography. Automatized pumps controlled specific flow rates of infused glucose and cyanide solutions, used to induce the oscillations. Flow and diffusion in the microfluidic devices were simulated to assure experimentally the desired coverage of the solutions across the yeast cells, a requirement for time dependent measurements. Using near infrared optical tweezers, yeast cells were trapped and positioned in array configurations, ranging from a single cell to clusters of various symmetries, in order to obtain information about cell-cell communications during the metabolic cycles. Confocal illumination of an entire focal plane using a spinning disk, will allow acquirement of NADH periodic fluorescence signals during glycolytic oscillations. This method permits an improvement of the 2D projection images obtained with wide field microscopy to a tomographic description of the subcellular propagation of the oscillations.
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| 2. |
- Mojica Benavides, Martin, 1983, et al.
(författare)
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Intercellular communication induces glycolytic synchronization waves between individually oscillating cells
- 2021
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:6
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Tidskriftsartikel (refereegranskat)abstract
- Many organs have internal structures with spatially differentiated and sometimes temporally synchronized groups of cells. The mechanisms leading to such differentiation and coordination are not well understood. Here we design a diffusion-limited microfluidic system to mimic a multicellular organ structure with peripheral blood flow and test whether a group of individually oscillating yeast cells could form subpopulations of spatially differentiated and temporally synchronized cells. Upon substrate addition, the dynamic response at single-cell level shows glycolytic oscillations, leading to wave fronts traveling through the monolayered population and to synchronized communities at well-defined positions in the cell chamber. A detailed mechanistic model with the architectural structure of the flow chamber incorporated successfully predicts the spatial-temporal experimental data, and allows for a molecular understanding of the observed phenomena. The intricate interplay of intracellular biochemical reaction networks leading to the oscillations, combined with intercellular communication via metabolic intermediates and fluid dynamics of the reaction chamber, is responsible for the generation of the subpopulations of synchronized cells. This mechanism, as analyzed from the model simulations, is experimentally tested using different concentrations of cyanide stress solutions. The results are reproducible and stable, despite cellular heterogeneity, and the spontaneous community development is reminiscent of a zoned cell differentiation often observed in multicellular organs.
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| 3. |
- Mojica Benavides, Martin, 1983
(författare)
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Metabolic communication between individual yeast cells
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With the recent progress in sensitive cell manipulation and microfabrication techniques, glycolytic oscillations in yeast have been observed at a single-cell level. It was shown that individually, oscillating cells could entrain their phases by periodic external perturbations. However, the mechanisms by which individual cells communicate to couple their glycolytic cycles within a heterogenic population remained an open question. The aims of the studies presented in this thesis focused on addressing the spatio-temporal dynamics present in the transitions from uncorrelated behavior of individual cells, to the emergence of synchronized subpopulations and traveling waves. In this work, it is reported the design, simulation and fabrication of microfluidic systems that allowed for the environmental control and experimental observation of glycolytic synchronization between individual yeast cells. Optical tweezers (for positioning), together with custom made microfluidic devices, were implemented to induce glycolytic oscillations in monolayered yeast cell arrangements. The developed diffusion-based chambers guaranteed the quasi-static conditions required for the intercellular exchange of chemical mediators. Subsequent image and signal processing, together with graph theory, served the purpose of evaluating the degree of synchrony among individual cells and the spatio-temporal distribution of the coupling. This study shows that synchronization communities are formed depending on the exposure ratios of cyanide and glucose, and the exchanged acetaldehyde. Moreover, those communities are also defined depending on the cell location in the monolayer. The relative phase delays between the glycolytic oscillations from different communities revealed the formation of glycolytic synchronization waves, which can overcome the existing heterogeneity in the system. The results presented in this work contribute to a further understanding on the experimental conditions required to achieve glycolytic synchronization in yeast for single-cell level studies. Furthermore, the spatio temporal characterization of the single-cell responses during cell-cell chemical interactions, explains the formation of traveling waves as a mechanism for glycolytic synchronization. These results, and the developed methodology, can be further optimized and extrapolated to study more complicated cell systems such as the pancreatic -cells, and the role of metabolic synchronization in the coordinated insulin secretion from the pancreas.
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| 4. |
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| 5. |
- van Niekerk, David, et al.
(författare)
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Phosphofructokinase controls the acetaldehyde induced phase shift in isolated yeast glycolytic oscillators.
- 2019
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Ingår i: The Biochemical journal. - 1470-8728. ; 476:2, s. 353-363
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Tidskriftsartikel (refereegranskat)abstract
- The response of oscillatory systems to external perturbations is crucial for emergent properties such as synchronization and phase locking, and can be quantified in a phase response curve. In individual, oscillating yeast cells, we characterized experimentally the phase response of glycolytic oscillations for external acetaldehyde pulses, and followed the transduction of the perturbation through the system. Subsequently, we analyzed the control of the relevant system components in a detailed mechanistic model. The observed responses are interpreted in terms of the functional coupling and regulation in the reaction network. We find that our model quantitatively predicts the phase dependent phase shift observed in the experimental data. The phase shift is in agreement with an adaptation leading to synchronization with an external signal. Our model analysis establishes that phosphofructokinase plays a key role in the phase shift dynamics as shown in the phase response curve, and adaptation time to external perturbations. Specific mechanism-based interventions, made possible through such analyses of detailed models, can improve upon standard trial and error methods, e.g. melatonin supplementation to overcome jet-lag, which are error prone, specifically, since the effects are phase and dose dependent.
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