- Buenos Aires, Distrito Federal, Argentina
<p>(<b>A</b>) When activated, IRE1 processes <i>Xbp1</i> mRNA by a non-conventional cytoplasmic splicing reaction, changing <i>Xbp1</i> open reading frame. HEK293T cells were treated with Akt-IV... more
<p>(<b>A</b>) When activated, IRE1 processes <i>Xbp1</i> mRNA by a non-conventional cytoplasmic splicing reaction, changing <i>Xbp1</i> open reading frame. HEK293T cells were treated with Akt-IV (10 µM), Akt-VIII (5 µM) or LY294002 (20 µM) for the indicated times. <i>Xbp1</i> mRNA splicing was detected by RT-PCR. <i>Xbp1s</i>: spliced form (activated IRE1); <i>Xbp1u</i>: unspliced form (inactive IRE1). (<b>B</b>) When activated, ATF6 translocates to the Golgi apparatus where it is cleaved to release a fragment that enters the nucleus where it functions as a transcription factor. HEK293 cells were transfected with ATF6-Flag plasmid and 24 h later they were treated with Akt-IV (10 µM), Akt-VIII (5 µM), LY294002 (20 µM), or thapsigargin (Tg; 100 nM) for the indicated times. Western blots (WB) using antibodies against FLAG and actin are shown for every case (B, upper panel). ATF6: uncleaved protein; ATF6f: cleaved form. (<b>C</b>) HEK293T cells were transfected with a plasmid that expresses the YFP-NLS-mATF6short reporter (top, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069668#pone.0069668.s001" target="_blank">Fig. S1A</a> for details). Forty-eight hours post-transfection cells were treated for the indicated times with Akt-IV and then fixed, DNA was stained with DAPI and cells were imaged (lower panel); Yellow, YFP-ATF6; Blue, DNA; scale bar, 5 µm. For all cases cells treated with DMSO were used as a control (Control). (<b>D</b>) When activated, PERK is autophosphorylated at multiple residues and activated to phosphorylate eIF2α. HEK293T cells were treated with Akt-IV (10 µM), Akt-VIII (5 µM) or LY294002 (20 µM) for the indicated times. Protein extracts were analyzed by WB using the indicated antibodies. Data in the plot corresponds to ratio of phosphorylated total abundance of each of the indicated proteins (normalized to the initial value) in cells treated with the indicated drugs for different times. Error bars correspond to the standard error of three independent experiments. (<b>E</b>) HEK293T cells were treated for 5 h with Akt-IV. peIF2α abundance was detected by immunofluorescence. Green, peIF2α; Blue, DNA; scale bar, 5 µm. Data are representative of at least three independent experiments.</p
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Research Interests: Signal Processing, Systems Biology, Biology, Mathematical Modelling, Medicine, and 15 moreG Protein Signaling, Signal Transduction, Saccharomyces cerevisiae, Humans, Molecular systems biology, GPCR, Yeast, Ciencias Biológicas, Robustness, Theoretical Models, Mapk Pathways, Protein Binding, fractional occupancy measurement, paradoxical components, and G Protein Coupled Receptors
In a companion paper, we carried out a high-throughput screen to identify genes that suppressed cell-to-cell variability in signaling in yeast. Two genes affected cytoplasmic microtubules that can connect the nucleus to a signaling site... more
In a companion paper, we carried out a high-throughput screen to identify genes that suppressed cell-to-cell variability in signaling in yeast. Two genes affected cytoplasmic microtubules that can connect the nucleus to a signaling site on the membrane. Here, we show that microtubule perturbations that affected polymerization and depolymerization, membrane attachment, and force generation increased variability. For some perturbations, "outlier" cells drove the increased variability. Bypass experiments that activated the PRS ectopically at downstream points indicated that microtubule-dependent processes might stabilize the membrane-recruited scaffold protein Ste5. The variability caused by microtubule perturbations required the MAP kinase Fus3. Microtubule perturbations hindered stable scaffold formation and decreased the accuracy of a polarity-dependent fate choice. Our experiments suggest that membrane-attached microtubules stabilize signaling by scaffold-bound Fus3, and ...
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In order to contribute to the design of crossfeeding systems, we modeled population control in a coculture of two crossfeeding strains of an organism, each of which secretes a metabolite the other strain requires to grow. Differential... more
In order to contribute to the design of crossfeeding systems, we modeled population control in a coculture of two crossfeeding strains of an organism, each of which secretes a metabolite the other strain requires to grow. Differential equations show that the steady-state population ratio can be tuned by varying the ratio of the metabolite secretion rates, as long as they fall within a range determined by the nature of the organism. Numerical simulations of Trp/His crossfeeding in budding yeast suggest that the time required to reach steady state populations critically depends on the capacity of the cells to uptake the crossfeeding amino acids. We also engineered and evaluated a novel genetic device that secretes tryptophan-rich peptides with a cell penetrating sequence. Experimental validation showed that the device increases tryptophan secretion and enables growth of atrp−strain in a coculture in synthetic medium lacking tryptophan.
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The gradient of Bicoid (Bcd) is key for the establishment of the anterior-posterior axis in Drosophila embryos. The gradient properties are compatible with the SDD model in which Bcd is synthesized at the anterior pole and then diffuses... more
The gradient of Bicoid (Bcd) is key for the establishment of the anterior-posterior axis in Drosophila embryos. The gradient properties are compatible with the SDD model in which Bcd is synthesized at the anterior pole and then diffuses into the embryo and is degraded with a characteristic time. Within this model, the Bcd diffusion coefficient is critical to set the timescale of gradient formation. This coefficient has been measured using two optical techniques, Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS), obtaining estimates in which the FCS value is an order of magnitude larger than the FRAP one. This discrepancy raises the following questions: which estimate is "correct''; what is the reason for the disparity; and can the SDD model explain Bcd gradient formation within the experimentally observed times? In this paper, we use a simple biophysical model in which Bcd diffuses and interacts with binding sites to show ...
Research Interests: Bioinformatics, Computer Science, Biophysics, Computational Biology, Biology, and 15 moreLife Sciences, Drosophila melanogaster, Development, Fluorescence Correlation Spectroscopy, Biological Sciences, Biomedical Research, Computer Simulation, Mathematical Sciences, Diffusion, Animals, Ciencias Biológicas, Body Patterning, Concentration gradient, Biofísica, and Biological System
Significance Many cell decisions depend on precise measurements of external ligands reversibly bound to receptors. Yeast cells orient in gradients of sex pheromone detecting differences in the amount of ligand-receptor complex. However,... more
Significance Many cell decisions depend on precise measurements of external ligands reversibly bound to receptors. Yeast cells orient in gradients of sex pheromone detecting differences in the amount of ligand-receptor complex. However, yeast can orient in gradients with nearly all receptors occupied. We describe a general systems-level mechanism, pre-equilibrium sensing and signaling (PRESS), which overcomes this saturation limit by shifting and expanding the input dynamic range to which cells can respond. PRESS requires that events downstream of the receptor be transient and faster than the time required for the receptor to reach equilibrium binding. Experiments and simulations show that PRESS operates in yeast and may help cells orient in gradients. Many ligand-receptor interactions are slow, suggesting that PRESS is widespread throughout eukaryotes.
Research Interests: Biophysics, Kinetics, Biology, Cell Signaling, Medicine, and 15 moreMultidisciplinary, Signal Transduction, Budding Yeast, Saccharomyces cerevisiae, Molecular systems biology, Cell Polarity, Ciencias Biológicas, Signaling, Receptor, Protein Binding, Ligands, Extracellular, Extracellular Space, Señalización, and G Protein Coupled Receptors
Ultrasensitive response motifs, which are capable of converting graded stimulus in binary responses, are very well-conserved in signal transduction networks. Although it has been shown that a cascade arrangement of multiple ultrasensitive... more
Ultrasensitive response motifs, which are capable of converting graded stimulus in binary responses, are very well-conserved in signal transduction networks. Although it has been shown that a cascade arrangement of multiple ultrasensitive modules can produce an enhancement of the system's ultrasensitivity, how the combination of layers affects the cascade's ultrasensitivity remains an open question for the general case. Here we introduced a methodology that allowed us to determine the presence of sequestration effects and to quantify the relative contribution of each module to the overall cascade's ultrasensitivity. The proposed analysis framework provides a natural link between global and local ultrasensitivity descriptors and is particularly well-suited to characterize and better understand mathematical models used to study real biological systems. As a case study we considered three mathematical models introduced by O'Shaughnessy et al. to study a tunable syntheti...
One goal of systems biology is to understand how genome-encoded parts interact to produce quantitative phenotypes. The Alpha Project is a medium-scale, interdisciplinary systems biology effort that aims to achieve this goal by... more
One goal of systems biology is to understand how genome-encoded parts interact to produce quantitative phenotypes. The Alpha Project is a medium-scale, interdisciplinary systems biology effort that aims to achieve this goal by understanding fundamental quantitative behaviours of a prototypic signal transduction pathway, the yeast pheromone response system from Saccharomyces cerevisiae. The Alpha Project distinguishes itself from many other systems biology projects by studying a tightly bounded and well-characterised system that is easily modified by genetic means, and by focusing on deep understanding of a discrete number of important and accessible quantitative behaviours. During the project, the authors have developed tools to measure the appropriate data and develop models at appropriate levels of detail to study a number of these quantitative behaviours. The authors have also developed transportable experimental tools and conceptual frameworks for understanding other signalling ...
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The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational... more
The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and 3H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in ...
Research Interests: Molecular Biology, Cancer, Biology, Cell Biology, Cell Signaling, and 13 moreAutophagy, Medicine, Signal Transduction, Cell Differentiation, Phosphorylation, Subcellular Localization, Ciencias Biológicas, Protein kinase B, Akt, Golgi, Palmitoylation, Enseñanza - Aprendizaje Ciencias Naturales Y Exactas, and Lysosomes
Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors... more
Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused α-bungarotoxin, a neurotoxin derived from the snake Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine ...
Research Interests: Biophysics, Chemistry, Neurotransmission, Cell Biology, Fluorescent Dyes and Reagents, and 14 moreMedicine, Ph, Acetylcholine, Synaptic Transmission, Molecular Mechanisms of Synaptic Transmission, Synaptic Vesicle Recycling, Green Fluorescent Protein, Ciencias Biológicas, Electrophisiology, Enseñanza - Aprendizaje Ciencias Naturales Y Exactas, Fluorescent Chemosensors, Neuromuscular Junction, Fluorescent Sensor, and Fluorescent Chemical Sensors
Cells make decisions based on a combination of external and internal signals. In yeast, the high osmolarity response (HOG) is a mitogen-activated protein kinase (MAPK) pathway that responds to a variety of stimuli, and it is central to... more
Cells make decisions based on a combination of external and internal signals. In yeast, the high osmolarity response (HOG) is a mitogen-activated protein kinase (MAPK) pathway that responds to a variety of stimuli, and it is central to the general stress response. Here we studied the effect of heat-stress (HS) on HOG. Using live-cell reporters and genetics, we show that HS promotes Hog1 phosphorylation and Hog1-dependent gene expression, exclusively via the Sln1 phosphorelay branch, and that the strength of the activation is larger in yeast adapted to high external osmolarity. HS stimulation of HOG is indirect. First, we show that HS causes glycerol loss, necessary for HOG activation. Preventing glycerol efflux by deleting the glyceroporin FPS1 or its regulators RGC1 and ASK10/RGC2, or by increasing external glycerol, greatly reduced HOG activation. Second, we found that HOG stimulation by HS depended on the operation of a second MAPK pathway, the cell-wall integrity (CWI), a well-k...
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Cells make accurate decisions in the face of molecular noise and environmental fluctuations by relying not only on present pathway activity, but also on their memory of past signaling dynamics. Once a decision is made, cellular... more
Cells make accurate decisions in the face of molecular noise and environmental fluctuations by relying not only on present pathway activity, but also on their memory of past signaling dynamics. Once a decision is made, cellular transitions are often rapid and switch-like due to positive feedback loops in the regulatory network. While positive feedback loops are good at promoting switch-like transitions, they are not expected to retain information to inform subsequent decisions. However, this expectation is based on our current understanding of network motifs that accounts for temporal, but not spatial, dynamics. Here, we show how spatial organization of the feedback-driven yeast G1/S switch enables the transmission of memory of past pheromone exposure across this transition. We expect this to be one of many examples where the exquisite spatial organization of the eukaryotic cell enables previously well-characterized network motifs to perform new and unexpected signal processing func...
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Crosstalk between MAPK pathways activated by a single sensory input enhances yeast responses to multiple simultaneous stimuli.
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Research Interests: Systems Biology, Biology, Cell Biology, Medicine, Signal Transduction, and 15 moreSaccharomyces cerevisiae, Computer Simulation, Mutation, Molecular systems biology, Osmotic pressure, Yeast, Phosphorylation, Signalling, Glycerol, Homeostasis, Physiological Stress Markers, DNA binding proteins, Intracellular Space, Mitogen- Activated Protein Kinases, and Stress Physiological
Research Interests: Kinetics, Biology, Biophysical Chemistry, Medicine, Biological Sciences, and 13 moreSaccharomyces cerevisiae, Ste, Physical sciences, Membrane, CHEMICAL SCIENCES, Ciencias Biológicas, Enseñanza - Aprendizaje Ciencias Naturales Y Exactas, Protein Binding, Dissociation Constant, Protein Transport, Cell Membrane, binding sites, and Mitogen- Activated Protein Kinases
Cells detect changes in their environment and generate responses, often involving changes in gene expression. In this paper we use information theory and a simple transcription model to analyze whether the resulting gene expression serves... more
Cells detect changes in their environment and generate responses, often involving changes in gene expression. In this paper we use information theory and a simple transcription model to analyze whether the resulting gene expression serves to identify extracellular stimuli and assess their intensity when they are encoded in the amplitude, duration or frequency of pulses of a transcription factor’s nuclear concentration (or activation state). We find, for all cases, that about three ranges of input strengths can be distinguished and that maximum information transmission occurs for fast and high activation threshold promoters. The three input modulation modes differ in the sensitivity to changes in the promoters parameters. Frequency modulation is the most sensitive and duration modulation, the least. This is key for signal identification: there are promoter parameters that yield a relatively high information transmission for duration or amplitude modulation and a much smaller value fo...
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Polarity decisions are central to many processes, including mitosis and chemotropism. InSaccharomyces cerevisiae, budding and mating projection (MP) formation use an overlapping system of cortical landmarks that converges on the small G... more
Polarity decisions are central to many processes, including mitosis and chemotropism. InSaccharomyces cerevisiae, budding and mating projection (MP) formation use an overlapping system of cortical landmarks that converges on the small G protein Cdc42. However, pheromone-gradient sensing must override the Rsr1-dependent internal polarity cues used for budding. Using this model system, we asked what happens when intrinsic and extrinsic spatial cues are not aligned. Is there competition, or collaboration? By live-cell microscopy and microfluidics techniques, we uncovered three previously overlooked features of this signaling system. First, the cytokinesis-associated polarization patch serves as a polarity landmark independently of all known cues. Second, the Rax1-Rax2 complex functions as a pheromone-promoted polarity cue in the distal pole of the cells. Third, internal cues remain active during pheromone-gradient tracking and can interfere with this process, biasing the location of MP...
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Polarity decisions are central to many processes, including mitosis and chemotropism. In S. cerevisiae, budding and mating projection (MP) formation use an overlapping system of cortical landmarks that converge on the small G-protein... more
Polarity decisions are central to many processes, including mitosis and chemotropism. In S. cerevisiae, budding and mating projection (MP) formation use an overlapping system of cortical landmarks that converge on the small G-protein Cdc42. However, pheromone gradient sensing must override the Rsr1-dependent internal polarity cues used for budding. Using this model system, we asked what happens when intrinsic and extrinsic spatial cues are misaligned. Is there competition, or collaboration? By live cell microscopy and microfluidics technics we uncovered three previously overlooked features of this signaling system. First, the cytokinesis-associated polarization patch serves as a polarity landmark independently of all known cues. Second, the Rax1-Rax2 complex functions as novel pheromone promoted polarity cue in the distal pole of the cells. Finally, we showed that internal cues remain active during pheromone gradient tracking and that they interfere with this process biasing the loc...
Populations of isogenic cells often respond coherently to signals, despite differences in protein abundance and cell state. Previously, we uncovered processes in the pheromone response system (PRS) that reduced cell-to-cell variability in... more
Populations of isogenic cells often respond coherently to signals, despite differences in protein abundance and cell state. Previously, we uncovered processes in the pheromone response system (PRS) that reduced cell-to-cell variability in signal strength and cellular response. Here, we screened 1,141 non-essential genes to identify 50 "variability genes". Most had distinct, separable effects on strength and variability of the PRS, defining these quantities as genetically distinct "axes" of system behavior. Three genes affected cytoplasmic microtubule function: and We used genetic and chemical perturbations to show that, without microtubules, PRS output is reduced but variability is unaffected, while, when microtubules are present but their function is perturbed, output is sometimes lowered, but its variability is always high. The increased variability caused by microtubule perturbations required the PRS MAP kinase Fus3 and a process at or upstream of Ste5, the me...
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We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein... more
We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell-cycle stages. Surprisingly, in S phase, where Ste5 recruitment should be blocked, we observed an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK modify shared sites, which are most extensively phosphorylated when both kinases are active and able to bind their docking sites o...
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Studies of cell-to-cell variation have in recent years grown in interest, due to improved bioanalytical techniques which facilitates determination of small changes with high uncertainty. Like much high-quality data, single-cell data is... more
Studies of cell-to-cell variation have in recent years grown in interest, due to improved bioanalytical techniques which facilitates determination of small changes with high uncertainty. Like much high-quality data, single-cell data is best analysed using a systems biology approach. The most common systems biology approach to single-cell data is the standard two-stage (STS) approach. In STS, data from each cell is analysed in a separate sub-problem, meaning that only data from the same cell is used to calculate the parameter values within that cell. Because only parts of the data are considered, problems with parameter unidentifiability are exaggerated in STS. In contrast, a related approach to data analysis has been developed for the studies of patient-to-patient variations. This approach, called nonlinear mixed-effects modelling (NLME), makes use of all data, when estimating the patient-specific parameters. NLME would therefore be advantageous compared to STS also for the study of...
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The process of embryo implantation requires extensive remodelling of the endometrial extracellular matrix, a function largely performed by matrix-degrading metalloproteinases (MMPs). In the present study, we used trophoblast cells... more
The process of embryo implantation requires extensive remodelling of the endometrial extracellular matrix, a function largely performed by matrix-degrading metalloproteinases (MMPs). In the present study, we used trophoblast cells isolated from human term placentas to study the regulation of MMPs by nitric oxide (NO). Using a combination of zymography, Western blot and indirect immunofluorescence, we showed that MMP-2 and MMP-9 are increased during the conversion from low-motile cytotrophoblast cells to the highly motile and differentiated syncytiotrophoblast multinucleated cells. We also observed an increase in NO production and NO synthase (NOS) expression during this cellular differentiation process. In addition, we demonstrated a positive regulatory role of NO on the activity and protein expression of MMP-2 and MMP-9, because NO donors (NOC-18 and spermine-NONOate) or the NOS substrate (L-arginine) stimulate, whereas NOS inhibitors (N(G)-nitro-L-arginine methyl ester and N(G)-mo...
Research Interests: Endocrinology, Genetics, Andrology, Art, Fertility, and 31 moreEnzyme Inhibitors, Cell Biology, Cryopreservation, Oogenesis, Western blotting, Embryo, Theriogenology, Biological Sciences, Cell Differentiation, Pregnancy, Humans, Spermatogenesis, Scientific, Educational, Nitric oxide, Reproduction and human fertility, Female, Transgenesis, Cloning, Fluorescent Antibody Technique, Fertilization, Embryonic Development, Nitric Oxide Synthase, Folliculogenesis, Arginine, Embryogenesis, Gametogenesis, Fertilisation, Embryo Development, Matrix Metalloproteinase, and Nitric oxide donors
This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor mRNA, play specific roles during follicular development. In particular, we... more
This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor mRNA, play specific roles during follicular development. In particular, we analyzed the presence of the ED-I region, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I + FN whereas total FN levels remained relatively constant. A negative correlation (P < 0.001) was detected between ED-I + FN and estradiol levels. This steroid was without effect on the alternative splicing of FN in primary cultures of bovine granulosa cells. However, cAMP produced a marked decrease in the incorporation of the ED-I region. In contrast, transforming growth factor beta (TGF-beta) elicited both a stimulation on overall FN synthesis and an increase in the inclusion of ED-I. This effect was evident...
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Much work has been done on the study of the biochemical mechanisms that result in ultrasensitive behavior of simple biochemical modules. However, in a living cell, such modules are embedded in a bigger network that constrains the range of... more
Much work has been done on the study of the biochemical mechanisms that result in ultrasensitive behavior of simple biochemical modules. However, in a living cell, such modules are embedded in a bigger network that constrains the range of inputs that the module will receive as well as the range of the module&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s outputs that network will be able to detect. Here, we studied how the effective ultrasensitivity of a modular system is affected by these restrictions. We use a simple setup to explore to what extent the dynamic range spanned by upstream and downstream components of an ultrasensitive module impact on the effective sensitivity of the system. Interestingly, we found for some ultrasensitive motifs that dynamic range limitations imposed by downstream components can produce effective sensitivities much larger than that of the original module when considered in isolation.
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This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over... more
This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposely defocused transmission image (sometimes referred to as bright-field or BF) is acquired to segment the image and locate each cell. Fluorescence images (one for each of the color channels to be analyzed) are then acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses the image-processing capabilities of Cell-ID and data analysis by the statistical programming framework R, which is supplemented with a package of routines for analyzing Cell-ID output. Both Cell-ID and the analysis package are open-source.
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We analyzed the presence of 3 beta-Hydroxysteroid Dehydrogenase/Delta(5--&gt;4)-isomerase enzyme (3 beta-HSD) activity, a key enzyme of the steroid metabolic pathway, the mRNA of this enzyme, and the steroid metabolism in in vitro... more
We analyzed the presence of 3 beta-Hydroxysteroid Dehydrogenase/Delta(5--&gt;4)-isomerase enzyme (3 beta-HSD) activity, a key enzyme of the steroid metabolic pathway, the mRNA of this enzyme, and the steroid metabolism in in vitro produced bovine embryos. 3 beta-HSD activity was detected in in vitro matured oocytes (74.4 +/- 1.4%), 1-cell (72.9 +/- 6.1%), 2-cell (61.8 +/- 7.4%), 8-cell (50 +/- 5%), morulae (50.8 +/- 2.6%), blastocysts (94.4 +/- 3%), and hatched blastocysts (100 +/- 0%) meanwhile the 4-cell stage showed a significant reduction (16.7 +/- 4.7%). When total embryonic RNA of different stages was subjected to RT-PCR assays, the mRNA of 3 beta-HSD was found to be present in all developmental stages of in vitro produced bovine embryos, from the oocyte to the blastocyst, with a marked decrease at the 4-cell stage. To determine whether the temporal pattern of enzyme activity was dependent on the maternal to zygotic transition, embryos were incubated in the presence of a transcription inhibitor, alpha-amanitin. The reappearance of the enzyme activity after the 4-cell stage was blocked in alpha-amanitin treated embryos, indicating the requirement of embryonic transcription. On the other hand, the embryonic steroid metabolism was tested by incubating blastocyst with tritiated pregnenolone. Analysis of the metabolites by TLC indicated the production of a compound with a mobility identical to progesterone. These results described the expression of the 3 beta-HSD and the activity of this metabolic enzyme in bovine oocytes and preimplantation embryos, suggesting that steroids may act as autocrine effectors on preimplantation embryo development.
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This unit describes a method to quantify, from sets of microscope images, various cellular parameters from individual cells, and includes procedures to track cells over time. For example, the user can measure cell volume, total and... more
This unit describes a method to quantify, from sets of microscope images, various cellular parameters from individual cells, and includes procedures to track cells over time. For example, the user can measure cell volume, total and subcellular localization (nuclear ...