Leonardo Martins

Increased cytoplasm viscosity hampers aggregate polar segregation in Escherichia coli

In Escherichia coli, under optimal conditions, protein aggregates associated with cellular aging are excluded from midcell by the nucleoid. We study the functionality of this process under sub‐optimal temperatures from population and time lapse images of individual cells and aggregates and nucleoids within. We show that, as temperature decreases, aggregates become homogeneously distributed and uncorrelated with nucleoid size and location. We present evidence that this is due to increased cytoplasm viscosity, which weakens the anisotropy in aggregate displacements at the nucleoid borders that is responsible for their preference for polar localisation. Next, we show that in plasmolysed cells, which have increased cytoplasm viscosity, aggregates are also not preferentially located at the poles. Finally, we show that the inability of cells with increased viscosity to exclude aggregates from midcell results in enhanced aggregate concentration in between the nucleoids in cells close to dividing. This weakens the asymmetries in aggregate numbers between sister cells of subsequent generations required for rejuvenating cell lineages. We conclude that the process of exclusion of protein aggregates from midcell is not immune to stress conditions affecting the cytoplasm viscosity. The findings contribute to our understanding of E. colis internal organisation and functioning, and its fragility to stressful conditions.

System for Posture Evaluation and Correction - Development of a Second Prototype for an Intelligent Chair

The sitting position has become one of the most common postures in developed countries. However, assuming a poor sitting posture leads to several health problems, namely back, shoulder and neck pain. In a previous work, an intelligent chair was developed and was shown to classify and correct the seating position. This work describes improvements on this intelligent chair prototype culminating with the development of a new prototype. The improvements of this new prototype are presented, resulting in new studies for posture identification. Pressure maps for 12 sitting postures were gathered in order to automatically detect user’s posture through a neural network algorithm, obtaining an overall posture classification of around 81%.

Sitting Posture Detection using Fuzzy Logic - Development of a Neuro-fuzzy Algorithm to Classify Postural Transitions in a Sitting Posture

In a previous work, a chair prototype was used to detect 11 standardized seating postures of users, using just 8 air bladders (4 in the chair’s seat and 4 in the backrest) and one pressure sensor for each bladder. In this paper we describe a new classification algorithm, which was developed in order to classify the postures using as input the Centre of Pressure, the Posture Adoption Time and the Posture Output from the existing Neural Network Algorithm. This new Posture Classification Algorithm is based on Fuzzy Logic and is able to determine if the user is adopting a good or a bad posture for specific time periods. The newly developed Classification Algorithms will prompt the improvement of new Posture Correction Algorithms based on

Optimization of sitting posture classification based on user identification

In a precursory work, an intelligent sensing chair prototype was developed to classify 12 standardized sitting postures using 8 pneumatic bladders (4 in the chairs seat and 4 in the backrest) connected to piezoelectric sensors to measure inner pressure. A Classification of around 80% was obtained using Neural Networks. This work aims to demonstrate how algorithmic optimization can be applied to a newly developed prototype to improve posture classification performance. The aforementioned optimization is based on the split of users by sex and use two different previously trained Neural Networks (one for Male and the other for Female). Results showed that the best neural network parameters had an overall classification 89.0% (from the 92.1% for Female Classification and 85.8% for Male, which translates into an overall optimization of around 8%). Automatic separation of these sets was achieved with Decision Trees with an overall classification optimization of 87.1%.

Dynamics of transcription of closely spaced promoters in Escherichia coli, one event at a time.

Many pairs of genes in Escherichia coli are driven by closely spaced promoters. We study the dynamics of expression of such pairs of genes driven by a model at the molecule and nucleotide level with delayed stochastic dynamics as a function of the binding affinity of the RNA polymerase to the promoter region, of the geometry of the promoter, of the distance between transcription start sites (TSSs) and of the repression mechanism. We find that the rate limiting steps of transcription at the TSS, the closed and open complex formations, strongly affect the kinetics of RNA production for all promoter configurations. Beyond a certain rate of transcription initiation events, we find that the interference between polymerases correlates the dynamics of production of the two RNA molecules from the two TSS and affects the distribution of intervals between consecutive productions of RNA molecules. The degree of correlation depends on the geometry, the distance between TSSs and repressors. Small changes in the distance between TSSs can cause abrupt changes in behavior patterns, suggesting that the sequence between adjacent promoters may be subject to strong selective pressure. The results provide better understanding on the sequence level mechanisms of transcription regulation in bacteria and may aid in the genetic engineering of artificial circuits based on closely spaced promoters.

An image generator platform to improve cell tracking algorithms: Simulation of objects of various morphologies, kinetics and clustering

Several major advances in Cell and Molecular Biology have been made possible by recent advances in live-cell microscopy imaging. To support these efforts, automated image analysis methods such as cell segmentation and tracking during a time-series analysis are needed. To this aim, one important step is the validation of such image processing methods. Ideally, the “ground truth” should be known, which is possible only by manually labelling images or in artificially produced images. To simulate artificial images, we have developed a platform for simulating biologically inspired objects, which generates bodies with various morphologies and kinetics and, that can aggregate to form clusters. Using this platform, we tested and compared four tracking algorithms: Simple Nearest-Neighbour (NN), NN with Morphology and two DBSCAN-based methods. We show that Simple NN works well for small object velocities, while the others perform better on higher velocities and when clustering occurs. Our new platform for generating new benchmark images to test image analysis algorithms is openly available at (http://griduni.uninova.pt/Clustergen/ClusterGen_vl.0.zip).

Optimization of Sitting Posture Classification based on Anthropometric Data

An intelligent chair prototype was developed in order to detect and correct the adoption of bad sitting postures during long periods of time. A pneumatic system was enclosed in the chair (4 air bladders inside the seat pad and 4 in the backrest) to classify 12 standardized sitting postures, with a classification score of 80.9%. Recently we used algorithmic optimization applied to the existing classification algorithm (based on Neural Networks) to split users (using Classification Trees) by their sex and used two different previously trained Neural Networks (Male and Female) to get an improved classification of 89.0% when the user was identified and 87.1% for unidentified users. In this work we aim to investigate the usage of the anthropometric information (height and weight) to further optimize our classification process. Here we use four Machine Learning Techniques (Neural Networks, Support Vector Machines, Classification Trees and Naive Bayes) to automatically split the users in 2 classes (above and below the specific anthropometric median value). Results showed that Classification Trees worked best on automatically separating the body characteristics (i.e. Height) with a global optimization of 88.3%. During the classification process, if the user is identified, we skip the splitting step, and this optimization increases to 90.2%.

Real-Time Fuzzy Monitoring of Sitting Posture: Development of a New Prototype and a New Posture Classification Algorithm to Detect Postural Transitions

In a previous work, a chair prototype was used to detect 11 standardized siting postures of users, using just 8 air bladders (4 in the chair’s seat and 4 in the backrest) and one pressure sensor for each bladder. In this paper we describe the development of a new prototype, which is able to classify 12 standard postures with an overall score of 80.9 % (using a Neural Network Algorithm). We tested how this Algorithm worked during postural transitions (frontal and lateral flexion) and in intermediate postures, identifying some limitation of this Algorithm. This prompted the development of a Posture Classification Algorithm based on Fuzzy Logic and is able to determine if the user is adopting a good or a bad posture for specific time periods, using as input the Centre of Pressure, the Posture Adoption Time and the Posture Output from the existing Neural Network Algorithm. This newly developed Classification Algorithms is advancing the development of new Posture Correction Algorithms based on Fuzzy Actuators.

Intelligent Chair Sensor

In order to build an intelligent chair capable of posture detection and correction we developed a prototype that gathers the pressure map of the chair’s seat pad and backrest and classifies the user posture and changes its conformation. We gathered the pressure maps for eleven standardized postures in order to perform the automatic posture classification, using neural networks. First we tried to find the best parameters for the neural network classification of our data, obtaining an overall classification of around 80% for eleven postures. Those neural networks were exported to a mobile application in order to do real-time classification of those postures. Results showed a real-time classification of around 70% for eleven standardized postures, but we improved the overall classification score to 93.4% when we reduced the posture identification to eight postures, even when this classification was done with unfamiliar users to the posture identification system.

The precision of the symmetry in Z-ring placement in Escherichia coli is hampered at critical temperatures

Cell division in Escherichia coli is morphologically symmetric due to, among other things, the ability of these cells to place the Z-ring at midcell. Studies have reported that, at sub-optimal temperatures, this symmetry decreases at the single-cell level, but the causes remain unclear. Using fluorescence microscopy, we observe FtsZ-GFP and DAPI-stained nucleoids to assess the robustness of the symmetry of Z-ring formation and positioning in individual cells under sub-optimal and critical temperatures. We find the Z-ring formation and positioning to be robust at sub-optimal temperatures, as the Z-rings mean width, density and displacement from midcell maintain similar levels of correlation to one another as at optimal temperatures. However, at critical temperatures, the Z-ring displacement from midcell is greatly increased. We present evidence showing that this is due to enhanced distance between the replicated nucleoids and, thus, reduced Z-ring density, which explains the weaker precision in setting a morphologically symmetric division site. This also occurs in rich media and is cumulative, i.e. combining richer media and critically high temperatures enhances the asymmetries in division, which is evidence that the causes are biophysical. To further support this, we show that the effects are reversible, i.e. shifting cells from optimal to critical, and then to optimal again, reduces and then enhances the symmetry in Z-ring positioning, respectively, as the width and density of the Z-ring return to normal values. Overall, our findings show that the Z-ring positioning in E. coli is a robust biophysical process under sub-optimal temperatures, and that critical temperatures cause significant asymmetries in division.