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Cellular and Molecular Biomechanics, Microscopy

Study of the mechanical properties of actin filaments of astrocytes infected by Trypanosome cruzi

Astrocytes are responsible of neuronal protection and of many key functions in the nervous system activity. Studies on the mechanical properties of astrocytes have shown that their Young modulus (elasticity modulus) decreases in the presence of drugs, specially, actin synthesis inhibitors. Chagas disease, generated by the Trypanosome cruzi parasite, compromises the central nervous system, including the astrocytes. We are interested in studying the changes in astrocyte rigidity when they are invaded by the parasite by the use of AFM microscopy, which should shed light on the effects of this parasite.

Super resolution with light sheet

We investigate new approaches in microscopy such as super resolution, light sheet and light field microscopy. We intend to develop novel, simple-to-implement techniques that can shed light on biological processes. We have designed and built our own light field microscope in our facility.

MECHANICAL CHARACTERIZATION OF TRYPANOSOMA CRUZI FLAGELLUM UNDER SHEAR STRESS CONDITIONS

The biophysical analysis of host pathogen interactions can offer insight of the adhesion processes and synthesis of new medicine. Parasites are subjected to intense shear of heterogeneous media in arteries and veins, thus, is interesting to understand their mechanism of adhesion. These mechanisms under force have been well characterized in bacteria and leukocytes, however, for parasites this mechanism is still unknown. In the following work, the behavior of the parasite is analyzed under the influence of numerous shear stresses

LIGHT SHEET FLUORESCENCE MICROSCOPY (LSFM) APPLIED TO DEVELOPMENT STUDIES IN ZEBRAFISH

The study of the mechanisms underlying both neural development and functioning in vivo is today possible thanks to light sheet fluorescence microscopy (LSFM) techniques, such as Single Plane Illumination Microscopy (SPIM). In this project, we develop a methodology to image developing zebrafish embryos using the SPIM. This allows us to identify neural structures expressing an extracellular matrix protein, F-spondin, and to characterize migration patterns of optic tectum (Tec) neurons. We image zebrafish embryos (transgenic line spon1b:GFP) from 24 to 70 hours post-fertilization (hpf), and we have identified possible primordia of olfactory bulbs (OB) and habenular (Hb) progenitors at 24 hpf. Additionally, we apply image processing and analysis to obtain single cell trajectories and the morphology, to determine the mechanisms that might be important during optic tectum development.

VISUALIZATION OF ESCHERICHIA COLI BIOFILMS USING SPIM

We are developing a system to follow biofilm formation, under conditions of continuous nutrient efflux, using Single Plane Illumination Microscopy. This type of microscopy offers increased spatial and temporal resolution in addition to low phototoxicity and photobleaching, and has been previously applied to other organisms such as zebrafish and C. elegans. It is of our interest, to apply it to bacteria in order to follow the process of biofilm formation and obtain a better understanding on how these communities develop.

Observation of the extension and retraction of bacterial type I pili under cutting flow

In this work we study the mechanical properties of type I pili in bacteria adhered to a surface and exposed to changing flow conditions. We compare our results with the ones obtained in previous studies with individual pili, and we suggest a hypothesis that explains the observed differences. We modeled the pili as a chain that follows the model of a 'freely jointed chain' and validate it with our experimental data. We finally make a parallel of the mechanical properties of bacterial pili with the properties of the mechanism used by mussels to stay adhered under marine waves.

Validation of Thick Calcification Method in Octocorals by fluorescence measurements

Nowadays, little is known about the physiology and action mechanisms after calcification processes in soft corals. In this project, we want to validate the use of experimental techniques to quantify the thick calcification in octocorals with the use of the SPIM (Selective Plane Illumination microscopy) and fluorescence microscopy.

Super resolution microscopy with 3B analysis

Beyond the optical limit –where it does’nt matter how good a microscope is- hide minuscule structures inside the cell. To be able to watch them and understand them better, super resolution techniques have been developed. These make use of the turning on and off of fluorescent molecules to produce images with nanometric scale detail. We work on the implementation of one of these techniques (3B analysis microscopy) in our lab. The following image (false color) shows the quality that can be reached. Frame 1 shows the fluorescent sample as seen on the microscope with a 100x objective. The scale (2000nm) is shown in white and the region of interest analyzed is shown in yellow. Frame 2 is a digital zoom of the region of interest shown in figure 1. Frame 3 shows the reconstruction in super resolution of the region of interest. The difference between the resolution of frames 2 and 3 (which show the same object) is notorious. This reconstruction in super resolution was obtained from 940 frames that were analyzed computationally during 1100 minutes.

Study of the mechanical properties of lipid membranes in the presence of antimicrobial peptides

Antimicrobial peptides (AP) have the purpose of killing bacteria without harming human cells. A subgroup of these AP includes short peptides with positive charge, which induce transmembrane pores after a concentration threshold of peptides has adhered to the membrane.

We are interested in studying if adhered peptides modify the mechanical structure of membranes before pores are formed, affecting membrane function from the initial adhesion. These mechanical properties (bending modulus, elastic modulus, Young modulus) will be obtained by force spectroscopy measurements using the AFM microscope.

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 Carrera 1 # 18-10

      Building Q, Laboratory 401/505

      Universidad de Los Andes

      Bogotá, Colombia

 (+57 1) 3394949

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