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Autoantibodies play an important role in the destruction of non-infected red blood cells (nRBCs) during malaria. However, the relationship between this clearance and ABO blood groups is yet to be fully enlightened, especially for Plasmodium vivax infections. Here we show that anti-RBC IgG and IgM are increased in anemic patients with acute vivax malaria. Furthermore, both antibodies are able to decrease the deformability of nRBCs, but only IgG can induce in vitro erythrophagocytosis. Such effects are enhanced in type O erythrocytes, suggesting that individuals from this blood group infected with P. vivax malaria may be more susceptible to develop anemia.

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not only attributed to the normal variability of cells, but also to the differences in methods and models of cell response. Here, an integrated protocol using optical tweezers and defocusing microscopy is employed to obtain the rheological features of red blood cells in the frequency range of 1 Hz to 35 Hz. While optical tweezers are utilized to measure the erythrocyte-complex elastic constant, defocusing microscopy is able to obtain the cell height profile, volume, and its form factor a parameter that allows conversion of complex elastic constant into complex shear modulus. Moreover, applying a soft glassy rheology model, the scaling exponent for both moduli can be obtained. The developed methodology allows to explore the mechanical behavior of red blood cells, characterizing their viscoelastic parameters, obtained under well-defined experimental conditions, for several physiological and pathological conditions.

Purpose: The purpose of this study was to investigate the vascular effects of photobiomodulation using a light-emitting diode on the chorioallantoic embryonic membrane of chicken eggs grouped into different times of exposure and to detect the morphological changes induced by the light on the vascular network architecture using quantitative metrics. Methods: We used a phototherapy device with light-emitting diode (670 nm wavelength) as the source of photobiomodulation. We applied the red light at a distance of 2.5 cm to the surface of the chorioallantoic embryonic membrane of chicken eggs in 2, 4, or 8 sessions for 90 s and analyzed the vascular network architecture using AngioTool software (National Cancer Institute, USA). We treated the negative control group with 50 μl phosphate-buffered-saline (pH 7.4) and the positive control group (Beva) with 50 μl bevacizumab solution (Avastin, Produtos Roche Químicos e Farmacêuticos, S.A., Brazil). Results: We found a decrease in total vessel length in the Beva group (24.96% ± 12.85%) and in all the groups that received 670 nm red light therapy (2× group, 34.66% ± 8.66%; 4× group, 42.42% ± 5.26%; 8× group, 38.48% ± 6.96%), compared with the negative control group. The fluence of 5.4 J/cm2 in 4 sessions (4×) showed more regular vessels. The number of junctions in the groups that received a higher incidence of 670 nm red light (4× and 8×) significantly decreased (p

The mechanical properties of erythrocytes have been investigated by different techniques. However, there are few reports on how the viscoelasticity of these cells varies during malaria disease. Here, we quantitatively map the viscoelastic properties of Plasmodium falciparum-parasitized human erythrocytes. We apply new methodologies based on optical tweezers to measure the viscoelastic properties and defocusing microscopy to measure the erythrocyte height profile, the overall cell volume, and its form factor, a crucial parameter to convert the complex elastic constant into complex shear modulus. The storage and loss shear moduli are obtained for each stage of parasite maturation inside red blood cells, while the former increase, the latter decrease. Employing a soft glassy rheology model, we obtain the power-law exponent for the storage and loss shear moduli, characterizing the soft glassy features of red blood cells in each parasite maturation stage. Ring forms present a liquid-like behavior, with a slightly lower power-law exponent than healthy erythrocytes, whereas trophozoite and schizont stages exhibit increasingly solid-like behaviors. Finally, the surface elastic shear moduli, low-frequency surface viscosities, and shape recovery relaxation times all increase not only in a stage-dependent manner but also when compared to healthy red blood cells. Overall, the results call attention to the soft glassy characteristics of Plasmodium falciparum-parasitized erythrocyte membrane and may provide a basis for future studies to better understand malaria disease from a mechanobiological perspective.

The azobenzene chromophore is used as a functional dye for the development of smart microfluidic devices. A single layer microfluidic channel is produced, exploiting the potential of a dye doped PDMS formulation. The key advantage of this approach is the possibility to control the fluid flow by means of a simple light stimulus. Furthermore, the deformation can be controlled in time, space and intensity, giving rise to several degrees of freedom in the actuation of the channel squeezing. A future perspective will be the implementation of the microfluidic platform with structured light, to have the possibility to control the flow in a parallel and reversible manner at several points, modifying the pattern in real time.

The red blood cell membrane-cytoskeleton is a complex structure mainly responsible for giving the cell rigidity and shape. It also provides the erythrocyte with the ability to pass through narrow capillaries of the vertebrate blood circulatory system. Although the red blood cell viscoelastic properties have been extensively studied, reported experimental data differ by up to three orders of magnitude. This could be attributed to the natural cell variability, to the different techniques employed, and also to the models used for the cell response, which are highly dependent on cell geometry. Here, we use two methodologies based on optical tweezers to investigate the viscoelastic behavior of healthy human red blood cells, one applying small cell deformations (microrheology) and another imposing large deformations (tether extraction). We also establish a defocusing microscopy-based method to characterize the cell geometry and thus the erythrocyte form factor, an essential parameter that allows comparisons among the viscoelastic properties at different conditions. Moreover, for small deformations, a soft glassy rheology model is used to discuss the results, while for large deformations two surface shear moduli and one surface viscosity are determined, together with the surface tension and bending modulus of the erythrocyte membrane lipid component. We also show that F-actin is not detected in tethers, although the erythrocyte membrane has physical properties like those of other adherent cells, known to have tethers containing F-actin inside. Altogether, our results show good agreement with the reported literature and we argue that, to properly compare the viscoelastic properties of red blood cells in different situations, the task of cell geometry characterization must be accomplished. This may be especially important when the influence of agents, like the malaria parasite, induces changes in both the geometry and chemical constituents of the erythrocyte membrane. Together, the new methodologies and procedures used in this study would allow the erythrocyte community to better explore the mechanical behavior of red blood cells and may be useful to characterize erythrocyte viscoelasticity changes in several blood diseases.

Defocusing microscopy (DM) is a bright-field optical microscopy technique often used to obtain structural parameters of objects with low difference in refractive index in relation to the surrounding medium (phase objects). We show a use of this technique to measure the refractive index (n) profile of waveguides produced by femtosecond laser micromachining inside the bulk of a sodalime glass. The results are used to analyze the influence of production parameters on n. The methodology requires only a bright-field optical microscope and has proved to be easily applied. Results provide important insights on the waveguide microfabrication process, since translation speed, rather than intensity, has shown to be more important for achieving greater variations in refractive indices. Index of refraction differences between the waveguide and the substrate of the order of 10-4 were measured for a series of straight waveguides fabricated with different parameters. Low sample scan speeds and pulse energies near 1.20 μJ used for fabrication showed the highest values of refractive index change for waveguides in sodalime glasses.

This manuscript describes the experimental observation of vasculogenesis in chick embryos by means of network analysis. The formation of the vascular network was observed in the area opaca of embryos from 40 to 55 h of development. In the area opaca endothelial cell clusters self-organize as a primitive and approximately regular network of capillaries. The process was observed by bright-field microscopy in control embryos and in embryos treated with Bevacizumab (Avastin ® ), an antibody that inhibits the signalling of the vascular endothelial growth factor (VEGF). The sequence of images of the vascular growth were thresholded, and used to quantify the forming network in control and Avastin-treated embryos. This characterization is made by measuring vessels density, number of cell clusters and the largest cluster density. From the original images, the topology of the vascular network was extracted and characterized by means of the usual network metrics such as: the degree distribution, average clustering coefficient, average short path length and assortativity, among others. This analysis allows to monitor how the largest connected cluster of the vascular network evolves in time and provides with quantitative evidence of the disruptive effects that Avastin has on the tree structure of vascular networks.

Estradiol (E2) prevents cardiac hypertrophy, and these protective actions are mediated by estrogen receptor (ER)α and ERβ. The G protein-coupled estrogen receptor (GPER) mediates many estrogenic effects, and its activation in the heart has been observed in ischemia and reperfusion injury or hypertension models; however, the underlying mechanisms need to be fully elucidated. Herein, we investigated whether the protective effect of E2 against cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) is mediated by GPER and the signaling pathways involved. Isolated neonatal female rat cardiomyocytes were treated with ET-1 (100 nmol/l) for 48 h in the presence or absence of E2 (10 nmol/l) or GPER agonist G-1 (10 nmol/l) and GPER antagonist G-15 (10 nmol/l). ET-1 increased the surface area of cardiomyocytes, and this was associated with increased expression of atrial and brain natriuretic peptides. Additionally, ET-1 increased the phosphorylation of extracellular signal-related protein kinases-1/2 (ERK1/2). Notably, E2 or G-1 abolished the hypertrophic actions of ET-1, and that was reversed by G-15. Likewise, E2 reversed the ET-1-mediated increase of ERK1/2 phosphorylation as well as the decrease of phosphorylated Akt and its upstream activator 3-phosphoinositide-dependent protein kinase-1 (PDK1). These effects were inhibited by G-15, indicating that they are GPER dependent. Confirming the participation of GPER, siRNA silencing of GPER inhibited the antihypertrophic effect of E2. In conclusion, E2 plays a key role in antagonizing ET-1-induced hypertrophy in cultured neonatal cardiomyocytes through GPER signaling by a mechanism involving activation of the PDK1 pathway, which would prevent the increase of ERK1/2 activity and consequently the development of hypertrophy.

Plasma membrane repair (PMR) is an important process for cell homeostasis, especially for cells under constant physical stress. Repair involves a sequence of Ca2+-dependent events, including lysosomal exocytosis and subsequent compensatory endocytosis. Cholesterol sequestration from plasma membrane causes actin cytoskeleton reorganization and polymerization, increasing cell stiffness, which leads to exocytosis and reduction of a peripheral pool of lysosomes involved in PMR. These changes in mechanical properties are similar to those observed in cells exposed to oxidized Low Density Lipoprotein (oxLDL), a key molecule during atherosclerosis development. Using a human umbilical vein endothelial cell line (EAhY926) we evaluated the influence of mechanical modulation induced by oxLDL in PMR and its effect in endothelial fragility. Similar to MβCD (a drug capable of sequestering cholesterol) treatment, oxLDL exposure led to actin reorganization and de novo polymerization, as well as an increase in cell rigidity and lysosomal exocytosis. Additionally, for both MβCD and oxLDL treated cells, there was an initial increase in endocytic events, likely triggered by the peak of exocytosis induced by both treatments. However, no further endocytic events were observed, suggesting that constitutive endocytosis is blocked upon treatment and that the reorganized cytoskeleton function as a mechanical barrier to membrane traffic. Finally, the increase in cell rigidity renders cells more prone to mechanical injury. Together, these data show that mechanical modulation induced by oxLDL exposure not only alters membrane traffic in cells, but also makes them more susceptible to mechanical injury, which may likely contribute to the initial steps of atherosclerosis development.

article i nfo A group of cysteine-proteolytic enzymes from C. candamarcensis latex, designated as P1G10 displays pharmaco- logical properties in animal models following various types of lesions. This enzyme fraction expresses in vitro fi- brinolytic effect without need for plasminogen activation. Based on this evidence, we assessed by intravital microscopy the effect of P1G10 on recanalization of microvessels after thrombus induction in the ear of hairless mice. Video playback of intravital microscopic images allowed measurement of blood flow velocity (mm/s) dur- ing the experimental procedure. Groups treated with 5 or 7.5 mg/Kg P1G10 showed thrombolysis between 7-15 min, without vessel obstruction. Ex vivo experiments demonstrated that platelet activation by ADP is im- paired in a dose dependent manner following treatment with P1G10. The P1G10 action on plasma coagulation also showed that prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (aPTT, μg/uL) are increased in a dose dependent manner. In addition, P1G10 displayed fibrinogenolytic and fibrinolytic activities, both ina dose dependent manner. Each of these effects was suppressed by inhibition of the proteolytic activityof thefraction.TheantithromboticactionofP1G10canbeexplainedbyproteolyticcleavage of fibrinogen and fibrin, both key factors during formation of a stable thrombus. These results combined with prior evidence suggest that P1G10 has potential as thrombolytic agent.

We use a quantitative phase imaging technique, defocusing microscopy (DM), to measure morphological, chemical, and mechanical parameters of individual red blood cells (RBCs) immersed in solutions with different osmolalities. We monitor the RBCs’ radius, volume, surface area, sphericity index, and hemoglobin content and concentration. The complete shape of cells is recovered and the effects of their adhesion to the glass substrate are observed. Finally, membrane fluctuation measurements give us information about the cells deformability.

Background Plasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated. Methods Concentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group. Results Anaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance. Conclusions These findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.

We study the interface morphology of a 2D simulation of an avascular tumor composed of identical cells growing in an homogeneous healthy tissue matrix (TM), in order to understand the origin of the morphological changes often observed during real tumor growth. We use the Glazier-Graner-Hogeweg model, which treats tumor cells as extended, deformable objects, to study the effects of two parameters: a dimensionless diffusion-limitation parameter defined as the ratio of the tumor consumption rate to the substrate transport rate, and the tumor-TM surface tension. We model TM as a nondiffusing field, neglecting the TM pressure and haptotactic repulsion acting on a real growing tumor; thus, our model is appropriate for studying tumors with highly motile cells, e.g., gliomas. We show that the diffusion-limitation parameter determines whether the growing tumor develops a smooth (noninvasive) or fingered (invasive) interface, and that the sensitivity of tumor morphology to tumor-TM surface tension increases with the size of the dimensionless diffusion-limitation parameter. For large diffusion-limitation parameters, we find a transition (missed in previous work) between dendritic structures, produced when tumor-TM surface tension is high, and seaweed-like structures, produced when tumor-TM surface tension is low. This observation leads to a direct analogy between the mathematics and dynamics of tumors and those observed in nonbiological directional solidification. Our results are also consistent with the biological observation that hypoxia promotes invasive growth of tumor cells by inducing higher levels of receptors for scatter factors that weaken cell-cell adhesion and increase cell motility. These findings suggest that tumor morphology may have value in predicting the efficiency of antiangiogenic therapy in individual patients.

Defocusing microscopy (DM) is a recently developed technique that allows quantitative analysis of membrane surface dynamics of living cells using a simple bright-field optical microscope. According to DM, the contrast of defocused images is proportional to cell surface curvature. Although, until now, this technique was used mainly to determine size and amount of membrane shape fluctuations, such as ruffles and small random membrane fluctuations, in macrophages, its applications on cell biology extend beyond that. We show how DM can be used to measure optical and mechanical properties of a living macrophage, such as cell refractive index n, membrane bending modulus K(c), and effective cell viscosity eta for membrane-actin meshwork relaxation. Experimental data collected from defocused images of bone marrow-derived macrophages were used to evaluate these parameters. The obtained values, averaged over several different macrophages, are n = (1.384 +/- 0.015), K(c) approximately 3.2 x 10(-19) J, and eta approximately 459 Pa.s. We also estimate the amplitude of the small fluctuations to be of the order of 3 nm, which is around the step size of a polymerizing actin filament.

Defocusing microscopy was used for real-time observation and quantification of membrane surface dynamics in murine bone marrow macrophages. Small random membrane fluctuations (SRMF), possibly metabolic driven, were detected uniformly over all membrane surface. Morphological and dynamical parameters of ruffles, such as shape, dimensions, and velocity of propagation, were analyzed. Optical tweezers were used to promote phagocytosis of single Leishmania amazonensis amastigotes by selected macrophages. Analysis of ruffling activity on the macrophages before and during phagocytosis of the parasites indicated that increased ruffling response near forming phagosomes, most likely induced by the parasite, accelerates phagocytosis. The effects of temperature decrease on the dynamics of membrane surface fluctuations and on the phagocytosis of parasites were used to determine the overall activation energies involved in these processes. The values obtained support the existence of strong correlation between membrane motility and phagocytic capacity.

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

We use the Glazier-Graner-Hogeweg model to simulate three-dimensional (3D), single-phenotype, avascular tumors growing in an homogeneous tissue matrix (TM) supplying a single limiting nutrient. We study the effects of two parameters on tumor morphology: a diffusion-limitation parameter defined as the ratio of the tumor-substrate consumption rate to the substrate-transport rate, and the tumor-TM surface tension. This initial model omits necrosis and oxidative/hypoxic metabolism effects, which can further influence tumor morphology, but our simplified model still shows significant parameter dependencies. The diffusion-limitation parameter determines whether the growing solid tumor develops a smooth (noninvasive) or fingered (invasive) interface, as in our earlier two-dimensional (2D) simulations. The sensitivity of 3D tumor morphology to tumor-TM surface tension increases with the size of the diffusion-limitation parameter, as in 2D. The 3D results are unexpectedly close to those in 2D. Our results therefore may justify using simpler 2D simulations of tumor growth, instead of more realistic but more computationally expensive 3D simulations. While geometrical artifacts mean that 2D sections of connected 3D tumors may be disconnected, the morphologies of 3D simulated tumors nevertheless correlate with the morphologies of their 2D sections, especially for low-surface-tension tumors, allowing the use of 2D sections to partially reconstruct medically-important 3D-tumor structures.

We report experimental results concerning observation of a pattern forming system, subject to directional viscous fingering (printer's instability). This system was excited by a time-dependent, periodic perturbation. A variety of spatiotemporal effects was observed, including pattern transient dynamics, wave vector selection, and morphological transitions. Detailed measurement of pattern shape and its associated Fourier modes assured the detection of a crossover between different regimes of the pattern evolution.

Mutalysin II (mut-II) is an a-fibrinogenase isolated from Lachesis muta muta (bushmaster) snake venom. The enzyme lyses fibrin clots in vitro, and this activity does not depend on plasminogen activation.The aim of this study was to assess by intravital microscopy the effect of Mutalysin II on the recanalization of microvessels after thrombus induction in the ears of hairless mice. Photochemical thrombus formation was induced after i.v. injection of 5% fluorescein isothiocyanate labelled dextran (FITC-dextran) followed by mercury light exposure of individual microvessels of the ear of five anesthetized animals. Video playback analysis of intravital microscopy images of the ear microcirculation permitted us to measure blood flow velocity (mm/s) under control conditions (before thrombus formation) in the ear microvessels. Thirty minutes after thrombus formation (blood flow velocity stopped completely), each animal (n ¼ 5) was infused with Mutalysin II (2.0 mg/kg, i.v.). All animals treated with Mutalysin II showed evident thrombolysis after approximately 12 min, followed by recanalization. A separate group of mice (n ¼ 5) which received urokinase type-plasminogen activator (u-PA, 250 U/mouse, i.v.) showed blood flow restoration within the same interval (12 min). These in vivo data suggest that Mutalysin II has the potential to be an effective thrombolytic agent. r 2007 Elsevier Ltd. All rights reserved.

Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional (2D) Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature (Laplacian of the local thickness) of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations (ruffles) on the surface of macrophages (cell of the innate immune system), and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.

We introduce Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total three-dimensional (3D) imaging of transparent objects. By total 3D imaging, we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic, and hypertonic solutions. For each situation, the shapes of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of red blood cell (RBC) surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine the volume, surface area, sphericity index, and RBC refractive index for each osmotic condition.

We present measurements of the bulk Young's moduli of early chick embryos at Hamburger-Hamilton stage 10. Using a micropipette probe with a force constant k ~0.025 N/m, we applied a known force in the plane of the embryo in the anterior-posterior direction and imaged the resulting tissue displacements. We used a two-dimensional finite-element simulation method to model the embryo as four concentric elliptical elastic regions with dimensions matching the embryo's morphology. By correlating the measured tissue displacements to the displacements calculated from the in-plane force and the model, we obtained the approximate short time linear-elastic Young's moduli: 2.4 ± 0.1 kPa for the midline structures (notocord, neural tube, and somites), 1.3 ± 0.1 kPa for the intermediate nearly acellular region between the somites and area pellucida, 2.1 ± 0.1 kPa for the area pellucida, and 11.9 ± 0.8 kPa for the area opaca.

Thin transparent objects (phase objects) can become visible in a bright-field light microscope, if the microscope is slightly defocused. Thick transparent objects, like red blood cells (RBC), are seen because some of their parts are always out of focus. By applying our recently developed defocusing microscopy technique to RBC, we are able to interpret RBC bright-field light microscopy images, an old standing problem. From the average image contrast we obtain RBC shape, size, and refractive index. From contrast fluctuations caused by the flicker phenomenon, we obtain RBC bending modulus and cytoplasm viscosity.

We present a simple bright-field optical microscopy technique, which we termed Defocusing Microscopy (DM) [1-3], able to actually reconstruct the 3-dimensional (3D) structure of phase objects. For this reconstruction it is only necessary to measure the object DM contrast at two different objective focal positions. We apply the developed method to red blood cells and obtain the shapes of the upper and lower surfaces, the last one deformed by the substrate. We reconstruct cells in isotonic, hypotonic conditions, as shown in the figure below. Additionally, we obtain the height fluctuations, with nanometer sensitivity, and the elastic modulii of each surface separately. For entire 3D imaging of phase objects in motion, one can use two cameras focused on two different positions. Therefore, DM technique can also be applied to obtain 3D reconstruction of cells flowing in micro-fluidic devices.[1] U. Agero, C.H. Monken, C. Ropert, R.T. Gazzinelli, and O.N. Mesquita, Phys. Rev. E 67, 051904 (2003).[2] L.G. Mesquita, U. Agero, and O.N. Mesquita, Applied Phys. Lett. 88, 133901 (2006).[3] G. Glionna, C.K. Oliveira; L.G. Siman, Moyses HW, D.M.U. Prado, C.H. Monken, and O.N. Mesquita, Applied Phys. Lett. 94, 193701 (2009).View Large Image | View Hi-Res Image | Download PowerPoint Slide

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A microscopia de desfocalização é uma técnica que vem sendo empregada no Laboratório de Física de Sistemas Biológicos da UFMG para caracterizar objetos transparentes, como células e guias de onda. Através dela podemos obter parâmetros morfológicos e biomecânicos de células vivas utilizando apenas um microscópio óptico operando em campo claro. A partir de duas imagens desfocalizadas, é possível realizar a reconstrução 3D da amostra por meio da resolução de duas equações diferenciais. Apesar de bastante conveniente para o estudo de amostras biológicas, a aplicação da técnica é limitada pelo custo computacional para a análise dos dados. Este trabalho teve como objetivo encontrar uma maneira mais eficiente para realizar a reconstrução 3D. Para isso, desenvolvemos programas em Python que recebem duas imagens desfocalizadas como entrada e retornam a espessura e assimetria da célula. Para o cálculo da assimetria, a parte mais demorada do processo, os programas utilizam o método de elementos finitos por meio da plataforma Fenics. Realizamos testes para três situações diferentes: reconstrução de objetos de fase simulados, reconstrução de uma hemácia e a reconstrução de um cardiomiócito. Para os dois primeiros casos pudemos comparar os resultados obtidos aqui com aqueles obtidos pelos métodos que já haviam sido estabelecidos em trabalhos anteriores. O principal resultado deste trabalho foi a diminuição substancial no tempo necessário para realizar a reconstrução. Os novos programas demoram cerca de 10 s para realizar a reconstrução de células partindo de imagens com 128×128 pixels enquanto, para a metodologia anterior a este trabalho, o processo demorava cerca 7200 s em hardware similar. Por fim, conseguimos realizar a reconstrução de um cardiomiócito, uma célula consideravelmente maior, também em cerca de 10 s. Esses resultados indicam que o método de elementos finitos é uma boa alternativa para o cálculo da assimetria, retornando resultados rapidamente mesmo para imagens com resolução mais alta. Esses resultados abrem perspectivas para o uso da microscopia de desfocalização para o estudo de processos celulares dinâmicos. Defocusing microscopy is a technique that has been used at the Physics Laboratory of Biological Systems at UFMG to characterize transparent objects, such as cells and waveguides. Through it we can obtain morphological and biomechanical parameters of living cells using only an optical microscope operating in bright field. From two defocused images, it is possible to perform the 3D reconstruction of the sample by solving two differential equations. Despite being quite convenient for the study of biological samples, the application of the technique is limited by the computational cost of data analysis. This work aimed to find a more efficient way to perform the 3D reconstruction. For this, we develop Python programs that take two defocused images as input and return the cell thickness and asymmetry. For the asymmetry calculation, the most time-consuming part of the process, the programs use the finite element method through the Fenics platform. We performed tests for three different situations: reconstruction of simulated phase objects, reconstruction of a red blood cell and reconstruction of a cardiomyocyte. For the first two cases, we were able to compare the results obtained here with those obtained by the methods that had already been established in previous works. The main result of this work was the substantial decrease in the time needed to perform the reconstruction. The new programs take around 10 s to perform cell reconstruction starting from images with 128×128 pixels while, for the methodology prior to this work, the process took around 7200 s on similar hardware. Finally, we were able to perform the reconstruction of a cardiomyocyte, a considerably larger cell, also in about 10 s. These results indicate that the finite element method is a good alternative for the asymmetry calculation, returning results quickly even for higher resolution images. These results open perspectives for the use of defocusing microscopy to study dynamic cellular processes.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior FINEP - Financiadora de Estudos e Projetos, Financiadora de Estudos e Projetos INCT – Instituto nacional de ciência e tecnologia (Antigo Instituto do Milênio) Esta tese aborda às aplicações de técnicas de óptica não linear em diferentes tipos de materiais. Sobretudo, abordamos as aplicações da geração de segundo harmônico e da mistura de quatro ondas em materiais bidimensionais,neste caso da família dos dicalcogenetos de metais de transição. Também, utilizamos técnicas de espectroscopia Raman e Raman estimulado para identificação de depósitos de proteína beta-amiloide no cérebro de camundongos geneticamente modificados para desenvolver a doença de Alzheimer. No primeiro caso, utilizamos a geração de segundo harmônico, que é um efeito de óptica não linear de segunda ordem, e da mistura de quatro ondas, fenômeno de terceira ordem, para identificar ressonâncias com os éxcitons de monocamadas de MoS2, MoSe2 e WS2. Somando-se a isso, medimos e caracterizamos a susceptibilidade elétrica desses materiais, dentro e fora, da ressonância dos éxcitons. Esses resultados foram comparados com valores experimentais medidos por diferentes técnicas ópticas disponíveis na literatura. Para o caso dos materiais biológicos, estudamos as placas formadas pelo acúmulo de beta-amiloide no cérebro dos camundongos. Realizamos além de medidas de Raman convencional, para caracterizar as placas, a geração de segundo harmônico, a autofluorescência por excitação de dois fótons e a fluorescência por excitação de dois fótons utilizando marcadores, com o intuito de observar as placas, além de determinar sua composição. Utilizamos, também, técnicas de Raman estimulado, nesse caso o SRS e o CARS, com a finalidade de reduzir o tempo de exposição do material em relação ao hiperespectro Raman convencional, com a finalidade de caracterzar as placas amiloide em relação à picos Raman específicos. Com base nos resultados destas diferentes técnicas, fomos capazes de diferenciar os animais por idade, utilizando a técnica de análise de componente principal. Um outro aspecto do estudo foi combinar as análises de SRS com medidas de fluorescência por excitação de dois fótons, marcando células da glia com o intuito de diferenciar as células que participam da formação da placa amiloide. This thesis addresses the applications of non-linear optics techniques in different materials. Above all, we approach the applications of second harmonic generation and four-wave mixing in two-dimensional materials, in this case of the transition metal dichalcogenides family. Also, we used Raman and Stimulated Raman spectroscopy techniques to identify beta-amyloid protein deposits in the brain of mice genetically modified to develop Alzheimer's disease. In the first case, we used second-harmonic generation, which is a second-order nonlinear optical effect, and four-wave mixing, a third-order phenomenon, to identify resonances with the excitons of MoS2, MoSe2, and WS2 monolayers. In addition, we measure and characterize the electrical susceptibility of these materials, inside and outside, to exciton resonance. These results were compared with experimental values ​​measured by different optical techniques available in the literature. In the case of biological materials, we studied the plaques formed by the accumulation of beta-amyloid in the brains of mice. In addition to conventional Raman measurements, to characterize the plates, we performed second harmonic generation, autofluorescence by two-photon excitation and fluorescence by two-photon excitation using markers, in order to observe the plates, in addition to determining their composition. . We also used stimulated Raman techniques, in this case, the SRS and CARS, in order to reduce the exposure time of the material in relation to the conventional Raman hyperspectral, in order to characterize the amyloid plaques in relation to specific Raman peaks. Based on the results of these different techniques, we were able to differentiate the animals by age using the principal component analysis technique. Another aspect of the study was to combine SRS analyzes with fluorescence measurements by two-photon excitation, labeling glial cells in order to differentiate the cells that participate in the formation of the amyloid plaque.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A quantificação da contratilidade dos cardiomiócitos é uma tarefa biomédica essencial e uma etapa fundamental na compreensão dos mecanismos envolvidos na patogênese da doença cardíaca. No entanto, medidas precisas e reprodutíveis são dificultadas por vários fatores inerentes às opções de software disponíveis atualmente. Por tanto, o presente trabalho tem por objetivo desenvolver uma tecnologia inovadora para quantificar a biomecânica dos cardiomiócitos por meio de um open-source software escrito em linguagem Python. Foram utilizadas imagens celulares obtidas em microscópio de campo claro e processadas pelo método de fluxo óptico denso. Para validar a eficiência do software, tratamos células de camundongos e ratos machos adultos (12 semanas), neonatais (1-3 dias) e cardiomiócitos derivados de células-tronco pluripotentes induzidas por humanos (hiPSCM) com drogas que possuem efeitos bem definidos nos parâmetros de contratilidade (isoproterenol ou verapamil). A análise estatística foi realizada por meio de ANOVA e Teste t student, utilizando p

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior O Imageamento Hiperespectral (IH) é uma técnica utilizada para obter a assinatura espectral de uma amostra com resolução espacial, permitindo a identificação de um ou mais espectros em uma mesma amostra. O IH é amplamente utilizado em escala macroscópica para a caracterização e estudo de amostras em diversas áreas de aplicação desde o sensoriamento remoto da superfície do planeta à conservação e restauração de obras de arte. Nos últimos anos apreciamos o desenvolvimento do IH em escala microscópica, isto é imagens com resolução espacial da ordem de micrometros com resolução espectral. A aplicação de técnicas ópticas em conjunto com o imageamento microscópico é de grande interesse para o estudo de materiais biológicos, especialmente as aplicações como ferramentas auxiliares para diagnósticos patológicos. Atualmente os métodos de diagnóstico se baseiam majoritariamente na análise visual dos tecidos, o que envolve procedimentos de preparação das amostras que implicam custos financeiros e demandam tempo para cortes e colorações para obtenção das lâminas de microscopia. A literatura aponta vantagens na utilização de técnicas ópticas, especialmente a espectroscopia óptica, como uma possível ferramenta de diagnóstico de patologias. Resultados recentes apontam a possibilidade da utilização da espectroscopia óptica em conjunto com o imageamento microscópico para o diagnóstico de pré-câncer, isto é, o estágio próximo do início do desenvolvimento do câncer em que a morfologia do tecido ainda não foi alterada o suficiente para o diagnóstico com os métodos amplamente utilizados. Neste trabalho apresentamos o desenvolvimento de um Microscópio Hiperespectral e a aplicação em materiais biológicos. O microscópio hiperespectral (MH) consiste em um microscópio óptico customizado com a adaptação de um interferômetro baseado em materiais birrefringentes que servem como a base para um espectrômetro por Transformada de Fourier (ETF). Apresentamos uma revisão dos processos físicos que possibilitam a utilização de elementos birrefringentes em um ETF. Discutimos detalhes da montagem, como resolução espacial e espectral e apresentamos as rotinas de calibração usadas. Apresentamos amostras de calibração, amostras de teste e a aplicação em cortes histológicos de biópsia em tecido de próstata. Hyperspectral Imaging (HI) is a technique used to obtain the spectral signature of a sample with spatial resolution, allowing the identification of one or more spectra from the sample. HI is widely used on macroscopic scale for characterization and study in a wide range of applications, from remote sensing of the planet's surface to art conservation and restoration. Recently, the development of HI on a microscopic scale has been achieved, that is, images with spatial resolution about micrometers and with spectral resolution. The application of optical techniques associated to microscopic imaging is a subject of interest for the study of biological materials, especially applications as acessorial tools for pathological diagnosis. Currently, diagnostic methods are mainly based on tissue visual evaluation, which leads to the need for sample preparation procedures that imply financial costs and time for staining and preparing microscopic slides. The literature points out advantages in the use of optical techniques, especially optical spectroscopy, as a possible tool for diagnosing pathologies. In particular, the possibility of using optical spectroscopy associated to microscopic imaging for pre-cancer diagnosis, the stage close to the beginning of cancer development where the tissue morphology does not present observable alterations by the methods widely used for diagnosis. The aim of this work is to present the development of a Hyperspectral Microscope (HM) and its applications for analysis of biological materials. HM is a custom-made microscope coupled to a birefringent-based interferometer used to perform Fourier Transform Spectroscopy (FTS). We present a review of the physical processes that allow the use of birefringent elements in an FTS. We show the experimental details, such as spatial and spectral resolution and calibration routines. We also present results for calibration samples, test samples and the application in histological sections of prostate tissue biopsy.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais O modelo Peyrard-Bishop (PB) é parte de um conjunto de modelos chamados mesoscópicos, que consiste em uma descrição clássica das interações moleculares com potenciais simples e que permite o cálculo da temperatura de desnaturação com eficiência em moléculas como o DNA e RNA. Nesse modelo, a dupla fita é considerada perfeitamente plana, ou seja, sem a caracterı́stica torção helicoidal e também não são considerados quaisquer parâmetros relaciconados à dimensão molecular. O modelo permite a integração trivial de um dos seus graus de liberdade, restando apenas um grau para ser resolvido por técnicas de integração numérica como a técnica de integral de transferência, razão pela qual é mais conhecido como modelo unidimensional (1D). Na Hamiltoniana original, o modelo produz uma curva de desnaturação muito suave enquanto seria esperado uma transição mais abrupta, o que requer a adição de termos não-lineares artificiais. As definições usadas na Hamiltoniana PB tornam bastante difı́cil a comparação dos seus resultados com os de técnicas microscópicas como dinâmica molecular atomı́stica. Desde a introdução do modelo em 1989, há um esforço em obter Hamiltonianas tridimensionais, mas, em geral, elas não são redutı́veis à equações 1D, isto é, a um modelo onde reste apenas uma dimensão a ser integrada pela técnica de integral de transferência. Obter um modelo tridimensional que possa ser reduzido a apenas uma dimensão integrável tem sido um problema em aberto. Nosso trabalho consistiu em fazer uma análise detalhada do modelo Peyrard-Bishop e elaborar um modelo tridimensional com caracterı́sticas geométricas mais próximas dos modelos atomı́sticos que o PB bidimensional. Desenvolvemos algumas aproximações que nos permitiram obter a redução da dimensão integrável, efetivamente obtendo um novo modelo 1D, e que nos permite utilizar a técnica de integral de transferência. Diferente do modelo oriundo da dimensão bidimensional, o novo modelo resulta em transições abruptas sem necessitar de potenciais artificialmente introduzidos. De fato, foi possı́vel demonstrar que as transições abruptas resultam diretamente por termos considerando três graus de liberdade. Para quantificar os efeitos das aproximações, realizamos a integração numérica da Hamiltoniana sem aproximação alguma. Com isso mostramos que o novo modelo é adequado à condição de pequenos ângulos que é similar à situação da fita dupla quando se encontra próximo à temperatura de desnaturação. Uma vantagem adicional do novo modelo é que pode ser aplicado sem modificação ao conjunto de técnicas desenvolvidas para sequências de DNA heterogêneas.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Este trabalho apresenta um estudo experimental da vasculogênese em embriões de galinha sob uma perspectiva física. Utilizamos conceitos de física estatística associados à ciência de redes para caracterizar a auto-organização dos aglomerados celulares em uma rede única de capilares. Para isso, acompanhamos por meio da microscopia ótica de campo claro a formação da rede vascular durante 15 horas do desenvolvimento embrionário. Avaliamos a auto-organização dos aglomerados de células endoteliais em embriões controles, sem tratamento, e em embriões tratados com bevacizumabe (Avastin), um anticorpo que inibe a sinalização do fator de crescimento de célula endotelial [do inglês vascular endothelial growth factor (VEGF)]. Neste trabalho estudamos a vasculogênese no embrião controle, ou seja sem restrição de VEGF, e caracterizamos a formação da rede vascular discutindo os princípios físicos que conduzem o comportamento coletivo dos aglomerados celulares na formação da rede vascular. Além disso, caracterizamos as topologias das redes vasculares diminuindo a concentração de VEGF livre. Para isso, desenvolvemos uma metodologia para extrair a partir de imagens capturadas ao longo da formação da rede vascular: os padrões binários, as topologias de rede e as matrizes de adjacência. A caracterizacão dos padrões binários é realizada medindo-se a densidade de vasos, o número de aglomerados celulares e a densidade do maior aglomerado conectado. A partir das topologias de rede extraímos as matrizes de adjacência e caracterizamos a formação da rede vascular por meio das métricas usuais de rede, tais como: distribuição de graus, coefciente de agrupamento médio, menor caminho médio e coefcientede correlação dos graus entre outros. Essas análises permitiram monitorar a emergência do maior aglomerado conectado fornecendo evidências quantitativas dos efeitos disruptivos que o Avastin exerce sobre as topologias do tipo árvore exibidas nas redes vasculares dos embriões controles. In this work we present an experimental study of vasculogenesis in chicken embryos from a physics point of view. We use statistical physics and the network science to characterize the self-organization of cell clusters forming a single vascular network. The vascular network formation was observed by means bright-field microscopy during 15 hours of embryonic development. We quantity the self-organization of endothelial cell clusters forming a single vascular network in control embryos and in embryos treated with bevacizumab (Avastin), an antibody that inhibits the signalization of the vascular endothelial growth factor, VEGF. In this work our aim is twofold: first we propose to study vasculogenesis on control embryos, it means without VEGF restriction, in order to characterize the vascular network formation and discuss physical principles that may be driven the collective behavior in the system. Second, we characterize the vascular network formation applying a drug that binds to VEGF decreasing the free VEGF in extracellular matrix. In order to get quantitative parameters from the original images sequence we developed a methodology to extract binary patterns, network topologies and the adjacency matrices. This methodology allows us to make an accurate characterization of the endothelial cell clusters forming a single vascular network in control embryos and in Avastin-treated embryos. This characterization accounts for statistical parameters such as vessels density, cord length, number of cell clusters, fractal dimension and the largest cluster density. Also we characterized the system by means of the usual network metrics suchas: the degree distribution, average clustering coeficient, average short path length, and assortativity among others. From these studies we were able to monitor the growth of the largest connected cluster in the system and provide quantitative evidences of the disruptive effects that Avastin has on the tree structure of vascular networks.

Atualmente não existe um método quantitativo para o diagnóstico de câncer de próstata. Para a realização do diagnóstico, os patologistas utilizam um padrão histológico de graduação que está relacionado à morfologia das glândulas (ácinos) presentes na biópsia e na organização do tecido conjuntivo no entorno da glândula, conhecido como padrão de Gleason. O método é subjetivo e depende muito da experiência e treinamento do patologista. Recentemente com o avanço no desenvolvimento de laser femtosegundos, as microscopias óptica não lineares, principalmente a microscopia por geração de segundo harmônico (SHG, do inglês second harmonic generation) se tornaram ferramentas importantes para aplicações em diagnósticos de doenças. Um desenvolvimento importante seria a utilização destas microscopias como uma ferramenta quantitativa para o diagnóstico de câncer. Neste trabalho apresentamos estudos de biópsias de próstata através de microscopias multifótons com o objetivo de obter um método quantitativo para diagnóstico de câncer. Mostramos resultados obtidos através de imagens SHG das fibras de colágeno e imagens de fluorescência em biópsias coletadas de pacientes tratados no Hospital das Clínicas da UFMG. As amostras foram extraídas de diferentes posições da próstata, com diagnósticos: normal, hiperplasia prostática benigna e várias graduações de malignidade de câncer. Apresentamos um método de análise de imagens baseado no cálculo da anisotropia das fibras de colágeno. As análises mostram diferenças no alinhamento das fibras de colágeno no entorno dos ácinos, que são alteradas com a agressividade do câncer. Medindo a anisotropia (grau de alinhamento) das fibras de colágeno obtemos que quanto maior o grau de malignidade do câncer de próstata maior a tendência das fibras de colágeno se alinharem em torno das glândulas. Estes resultados correlacionam diretamente com o escore de Gleason, que é o padrão utilizado pelos patologistas para diagnóstico. There is currently no quantitative method for the diagnosis of prostate cancer. In order to make the diagnosis, pathologists use a histological pattern of graduation that is related to the morphology of glands (acini) present in the biopsy and in the organization of the connective tissue around the gland that is known as the Gleason pattern. The method is subjective and depends heavily on the experience and training of the pathologist. With the recent advance in the development of femtosecond laser, non-linear optical microscopy, in special Second Harmonic Generation (SHG), has became a powerful tool in helping to diagnose diseases. One important development would be the use of the SHG microscopy as a quantitative tool for the diagnosis of cancer. In this work we present studies of prostate biopsies through multiphoton microscopies with the purpose of obtaining cancer diagnosis. We show results obtained through SHG images of collagen fibers and fluorescence images in biopsies collected from patients treated at Hospital das Clínicas da UFMG. The samples were drawn from different positions of the prostate, with diagnosis as follows: normal, benign prostatic hyperplasia and several malignancy of cancer. We present a method of image analysis based on the calculation of anisotropy of collagen fibers. The analysis show differences in the alignment of collagen fibers around the acini which are altered with the aggressiveness of cancer. Measuring the anisotropy (degree of alignment) of the collagen fibers we show that the higher the degree of malignancy of prostate cancer the greater the tendency of collagen fibers to become aligned around the glands. These results correlate directly with the Gleason score, which is the standard method used by pathologists for diagnosis.

O entendimento de estruturas de RNAs não canônicos como despareamentos, protuber âncias, bases triplas, laços internos, hairpins e pseudo-nós, vem crescendo devido a seus pap éis biológicos em que são submetidas e as amplitudes de movimento realizadas. Em particular, estruturas contendo protuberâncias (bulges) tem sido importantes em interações RNA-RNA, RNA ligantes e também são sítios especícos para as ligações de proteínas. Diversos modelos físicos estão sendo utilizados nesse tipo de estudo. O modelo termodinâmico mais utilizado no estudo de estruturas de RNAs não canônicos desse tipo é o modelo de primeiros vizinhos (NN). Entretanto, apesar da quantidade de trabalhos publicados utilizando o modelo NN, o modelo não consegue fazer uma ligação física das energias termodinâmicas calculadas com as propriedades estruturais que essas moléculas assumem. É necessário a procura de modelos alternativos para um melhor entendimento de duplexos com esses defeitos. Modelos mesoscópicos tipo Peyrard-Bishop (PB) ainda não foram utilizados no estudo das protuberâncias. O modelo PB consegue descrever de forma intuitiva as interações contidas em duplexos. Ao longo dos últimos anos, variações do modelo PB tem sido utilizados em diversas aplicações físicas e alcançando resultados importantes no estudo das propriedades termodinâmicas de duplexos canônicos. Entretanto, o mesmo não foi aplicado a estruturas assim étricas antes. Neste trabalho, mostramos que é possível o uso do modelo Peyrard-Bishop no estudo de estruturas assimétricas de DNAs e RNAs com protuberâncias de uma base ( singlebulges). Na primeira parte do trabalho, adaptamos a Hamiltoniana do modelo PB de forma que ela descreva o empilhamento (stacking) para diferentes monômeros. Através de um tratamento algébrico adequado, mostramos que o modelo Peyrard-Bishop com empilhamento harmônico descreve estruturas assimétricas com uma constante elástica efetiva, de natureza similar a de um oscilador harmônico com molas em série. A função de partição para esse sistema também pode ser resolvida numericamente pela técnica de integral de transferência (IT). Buscamos o mesmo tratamento para o potencial com empilhamento anarmônico. Nesse caso, chegamos a uma equação de Fredholm de segunda ordem, que torna o método IT impraticável. Para a 5 solução da equação de Fredholm de segunda ordem, sugerimos o uso de blocos de funções de pulso (2D-BPFs) e as aplicamos ao modelo Peyrard-Bishop em um polímero de DNA contendo somente pares de base CG. Em seguida, usamos com sucesso o resultado da assimetria com potenciais harmônicos para o estudo de single-bulges do Grupo I em moléculas de RNA. Para isso, criamos uma pseudo-base para denotar a posição do bulge no duplexo na ta oposta e dividimos as interações locais próximas ao bulge em subgrupos. Os parâmetros do modelo PB pertencentes a cada subgrupo foram calculados através de uma minimização entre as temperaturas preditas e experimentais. Para uma melhor compreensão dos resultados, estudamos os pers de aberturas de sequências experimentais que mostram uma forte pertubação para bulges guanosinas, não relatada em estudos anteriores. Os cálculos feitos para as sequências experimentais contendo single-bulges adenosinas e uridinas estão em acordo com medidas de ressonância magnética nuclear (NMR). Como no caso experimental, notamos uma certa prefer ência de bulges uridinas possuírem a conguração para fora da hélice (ipped out). Os resultados encontrados aqui no estudo de bulges demonstram a correlação entre propriedades termodinâmicas e propriedades estruturais físicas. There is a growing understanding of the biological role of non-canonical RNAs such as mismatches, bulges, triple bases, internal loops hairpins and pseudo-knots, and the nonlinear behavior associated with these processes. In particular, structures containing bulges are important for RNA-RNA interactions, RNA ligands and specic sites of protein bondings. Several physical models are being used for this kind of study. The most frequently used thermodynamic model for non-canonical RNAs of this kind is the nearest-neighbor model (NN model). However, despite the large number of studies using this model they do not obtain a physical relationship between the thermodynamic energies and the structural properties that these molecules assume. Therefore, it is necessary to nd alternative models for a better understanding these kind of defects. The mesoscopic models such as the Peyrard-Bishop (PB) model have not been used in the study of bulges. The PB model can describe in an intuitive way the interactions contained in these duplexes. In the last few years, several variants of this model were used in dierent physical applications and achieved important results with respect to the thermodynamic properties of canonical duplexes. However, the PB model has not been applied to asymmetric structures before. In this work, we show that it is possible to use the Peyrard-Bishop model in asymmetric structures of DNA and RNA containing single-bulges. In the rst part of this work, we adapted the Hamiltonian of the Peyrard-Bishop model to describe the stacking of dierent monomers. We show through the algebraic treatment that the PB model with harmonic stacking describes asymmetrical structures with an eective elastic constant, similar to harmonic oscillators in series. The partition function of this system can be solved numerically by the transfer integral (TI) method, as in the original model. We tried the same treatment with the Hamiltonian with an anharmonic stacking. In this case however, we obtained a Fredholm equation of the second type, that makes it impossible to use the TI technique. For the solution of the Fredholm equation of the second type, we suggest the use of two-dimensional block pulse functions (2D-BPFs) and we apply these functions to the original model for a polymer of the DNA 7 containing only CG base pairs. After that, we apply successfully the asymmetry results to the study of Group I single-bulges in RNA molecules. For this purpose, we created a pseudo-base to describe the absence of the base in the opposite direction of the bulge and divide the local interactions into subgroups of parameters. The parameters of each subgroup were calculated through the minimization between the predicted and experimental temperatures. For a better understanding of the results, we study the opening proles of the experimental sequences and we found a strong perturbation of the guanosine bulges in the sequences that were not related before. The calculations for the experimental sequences containing adenosine and uridine bulges are in agreement with the nuclear magnetic resonance (NMR) measurements. As in the experiment, we note a preference of the uridine bulges for a ipped out conguration. The results found here in the study of bulges showed the correlation of thermodynamic properties with the structural properties of these molecules.

Polímeros estão presentes em nosso cotidiano como plásticos, próteses e em um grande número de outras utilidades. Em seres vivos executam diversas funções para a criação e a manutenção da vida, através das proteínas que representam as moléculas orgânicas mais abundantes do corpo humano. Portanto, entender as propriedades físicas de um polímero bem como o mecanismo de interação de uma tal cadeia polimérica com outras substâncias é de grande importância, além de um desao para a física fundamental. Compreender os mecanismos de interação entre uma cadeia polimérica e solventes pode proporcionar um grande avanço na medicina, principalmente, aplicado às terapias gênicas. Estas moléculas, que denominaremos ligantes (ligands) formam complexos com os polímeros, que em casos de bio-polímeros podem ter propósitos biológicos bem específicos. O objetivo deste trabalho é o estudo de uma cadeia polimérica quando ela está presa por suas terminações e imersa em uma solução rica em ligantes. Nós queremos entender qual o efeito nas propriedades mecânicas do polímero quando ligantes são adicionados ao sistema. A propriedade estudada é a correlação entre dois segmentos do polímero, que pode nos informar sobre o comportamento da cadeia. Em particular, a função de correlação que pode ser associada ao comprimento de persistência. Usamos o método de Monte Carlo para obter as correlações para a cadeia em diversas temperaturas e em um banho térmico com diferentes concentrações de ligantes. Polymers are present in our daily lifes as plastics, prosthetics and as well as in a swelling number of commodities. In living organisms they execute essential functions, and proteins represent the most abundant organic molecules of the human body. Therefore, understanding the physical properties of a polymeric molecule along with the mechanisms of interaction of a such polymeric chain with other substances is of great importance, constituting a topic of fundamental scientic interest. Understanding the interaction mechanisms between a polymeric chain and solvent molecules can advance medicine, mainly, specially applied to gene therapies. These molecules, we denote as ligands, form complexs with the polymeric molecules, which in cases of biopolymers may have useful biological applications. The objective of this work is the study of a polymeric chain pinned at both ends and surrounded by ligands. We want to understand how ligands aect the mechanical properties of the polymer. The property we focused on is the correlation among two segments of the polymer, which provides usefull information about the behavior of the chain. In particular, the correlation function can be associated to the so called persistence length. We used the Monte Carlo method to obtain the correlations for the chain at various temperatures and dierent ligand concentrations.

A função e a forma de proteínas estão intimamente ligadas. A compreensão da estrutura tridimensional delas se faz então necessária na elucidação detalhada dos processos biológicos nos quais elas participam. A cristalografia é uma poderosa técnica empregada na determinação estrutural de proteínas. Vasto conhecimento de física e bioquímica são necessários na capacitação de um profissional apto a trabalhar em todas as etapas do processo envolvido na determinação estrutural por cristalografia de proteínas. O objetivo do presente trabalho foi visitar todas essas etapas, que incluem expressão proteica por técnicas de DNA recombinante, a purificação e a cristalização da proteína, experimentos de difração de raios X por monocristal (XRD), processamento de dados, solução da estrutura e análises do modelo proposto. Para realizar todas as etapas, três proteínas foram estudadas: uma efingomielinase D (SMase D), o domínio E2 do papiloma vírus bovino tipo 1 (E2 de BPV-­¿1) e o complexo NahK/NahL. O trabalho com cada uma das proteínas cobriu partes distintas do processo de determinação estrutural. Foi feita a expressão, purificação, cristalização e experimentos de XRD com cristais da SMase D; a estrutura do domínio E2 de BPV-­¿1 foi elucidada a partir de dados de XRD e o modelo foi devidamente analisado; e os dados de XRD de um cristal de NahK/NahL, que indicaram uma desordem no mapa de densidade eletrônica experimental, foram devidamente reprocessados e analisados, resultando na proposta de um novo modelo para a estrutura do complexo. Proteins¿ shape and function are intimately related. Hence, the comprehension of their tridimensional structures is necessary for the detailed elucidation of the biological processes in which they participate. Crystallography is a powerful technique applied on protein structure determination. Great knowledge of Physics and Biochemistry is needed when capacitating a professional to be able to work on every stage of the process of structure determination by protein crystallography. The present work aimed at visiting every one of those stages, including recombinant protein expression, protein purification and crystallization, X-ray diffraction experiments (XRD), data processing, structure solution and analyses. To cover the whole process, three proteins were studied: a sphingomyelinase D (SMase D), the bovine papillomavirus-1 E2 DNA binding domain (BPV-1 E2) and the NahK/NahL complex. Different parts of the process of structure determination were covered working with each of the proteins. SMase D was expressed, purified and crystallized and XRD experiments were performed with its crystals; the structure of BPV-1 E2 was elucidated from XRD data and the proposed model was properly analyzed; XRD data from a crystal of the NahK/NahL complex, which indicated an unordered region on the experimental electron density map, were reprocessed and analyzed, resulting on the proposition of a new structural model of the complex.

A técnica de escrita direta para a fabricação de guias de onda em vidro é promissora para as áreas de Informação e Computação Quântica, pois permite a fabricação rápida e tridimensional de dispositivos fotônicos integrados. Essa técnica consiste na focalização de um laser ultra-rápido no interior de um vidro que se desloca, deforma que seja possível alterar o índice de refração na região focal, formando guias de onda. O objetivo desta dissertação foi estudar o método de escrita direta na fabricação de algumas amostras, realizadas no Laboratório de Fotônica do Instituto de Física de São Carlos, da Universidade de São Paulo (IFSC-USP). Caracterizamos os guias fabricados em relação a sua geometria usando microscopia Confocal, eciência no guiamento da luz por meio do acoplamento da luz de um laser, e índice de refração com o auxílio da técnica de Microscopia de Desfocalização. Essa técnica foi desenvolvida pelo Laboratório de Física de Sistemas Biológicos da UFMG, e possibilita uma medida direta do índice de refração de um objeto de fase colocado em microscópio desfocalizado. Trabalhamos com duas amostras em dois tipos de vidro diferentes, Gorilla Glass e sodalime: 11 guias foram fabricados no primeiro, e 44 no segundo. Nossos resultados mostram que os guia sem Gorilla Glass foram mais ecientes no guiamento da luz do laser, comparado aos guias em sodalime, mas ambos apresentaram transmitâncias menores que15%. Com a maior abundância de guias na amostra de sodalime, foi possível perceber que a potência do laser de femtossegundo e a velocidade de translação da amostra no momento da fabricação têm papel fundamental na modicação do índice de refração na região focal. Em posse desses dados, somos capazes de direcionar fabricações futuras para atender melhor aos objetivos de uso desses dispositivos em aplicações com sistemas quânticos fotônicos. The direct-writing technique for fabrication of waveguides in glass is a promising advance for the areas of Quantum Information and Quantum Computation, since it allows for rapid, three-dimensional fabrication of integrated photonic devices. This technique consists in focusing an ultra-fast laser inside a moving glass substrate, causing a change inrefractive index on the focal region, forming the waveguides. The aim of this dissertation was to study the direct-writing method in the fabrication of some samples, made at the Photonics group of Universidade de São Paulos Instituto de Física de São Carlos (IFSC-USP). We characterized the fabricated waveguides regarding their geometry using Confocal microscopy, their efficiency on light guiding by means of coupling a lasers light, and their refractive index using Defocusing Microscopy technique. This technique was developed by UFMGs Laboratório de Física de Sistemas Biológicos, and allows a direct measurement of the refractive index of a phase object in a defocused microscope. We worked with two samples in two dierent kind of glasses, Gorilla Glass and sodalime: 11 waveguides were fabricated inside the former, and 44 in the latter. Our results show waveguides inside Gorilla Glass were more efficient in guiding the light compared to waveguides inside sodalime sample, but both showed transmittance lower than 15%. With the larger amount of waveguides inside sodalime, it was possible to realize thatfemtosecond laser power and samples translation speed at fabrication have main role in the modication of refractive index on the focal region. In possession of this data, we are capable of leading further fabrication to better suit the objectives of these devices on applications in photonic quantum systems.

No mundo microscópico, a luz pode ser usada como uma ferramenta mecânica, podendo fazer muito mais do que simplesmente iluminar um objeto. Assim, utilizando-se duas pinças ópticas é possível criar um poço de potencial duplo, o qual permite aprisionar uma microesfera e analisar a estatística de suas transições entre os poços. Nesse sentido, este estudo vericou, experimentalmente, a relação das durações de transições com a distância entre os poços de potenciais, bem como, se essas durações são distribuições que obedecem a uma lei de potência como previsto teoricamente. Assim, a partir de um um estimador escravo denotado por , referente a uma equação escravo, foi analisado o equilíbrio da suscetibilidade do processo de Lagevin em uma dimensão num potencial (t). Em seguida, montou-se um setup óptico com uma pinça óptica dupla para realizar medições das estatísticas, a partir de uma microesfera de poliestireno com diâmetro de 3 m para três distâncias entre os poços de potencial (2,87, 3,00 e 3,13 m). Os resultados apontaram que o comportamento de obedece uma lei de potência, com o expoente , sendo que o valor de cresce para distâncias maiores e diminui para distâncias menores. Comparando-se os resultados experimentais com a simulação realizada, constatou-se o mesmo comportamento em ambos os casos. In the microscopic world, light can be used as a "mechanical tool". It can do much more than simply illuminate an object, such as, transmitting energy in the form of heat or moment. Therefore, using two optical tweezers it is possible to create a double potential well, which allows you to trap a microsphere and analyze the statistics of its transitions between wells. In this sense, this study veried, experimentally, the dependence of durations with the distance between potentials wells, as well as if these durations are distributions that obey a power law as theoretically envisaged. Thus, from a slave estimator denoted by , referring to a slave equation, the equilibrium of susceptibility of the Lagevin process was analyzed in one dimension x(t) and a potential (t). Then, we build an optical setup with a double optical tweezer to carry out statistical measurements, from a polystyrene microsphere with a diameter of 3 m for three distances between the potential wells (2.87, 3.00 and 3.13 m). The results showed that the behavior of obeys a power law, with exponent , with values growing with longer distances between wells. Comparing the experimental results with the simulation performed, we obtained the same behaviour in both cases.

Modelos mesoscopicos como o proposto por Peyrard e Bishop apresentam uma relevancia significativa no estudo da estabilidade termica de DNA e RNA. A sua simplicidade computacional permite a analise desses oligonucleotideos por periodos mais longos e de importancia fisiologica e experimental inacessiveis por tecnicas mais sofisticadas. Recentemente houve bastante progresso na parametrizacao dos pares de bases canonicos CG e AT em DNA e CG e AU em RNA para este modelo. No entanto, por serem mais estaveis do que pares nao-canonicos, essas bases sao relativamente simplesde modelar. Nosso estudo viabilizou a aplicacao do modelo PB alem das bases canonicas, bem como a investigacao de parametros estruturais. O primeiro sistema que foi estudado e o de guanina-uracila (GU) em RNA. GU desempenha um papel biologico importante, atuando como um local de reconhecimento para biomoleculas, alem de ser o par nao complementar mais comum em RNA. Devido a nao isostericidadedo par GU, sua estabilidade e influenciada pelo contexto da sequencia. GU apresenta diversas possibilidades conformacionais, inclusive assumindo um numero diferente de ligacoes hidrogenio dependendo das bases vizinhas. Para considerar todas as possibilidades de contexto de GU em RNA e necessario lidar com uma quantidademuito grande de parametros. Esse problema e contornado ao realizarmos a otimizacao de parametros correlacionando com dados de temperatura de desnaturacao obtidos da literatura. Desenvolvemos uma estrategia de reducao do espaco de busca de parametros que possibilitou a determinacao de grupos de configuracao de GU ordenados por intensidade de ligacao de hidrogenio. Em comparacao com dados experimentais da literatura obtivemos uma grande concordancia nas ligacoes de hidrogenio, em especial com as tecnicas de NMR. Assim, pudemos nao apenas concluir a parametrizacao deGU em RNA mas tambem demonstrar que a tecnica permite prever ligacoes de hidrogenio de pares de base nao-canonicos. No segundo tema nos abordamos a limitacao da Hamiltoniana 2D Peyrard-Bishop que nao inclui qualquer parametro estrutural. Mostramos que e possivel partir de uma formulacao Hamiltoniana helicoidal e obter uma Hamiltoniana modificada em 2D que inclui a informacao sobre o passo da helice de DNA, tecnicamente chamado de rise. Nesse estudo tambem usamos o metodo de otimizacao correlacionando com dados de desnaturacao disponiveis na literatura para obter valores de rise. Para realizar a comparacao dos nossos resultados com os obtidospor medidas experimentais, desenvolvemos uma ferramenta de pesquisa que acessa a base de dados no Nucleic Acids Database (NDB) e seleciona as sequencias de interesse para obter valores de rise oriundos de raios-x e NMR. A concordancia dos nossos resultadosde rise foi em geral semelhante com os do NDB com excecao de AT seguido de TA em DNA. Alem disto, pudemos estudar a variacao do rise em funcao de concentracao salina. Os nossos resultados evidenciam a possibilidade de realizar estudos estruturais em oligonucleotideos usando temperaturas de desnaturacao. Mesoscopic models, like the one proposed by Peyrard and Bishop, are important for the study of the thermal stability of DNA and RNA. Its computational simplicity allows to analyse these oligonucleotides over longer time scales of physiological and experimental importance which are not accessible by more sophisticated techniques. Recently, there has been progress in the parametrization of canonical base pairs for this model. However, as they are more stable than non-canonical pairs, these bases are relatively easy to model. Our study aimed at applying the PB model beyond the canonical bases, as well as to investigate structural parameters. The first system we analysed was guanine-uracil (GU) in RNA. GU has an important biological role, acting as a recognition site for biomolecules and is also the most common non-complementarybase pair in RNA. Due to the non-isostericity of GU, its stability is influenced by the sequence context. GU has several conformational possibilities and may even have different hydrogen bonds depending on neighbouring bases. To consider all context possibilities for GU in RNA it becomes necessary to deal with a very large number of parameters. This problem is dealt with by optimizing the parameters correlatingwith melting temperature data from the literature. We developed a strategy to reduce the parameter search space which allowed us to determine GU configuration groups sorted by hydrogen bond intensity. When comparing with experimental results from the literature we obtain a good agreement for the hydrogen bonds, especially from NMR. Therefore, we not only were able to conclude the parametrization of GU inRNA but also to demonstrate that the technique allows to predict hydrogen bonds for non-canonical base pairs. In the second subject we approached a limitation of the 2D Peyrard-Bishop Hamiltonian which is the lack of structural parameters. We showed that it is possible to start from a helicoidal Hamiltonian and obtain a modified 2D Hamiltonian which includes information about the helix step, technically known asrise. In this study we also use the optimization method where we correlate melting temperature data from the literature to obtain the values for rise in DNA. To perform the comparison of our results with those obtained from experimental measurements we developed a query tool which accesses the Nucleic Acids Database (NDB) and selectssequences of interest to obtain values of rise coming from X-ray and NMR. In general our results agreed well with those from the NDB except for AT followed by TA in DNA. Furthermore, we were able to study the variation of rise with salt concentration. Our results show the possibility of performing structural studies in oligonucleotides using melting temperatures.

O modelo Peyrard-Bishop é uma aproximação clássica para descrever a interação molecular em DNA e RNA, onde a dupla fita é considerada perfeitamente plana, sem a característica torção helicoidal. A Hamiltoniana do modelo contém dois termos de potencial, um que descreve a ligação de hidrogênio entre as bases e outro que leva em conta o empilhamento entre os pares de bases. A função partição, para uma cadeia de N pares de base no DNA, resulta em uma integral múltipla de 2N variáveis. Usando a técnica de integral de transferência, esta integral múltipla pode ser reduzida para um problema de autovalores de dimensão N com apenas uma integral de uma variável. No entanto, para a Hamiltoniana Peyrard-Bishop, a forma dos potenciais causa um problema de divergência numérica, que usualmente é contornado truncando os limites da integral. Aqui nós estudamos em detalhe o problema numérico de diversas variantes do Hamiltoniano e também uma proposta formulada recentemente para contornar a divergência mapeando a integral em um espaço de integração 3D. Mostramos que a adição de um termo de torc¸ ao no potencial de empilhamento pode eliminar a divergência. Na análise da integral de transferência, obtemos as condições necessárias que devem ser satisfeitas pelo potencial que simula as ligações de hidrogênio. The Peyrard-Bishop model is a classical approach to describe the molecular interaction in DNA and RNA, where the double strand is considered as perfectly flat, without the characteristic helical torsion. The model Hamiltonian has two potential terms, one which describes the interaction between the bases and another taking into account the stacking between base pairs. The partition function, for a string of N pairs in DNA, results in a multiple integral of 2N variables. Using the transfer integral technique, this multiple integral can be reduced to an eigenvalue problem of dimension N with just one integral of one variable. However,for the Peyrard-Bishop Hamiltonian the form of the potentials causes a numerical divergence problem which is usually circumvented by simply truncating the integral limits. Here, we study in detail the numerical divergence of several variants of the Hamiltonian and also a mathematical method proposed recently to bypass the divergence by mapping the integral to 3D. We show that the addition of a term of torsion in the stacking potential eliminates the divergence. The conditions for the potentials to be convergent were obtained from the analysis of the transfer integral.

O objetivo geral desta tese foi o desenvolvimento de novos métodos experimentais e computacionais em microscopia óptica, em particular, utilizando a técnica de Microscopia de Desfocalização (MD) no estudo do contraste de objetos de fase (objetos transparentes) quando visualizados em um microscópio óptico desfocalizado operando em campo claro, o qual denominamos de contraste MD. A MD foi desenvolvida no Laboratório de Física de Sistemas Biológicos da UFMG e possibilita a medida direta da curvatura local de objetos de fase e a discriminação das interfaces que compõem estes objetos. Como objetivos secundários, a tese visou empregar os métodos desenvolvi-dos na caracterização de parâmetros morfológicos e elásticos de hemácias, os quais são influenciados diretamente pela ação de agentes químicos e físicos e por várias doenças hematológicas. Para tanto, a metodologia desenvolvida foi empregada em 25 hemácias sujeitas a soluções com diferentes osmolalidades: isotônica 300 mOsm/kg, hipotônica - 200 mOsm/kg e hipertônica 400 mOsm/kg. Adicionalmente, outras formas de hemácias (estomatócito e equinócito) foram avaliadas. Os métodos foram desenvolvidos a partir de dois grupos de medidas experimentais: contraste e flutuação do contraste MD de hemácias. A partir do contraste MD foi possível a determinação de parâmetros relacionados à morfologia da hemácia, tais como: raio, área superficial, volume, índice de esfericidade, índice de refração, perfil de espessura, perfil de altura de cada superfície da membrana (bicamada lípidica/citoesqueleto) e a imagem 3D total da hemácia. Através destes, as alterações na superfície das membranas superior (livre) e inferior (em contato com o substrato) das hemácias, tanto devido às diferentes soluções nas quais foram imersas quanto à adesão da célula ao substrato, foram aferidas. A resolução axial da MD nos permitiu obter imagens das superfícies das hemácias separadas por uma distância de até 300 nm. A partir das flutuações do contraste MD de hemácias, foi possível a determinação dos perfis radiais das flutuações em altura e dos comportamentos radiais dos parâmetros elásticos: módulo de curvatura (k), tensão superficial (a) e potencial de confinamento (7) para cada superfície ao longo da extensão da célula. Estas resultados permitiram uma discussão dos aspectos físicos das membranas de hemácias, incluindo novas interpretações para estes parâmetros. The general aim of this thesis was the development of new experimental and computational methods in light microscopy, in particular, using Defocusing Microscopy (DM) technique in the study of contrast of phase objects (transparent objects) when observed by an optical defocused microscope operating in bright _eld, which we call contrast MD. The MD was developed at the Physics Biological Systems Laboratory of UFMG and allows the direct measurement of local curvature of phase objects and the discrimination of interfaces that compose these objects. As secondary objectives, the thesis aimed to employ the developed methods in the characterization of morphological and mechanical changes of red blood cells (RBCs), which are in_uenced directly by the action of chemical and physical agents and various hematologic diseases. Therefore, the methodology was applied in 25 red blood cells subjected to di_erent osmolality solutions: isotonic - 300 mOsm/kg, hypotonic - 200 mOsm/kg and hypertonic - 400 mOsm/kg. Additionally, other forms of red blood cells (stomatocytes and echinocyte) were evaluated. The methods were based on two groups of experimental measurements: DM contrast and contrast _uctuation of red blood cells. From the DM contrast it was possible to determine parameters related to the red blood cell's morphology, such as radius, surface area, volume, sphericity index, refractive index, thickness, shape pro_le of each surface-membranes (lipid bilayer/cytoskeleton), besides the image 3D total of the cell. Through these, the changes in the upper (free) and lower(in contact with the substrate) surface-membranes of red blood cells, either due to di_erent solutions in which they were immersed or due the cell's adhesion to the substrate, were measured. The DM axial resolution allowed us to obtain images of the red blood cells' surfaces separated by a distance of down to 300 nm. From DM contrast _uctuations of red blood cells it was possible to determine the radial pro_les of _uctuations in height and the radial behaviors of the elastic parameters: bending modulus (kc), surface tension and con_ning potencial () for each surface along the cell extension. These results allowed a discussion of physical aspects of red blood cells membranes, including new interpretations for these parameters.

Misturas liotrópicas de laurato de potássio (KL)/ sulfato de potássio (K2SO4)/ álcool (CnH2n+1OH)/ água (H2O) apresentam fases no estado líquido cristalino nemático. Essas fases nemáticas foram colesterizadas através da adicão do agente quiral brucina à mistura. Este estudo foi realizado mantendo as frações molares de todos os constituintes das misturas constantes e variando o comprimento da mol´ecula de álcool entre oito (1-octanol) e dezesseis (1-hexadecanol) átomos de carbono. Três fases colestéricas foram identificadas: ChD (colestérica discótica), ChB (colestérica biaxial) e ChC (colestérica calamítica). O diagrama de fases foi construído em função do número de átomos de carbono n na molécula do álcool. As transições entre as fases colestéricas foram investigadas por medições das birrenfringências ópticas usando microscopia óptica de luz polarizada. As misturas com 9 n 12 apresentaram as três fases colestéricas como função da temperatura e uma região de crossover entre as fases ChD e ChB, com comprimento de correlação a temperatura nula maior do que as dimensões micelares típicas. Misturas com n=8 e n=13 apresentaram transição de fase de primeira ordem entre as fases ChD e ChC, sem a presença da fase ChB intermediária a elas. As misturas com n=14, 15 e 16 apresentaram somente a fase ChC como função da temperatura. Os resultados foram interpretados como consequência da nanosegregação das moléculas de álcool nas micelas com relação às moléculas do anfifílico principal. Lyotropic mixtures of potassium laurate (KL)/ potassium sulphate (K2SO4)/ alcohol (CnH2n+1OH)/ water (H2O) present nematic liquid crystal phases. These nematic phases were cholesterized by the doping of the mixtures with the chiral agent brucine. This study was conducted by keeping constant the molar fractions of all constituents of the mixtures and varying the length of the alcohol molecule between eight (1-octanol) and sixteen (1-hexadecanol) carbon atoms. Three cholesteric phases were identified: ChD (discotic cholesteric), ChB (biaxial cholesteric), and ChC (calamitic cholesteric). The phase diagram was constructed as a function of the number of carbon atoms n in the alcohol molecule. The cholesteric-cholesteric phase transitions were investigated by measurements of the optical birefringences via polarized light microscopy. The mixtures with 9 n 12 presented the three cholesteric phases as a function of temperature and a crossover between the ChD and ChB phases, with a bare correlation length larger than the typical micellar dimensions. Mixtures with n =8 and n =13 exhibited first order phase transitions among the ChD and the ChC phases, without the presence of the intermediate ChB phase. Mixtures with n =14, 15 and 16 showed only the ChC phase as a function of temperature. These results were interpreted as a consequence of the nanosegregation of the alcohol molecules in the micelles with respect to the main amphiphilic molecules.

A tese teve como objetivos o desenvolvimento de novas metodologias experimentais para auxiliar no entendimento de processos básicos presentes em células e moléculas únicas. Foram desenvolvidos dois métodos em Microscopia Óptica: o primeiro (Método 1) relacionado ao estudo da interação de moléculas únicas de DNA com fármacos, e o segundo (Método 2) relacionado ao estudo das propriedades mecânicas de hemácias. A ligação de ligantes, como fármacos e proteínas, à moléculas de DNA podem ser estudadas via experimentos de moléculas únicas, medindo-se as mudanças no comprimento de persistência (A) do complexo formado. Nesta tese é proposta uma metodologia para a análise do comprimento de persistência baseada em um modelo estatístico de desordem congelada (quenched), e descrevendo a isoterma de ligação por uma equação do tipo Hill. Uma expressão geral para o comprimento de persistência efetivo do complexo DNA/ligantes em função da concentração total de ligantes é obtida e aplicada a dados experimentais. Ao ajustar os dados de DNA/-ciclodextrina e DNA/proteína HU, o primeiro obtido durante o mestrado da doutoranda e o último retirado da literatura, os valores para comprimentos de persistência local, constante de dissociação e grau de cooperatividade da interação foram determinados. Em ambos os casos o comprimento de persistência se comporta de forma não monotônica em função da concentração de ligantes e os resultados obtidos possibilitaram a discussão de aspectos físicos da interação entre elasticidade-DNA e cooperatividade entre a ligação dos ligantes. O desenvolvido do método 1 deu origem ao artigo "Quantitative assessment of the interplay between DNA elasticity and cooperative binding of ligands". O segundo método apresentado na tese envolve medidas do contraste de hemácias obtidas pela técnica de Microscopia de Desfocalização (MD). A MD é uma técnica de microscopia óptica, desenvolvida no Laboratório de Física de Sistemas Biológicos da UFMG, e que possibilita a obtenção de informação quantitativa sobre objetos de fase. Nesta tese a equação de contraste para um objeto desfocalizado é deduzida, e a partir dela são apresentadas metodologias simples para obtenção do perfil de espessura, volume, reconstrução tridimensional e índice de refração de hemácias. Além disso, como uma novidade da técnica, o perfil de altura de cada superfície da célula (membranas superior e inferior, a ultima aderida ao substrato) é acessado, separadamente. A flutuação quadrática média do contraste também é apresentada, e sua relação com a flutuação de altura de membranas (efeito flickering) é mostrada. Modelando o espectro de flutuações como dependente de um termo de curvatura kc, de uma tensão superficial, e de um potencial de confinamento , todos os três variando lentamente ao longo do raio da célula, as flutuações em altura para cada membrana celular são determinadas, separadamente. Além disso, os comportamentos radiais dos parâmetros elásticos kc, e cada superfície da célula são monitorados, permitindo a discussão de aspectos físicos da membrana de hemácias. Finalmente, todos os métodos desenvolvidos foram aplicados a um conjunto de dados experimentais de 42 hemácias, coletados durante o doutorado da estudante. O artigo "Shape reconstruction and height fluctuation of phase objects using DM: an application to red blood cells" contendo todos os resultados do Método 2 está em fase de submissão. The aim of this thesis is the development of new experimental tools to improve the understanding of single cells and molecules basic processes. Two dierent methods in Optical Microscopy have been developed: the rst (Method 1) regarding the interaction between DNA molecules and drugs, and the second (Method 2) regarding mechanical properties of red blood cells. Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. A methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation is proposed. An general expression for the eective persistence length as a function of the total ligand concentration is obtained and applied to the experimental data of the DNA-cationic -cyclodextrin and to the DNA-HU protein data, the rst one obtained by the Ph. student during her master degree and the last one gotten from the literature, determining the values of the local persistence lengths, the dissociation constant and the degree of cooperativity for each set of data. In both cases the persistence length behaves non-monotonically as a function of ligand concentration and based on the results obtained some physical aspects of the interplay between DNA-elasticity and cooperative binding of ligands are discussed. The development of Method 1 generated the paper Quantitative assessment of the interplay between DNA elasticity and cooperative binding of ligands . The second method presented is based on contrast measurements of red blood cells (RBC) using Defocusing Microscopy (DM) technique. DM is a bright-eld microscopy technique developed in the Laboratory of Biological Systems of UFMG, used to obtain quantitative information of phase objects. In this thesis the contrast equation for a defocused microscope is deduced, and based on it very simple methodologies to obtain refractive index, thickness prole, volume and three dimensional (3D) reconstruction of red blood cells (RBC) are presented. In addition, as a new feature of the technique, the shape prole of each of the red cell surfaces (upper and lower membranes, the last one adhered to the substrate) are obtained, separately. The DM mean square contrast uctuation equation is also presented, and its relation to red cell surface membrane uctuations (RBC ickering) is shown. Modeling themembrane height uctuation spectrum as dependent of a bending modulus (c), a surface tension () and a conning potential (), all of them slowly varying along the cell radius, the surface uctuations of the cell free interface and of the one in contact with the substrate is obtained, separately. Moreover, the behaviour of the elastic parameters c, and for each interface along the cell radius is monitored and some physical aspects of the red cell membranes are discussed. Finally, all the developed methods are applied to the experimental data of 42 RBC and the average biomechanical properties are shown. The development of Method 2 generated the paper Shape reconstruction and height uctuation of phase objects using DM: application to red blood cells, which is in submission process.

No presente trabalho, investigamos a importância do colesterol na modulação das propriedades mecânicas celulares e como isto interfere na entrada do parasito flagelado, Trypanosoma cruzi, em sua célula hospedeira. Para isto, realizamos estudos de dinâmica celulares, utilizando experimentos de pinça óptica (OT) e de microscopia dedesfocalização (DM), bem como ensaios de exocitose lisossomal e invasão celular pelo T. cruzi em fibroblastos imortalizados de camundongos (WTCl3) e culturas primárias de cardiomiócitos murinos.Verificamos, através da extração de amarras celulares em células WTCL3, pela técnica de OT que tanto a rigidez de flexão quanto a tensão superficial eram maiores quanto menor a quantidade de colesterol remanescente nos fibroblastos. Já nos experimentos de DM, verificamos que o tratamento com a ciclodextrina capturadora de colesterol diminuiu a amplitude de oscilação da superfície celular, mostrando um enrijecimento celular. Já o tempo de relaxação da mesma aumentou, nos primeiros estágios do tratamento, retornando para valores semelhantes aos valores controle. Essa mudança na dinâmica de celular mediada pelo sequestro de colesterol foi gerada tanto pela reorganização do citoesqueleto cortical de actina quanto pela sua polimerização de novo, com formação de fibras de estresse viaativação de Rho. Demonstramos, em cardiomiócitos e em fibroblastos, que o tratamento com MCD, mas não com uma ciclodextrina não-depletora de colesterol, promove a exocitose de lisossomos nestas células e que este processo, em cardiomiócitos, era independente do cálcio intra e extracelular. Em células epiteliais imortalizadas, NRK, verificamos que a exocitose lisossomal induzida pela diminuição do colesterol ocorria mesmo na ausência da proteína sensora de cálcio presente nesta organela, a sinaptotagmina- VII, mostrando que este evento realmente era não dependente deste íon. A exocitose mediada pela diminuição do colesterol de membrana levou à secreção principalmente de lisossomos localizados próximos ao córtex celular, diferente do pool recrutado mediante o tratamento com drogasdespolimerizadoras do citoesqueleto, como a latrunculina-A. Estes dados sugerem a existência de dois pools distintos de lisossomos com diferentes dinâmicas de exocitose. Sabemos que o T. cruzi necessita tanto de um citoesqueleto cortical relativamente maleável quanto delisossomos localizados na região próxima ao córtex celular para que consiga infectar a sua célula hospedeira de forma irreversível. Embora haja estudos que apontem para a importância do colesterol de membrana na infectividade do T. cruzi na célula hospedeira, a qual diminui consideravelmente após tratamento com ciclodextrinas, em nenhum deles foi determinado o mecanismo pelo qual estes microdomínios de membrana contribuem para a invasão e estabelecimento da infecção pelo T. cruzi. Neste trabalho, utilizando cardiomiócitos, células importantes durante a colonização do hospedeiro por este parasito, mostramos que a depleção de colesterol diminui tanto a entrada dos tripomastigotas quanto a associação desses a lisossomos e sugerimos que essa redução da infectividade do parasito está diretamente relacionada à uma diminuição de uma população de lisossomos corticais e a um aumento da rigidez celular dos cardiomiócitos. In the present work we have investigated the importance of cholesterol in controlling mechanical properties of cells and how this modulates Trypanosoma cruzi entry in host cells. For this we performed studies of cell membrane dynamics using tether extraction with optical tweezers (OT) and defocusing microscopy (DM), as well as lysosomal exocytosis and T. cruzi invasion assays in a mouse fibroblast cell line (WTCL3) and primary cultures of neonatal murine cardiomyocytes. We observed, by OT, that both bending rigidity and surface tension increased when WTCL3 was treated with MCD, a drug that sequesters cholesterol from cell membranes. Additionally, by DM, we demonstrated that membrane-cytoskeleton relaxation time increased in the beginning of MCD treatment and decreased in the end, assuming values similar to control. With this technique we have also proved that the amplitude of oscillation diminished during cholesterol depletion, showing that these cells become stiffer. Together, these changes in membrane dynamics involved not only the actin cytoskeleton rearrangement, but also its polymerization de novo and stress fiber formation through Rho activation.Additionally, for cardiomyocytes and fibroblasts, we have proved that treatment with MCD, but not with a similar cyclodextrin with less affinity for cholesterol, triggered lysosome secretion and that this phenomenon was independent of extracellular and intracellular calcium, at least for cardiomyocytes. By using NRK cells we have also shown that lysosome exocytosis, in the absence of cholesterol, continued to happen even in the absence of synaptotagmin-VII, the calcium sensor protein present in these organelles. Exocytosis triggered by cholesterol removal led to the secretion of lysosomes located near the cell cortex, different from the pool recruited by actin depolymerizing drugs, such as latrunculin-A. These data suggest the existence of at least two different pools with different exocytosis dynamics. It is well known that T. cruzi needs both a relatively malleable actin cytoskeleton and a reservoir of cortical lysosomes in order to invade cells successfully. Although there are evidences that plasma membrane cholesterol is important for T. cruzi invasion, since it diminishes severely after cyclodextrin treatment, in none of them wasestablished a mechanism by which cholesterol interferes with T. cruzi entry. In this work we showed that for cardiomyocytes, an important cell during host infection, cholesterol depletion not only diminishes T. cruzi invasion, but also its association with lysosomal markers at the initial steps of host cell entry. The membrane dynamics change (increasing cortical energy barrier) and lysosomal exocytic events (depleting the cortical lysosome reservoir), triggered by cholesletrol sequestration, are most likely the mechanisms y which cholesterol depletion interferes withparasite host cell invasion.

Utilizamos a técnica de fluorescência por absorção de dois fótons para estudar alguns corantes importantes para aplicações em imagens biológicas. Algumas amostras biológicas são autofluorescentes, ou seja, elas possuem alta seção transversal de absorção e absorvem e emitem radiação. Entretanto a maioria das amostras não são autofluorescentes e necessitam serem marcadas por algum corante fluorescente para visualização. O objetivo do nosso trabalho foi estudar alguns destes corantes para marcação biológica. Estudamos o análogo da lei de Lambert Beer para um processo de absorção de dois fótons e apresentamos as técnicas de microscopia e espectroscopia de fluorescência utilizadas para a obtenção dos resultados. Para as medidas de espectroscopia de fluorescência por absorção de dois fótons, implementamos uma montagem experimental utilizando para a excitação das amostras um laser pulsado de Ti-safira. O laser é sintonizável na faixa de comprimentos de onda de 680 até 1080 nm e permite pulsos ultra curtos, da ordem de 140 fs, altas taxas de repetição, alta potência de pico e grandes larguras de banda espectral. A fluorescência é coletada por uma objetiva e através de uma fibra óptica é direcionada a um espectrômetro acoplado a uma câmera CCD para análise espectral. Através de um modulador acusto-óptico programável controlamos a dispersão temporal do pulso. A dispersão é responsável pelo atraso temporal das componentes da banda espectral e consequentemente pelo alargamento do pulso ao percorrer um meio dispersivo. Medimos os espectros de fluorescência para vários corantes e obtivemos seus espectros de fotoluminescência de excitação por dois fótons (TPE). Para a rodamina 6G comparamos o espectro de TPE com dados da literatura. Obtivemos também espectros de absorção por 1fóton (1PA) para os corantes rodamina 6G, DAPI e ER-Tracker Red, além de mostrarmos uma comparação entre os espectros de 1PA e TPE para a rodamina 6G. Mostramos os resultados da dependência da fluorescência com a potência para os corantes rodamina 6G e ER-Tracker Red. Usando microscopia multi-fótons, colaborei na obtenção de imagens de cortes de tecido biológico, parte do projeto de mestrado de Paulo Campos- Júnior, em pesquisa sobre como ascélulas de Leydig se distribuem no testículo de um porco selvagem, Caititu, Tayassu tajacu. We use the technique of fluorescence by two photon absorption to study some dyes that are important for applications in biological imaging. Some biological samples are autofluorescent, i.e. they have a high absorption cross section and absorb and emit radiation. However mostsamples are not autofluorescent and need to be marked by a fluorescent dye for visualization. The aim of our study was to investigate some of these dyes for biological labeling. We studied the analog of the Lambert Beers law for a process of two-photon absorption and present the techniques of fluorescence microscopy and spectroscopy that we used to obtain the results. We implemented an experimental setup for the two-photon absorption fluorescence spectroscopy using a pulsed Ti-sapphire laser. The laser is tunable in the wavelength range of 680 to 1080 nm and allows ultra short pulses, on the order of 140 fs, high repetition rates, high peak power and large spectral bandwidth. The fluorescence is collected by a lens and through an optical fiber is directed to a spectrometer coupled to a CCD camera for spectral analysis. We used a programmable acousto-optic modulator control the temporal dispersion of the pulse. The dispersion is responsible for the time delay of the spectral components on the pulse bandwidth and consequently introduces a pulse broadening as the beam passes through a dispersive medium. We measured the fluorescence spectra for various dyes and obtained their two-photon excitation photoluminescence spectra (TPE). For rhodamine 6G we compared the measured TPE spectrum with data on the literature. We also obtained the one photon absorption spectra(1PA) for rhodamine 6G dye, DAPI and ER-Tracker Red, and we show a comparison between the 1PA and TPE spectra for rhodamine 6G. We show also results for the dependence of the fluorescence with the excitation power, for the dyes rhodamine 6G and ER-tracker red. Using multi-photon microscopy, I collaborated in the imaging of biological tissue sections, as part of the Masters research work of Paulo Campos-Junior, on studies of how the Leydig cells are distributed in the testis of a wild pig, Caititu, Tayassu tajacu.