Orientador: Carlos Lenz Cesar / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-11T02:36:11Z (GMT). No. of bitstreams: 1 Neves_AntonioAlvaroRanha_D.pdf: 9709693 bytes, checksum: 0a918ae7eeaa5895a391c7465942bdd2 (MD5) Previous issue date: 2006 / Resumo: A pinça óptica é um instrumento capaz de manipular e aprisionar partículas dielétricas por meio da pressão de radiação. Suas aplicações nas ciências da vida e biofísica cresceram exponencialmente após a demonstração de que ela permitia manter vivos microorganismos capturados por longos tempos. Informações obtidas destes experimentos requerem um transdutor de força, para o qual se utiliza o deslocamento de uma microesfera capturada. Estamos portanto trabalhando no limite dos regimes de óptica geométrica e Rayleigh, que geralmente são utilizados para simplificar a força óptica. Até hoje não existe consenso entre as teorias das forças, para um regime de tamanho arbitrário, nas pinças ópticas nem para sistemas de alta simetria como microesferas, muito menos em geometrias mais complicadas. Uma das maiores dificuldades encontradas nesse aspecto é a ausência de boas medidas experimentais das forças ópticas, independentes de modelos. Por isso grande parte do trabalho dessa tese foi o desenvolvimento de um sistema de medidas de forças ópticas utilizando pinças duplas para obtenção de toda uma curva da força em função do deslocamento tridimensional da partícula capturada, e não apenas os valores da força em posições fixas. A segunda grande contribuição vem da descrição teórica da força óptica. A grande dificuldade nesse aspecto é a descrição de um feixe incidente de grande abertura numérica e sua decomposição em ondas parciais. É nesse contexto que se encaixa esse trabalho de tese. Acreditamos ter dado uma contribuição extremamente valiosa resolvendo de forma analítica e exata o problema da decomposição de um feixe convergente em ondas parciais relativas a qualquer origem do sistema de coordenadas em três dimensões. / Abstract: The optical tweezers is an instrument capable of manipulating and trapping dielectric particles through the radiation pressure. Their applications in the life sciences and biophysics increased exponentially after it has been demonstrated that it allowed to microorganisms to be maintained alive trapped for long times. Information obtained from these experiments requires a force transducer, for which the displacement of the captured micro sphere is used. We are therefore working in the limit of the geometrical optics and Rayleigh regime, which are usually used to simplify the optical force. Until today consensus fails to exist among the theories of forces for optical tweezers, of an arbitrary size regime, neither for systems of high symmetry as micro spheres, much less in more complicated geometries. One of the greatest difficulties encountered in this aspect is the absence of good experimental measurements of optical forces, independent of models. Therefore great part of this thesis was the development of a system capable of measuring optical forces using a double tweezers setup to obtain an entire curve of the force as a function of the three-dimensional displacement of the trapped particle, and not just the values of the force for fixed positions. The next grand contribution comes from the theoretical description of the optical force. The large difficulty in this aspect is the description of incident beams of great numerical aperture and its decomposition in partial waves. It is in this context that this thesis fits in. We believed to have given an extremely valuable contribution, solving in an analytical and exact way the problem of the decomposition of a convergent beam partial waves relative the any origin of the three dimensional coordinate system. / Doutorado / Física / Doutor em Ciências

Orientador: René Alfonso Nome Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T04:23:53Z (GMT). No. of bitstreams: 1 Zornio_BrunoFedosse_M.pdf: 1493558 bytes, checksum: 07565e7077eefa5f9f91f03dc9cc90d4 (MD5) Previous issue date: 2014 / Resumo: No final do século XIX Van¿tHoff empiricamente estabeleceu que a constante de velocidade de uma reação química é função exponencial da razão entre energia de ativação da reação pela energia térmica do ambiente (e portanto função da temperatura). Há uma variada ordem nas escalas de tempos reacionais, em especial, a constante de velocidade de reações lentas (por exemplo, reações bioquímicas não catalisadas) é difícil de determinar. Uma partícula difundindo em um meio viscoso que apresenta movimento aleatório ¿ com posição média (em um intervalo de tempo suficientemente grande) nula, e a variância da posição linearmente dependente em função do tempo ¿ é dita browniana, e quando submetida a um potencial bi quadrático é um bom modelo para descrição de reações químicas. A partir da dinâmica de Langevin (que serve para descrever a dinâmica de uma partícula browniana) é derivada a teoria de Kramers para meio viscosos - que relaciona o formato da curva potencial com a constante de taxa de reações químicas -. Experimentalmente pode-se recriar esse modelo utilizando pinças ópticas. Pinças ópticas são capazes de aprisionar partículas da ordem micrométricas em suspensão, pode-se recriar um potencial bi estávelutilizando uma pinça óptica dupla (com dois pontos de aprisionamento). Este estudo tem como objetivo avaliar a constante de taxa de um processo de transição entre poços de potencial de partículas brownianas teoricamente utilizando uma simulação de dinâmica de Langevin para sistemas em equilíbrio tanto quanto para sistemas antes de atingir o equilíbrio, assim como determinar experimentalmente utilizando microscopia ocoeficiente de difusão a partir da trajetória temporal de uma única partícula. Os resultados teóricos obtidos são bastante condizentes com os resultados experimentais descritos na literatura, assim como as predições da constante de taxa para tempos antes do equilíbrio apresentam correlação com o sistema em equilíbrio. Com relação à estimativa do coeficiente de difusão apresenta um erro sistemático associado ao tamanho da trajetória temporal de uma única partícula / Abstract: By the end of the XIX century, Van¿t¿ Hoff has empirically established that the rate constant of some chemical reaction is exponentially dependent by the ratio between the reaction activation energy and the environment thermal energy (and so on function of temperature). There is a wide variety in the reaction time scales, in particular, the rate constant of slow reactions (such as uncatalysed biochemical reactions) is difficult to determine. A diffusing particle in a viscous media which exhibit random motion ¿ with mean position (in a sufficiently large time series) is zero, and the position variance is linearly time dependent ¿ is called Brownian, and when is submitted in a biquadratic potential it¿s a good model to describe chemical reactions. By the Langevin dynamics (which serves to describe the Brownian particle motion) the Kramers theory for viscous media is derived ¿ that theory connects the potential energy shape with the chemical rate constant -. Experimentally it is possible to create this model using optical tweezes. Optical tweezers where capable to trap micrometrical beads in suspension, it can generate a bi-stable using a double optical tweezers (that is with two trapping points). The main objective of this essay is evaluate the rate constant a Brownian particles jumping between potential wells theoretically using Langevin dynamics simulations for the system at equilibrium and before reach the equilibrium, as determinate experimentally the diffusion coefficient of single particle time path using microscopy. The theoretical results is very consistent with experimental results described in literature, as well as the prediction of the rate constant for the system before reaches equilibrium are correlated with the rate constant for the system at equilibrium. For the diffusion coefficient estimative it was observed that there is a systematical source of errors, and its is related with the length of the time series of the single particle path / Mestrado / Físico-Química / Mestre em Química

Une famille de pinces moléculaires commutables par coordination métallique basées sur une architecture modulaire de type bis(M-salphen) terpyridine (salphen = N,N’-disalicylidene-o-phenylenediamine) a été synthétisée dans le but de moduler, par un effet mécanique, des propriétés physiques ou chimiques à l’échelle moléculaire. Deux pinces fonctionnalisées par des complexes phosphorescents de platine(II) ont été synthétisées par une approche modulaire. Un mouvement réversible de fermeture et d’ouverture par coordination / décoordination a été obtenu ainsi qu’une extinction de la luminescence via un contrôle allostérique de la reconnaissance de Hg2+. Une pince comportant des complexes paramagnétiques de cuivre (II) a permis l’observation par RPE et SQUID d’une commutation réversible de l’interaction d’échange à travers l’espace entre les deux centres métalliques. Du manganèse(III) a également été utilisé afin de permettre la commutation de l’interaction d’échange via un ligand pontant cyanure grâce à un contrôle allostérique de l’intercalation. De manière similaire, la synthèse d’une pince moléculaire incorporant des unités molécule aimant a été mise au point. D’autre part les propriétés rédox d’une pince à base de nickel(II) ont aussi pu être contrôlées par le mouvement et l’intercalation de pyrazine dans la forme oxydée nickel(III). Finalement, la synthèse d’une pince catalytique a été mise au point, ouvrant la voie à des catalyseurs commutables biomimétiques fonctionnant par effet allostérique. / A family of switchable molecular tweezers based on a bis(M-salphen) terpyridine (salphen = N,N’-disalicylidene-o-phenylenediamine) structure has been developed to study the influence of a mechanical motion on physical properties at the molecular level. Two tweezers functionalized by phosphorescent platinum (II) complexes were synthesized using a modular approach. The reversible mechanical motion by coordination / decoordination of a first metal cation allowed reversible quenching of the luminescence thanks to mercury (II) intercalation with allosteric control. Similar tweezers bearing paramagnetic copper (II) permitted to reversibly switch through-space exchange interactions between the two metallic centers according to EPR and SQUID measurements. manganese (III) was also used in order to switch the exchange interaction through a cyanide bridging ligand whose intercalation can be controlled by allosteric effect. With a similar strategy, the synthetic route towards tweezers showing single-molecule-magnet behavior has beendeveloped. The redox property of nickel(II) based tweezers was also controlled by the mechanical motion and the intercalation of pyrazine in the oxidized nickel (III) closed form. Finally, the synthesis of catalytic tweezers was developed, opening the way to switchable biomimetic catalysts operating with allosteric control.

Les récepteurs purinergiques P2Xs sont impliqués dans de nombreux processus physiopathologiques et représentent des cibles thérapeutiques majeures. Leur étude souffre néanmoins d’un manque de molécules pharmacologiques sélectives des différents sous-types et les mécanismes impliqués dans les transitions allostériques restent putatifs.Nous avons développé des outils optochimiques innovants, dérivés d’azobenzènes permettant une maîtrise spatiale et temporelle inégalée de la fonctionnalité du récepteur : d’une part,un outil dérivé d’une stratégie de pharmacologie optogénétique contrôlant l’activité d’un récepteur ingéniéré et d’autre part, une sonde moléculaire capable d’induire des mouvements entre des résidus au sein de la protéine et d’étudier les mécanismes lors de l’activation. Ces travaux nous ont permis de proposer un nouveau mécanisme d’activation du récepteur. Ces deux outils pourraient être utilisés sur d’autres cibles pour des investigations moléculaires et physiologiques. / Purinergic P2X receptors are implicated in a diverse range of physiopathological processes and are therefore promising therapeuthic target. Their study suffers from the lack of pharmacological tools selective of one subtype only and mechanisms by which the receptor switches between different conformational states remain elusive.We have developed novel optochemical tools based on azobenzene derivatives to obtain a spatio temporal control of the functionality of the receptor. Inspired by optogenetic pharmacology, we have designed an engineered receptor to control electrical activity of cultured neurons. Molecular photo-switchable tweezers have been developed to explore allosteric transitions of the protein and giving new insights into the P2X pore gating mechanism. This approach provides data enabling us to purpose a new model of the active state. The versability of the two strategies makes these tools promising for molecular and physiological studies of other membrane proteins.

To respond to rapidly-changing stresses in their environment, bacterial cells must be able to sense a variety of chemical cues and respond to them by activating the relevant genes. The lysine riboswitch is a short RNA motif, located just upstream of a gene encoding a lysine biosynthesis protein, that suppresses the expression of that gene when sufficient lysine is present in the cell. It acts by binding a lysine monomer in a region called the aptamer, which in turn rearranges an adjacent domain called the expression platform, sequestering the ‘start' sequence of the gene and preventing it from being transcribed. In this thesis, the lysine riboswitch's ligand-binding transition is studied using single-molecule fluorescence microscopy, optical tweezers, and a hybrid optical force/fluorescence technique. Förster Resonance Energy Transfer (FRET) is used with a fluorescently-labeled aptamer to show that it has a previously-undescribed, partially-folded structural state with enhanced ligand affinity compared to the unfolded structure. The Mg²⁺ dependence of the transition between these states is shown to resolve existing debates in the literature about the sensitivity of the riboswitch. The kinetics of the folding transition are explored using FRET, optical force, and hybrid ‘Fleezers' to map the free energy landscape of ligand binding and show that the ligand itself promotes transitions into the aptamer's folded state, a so-called ‘induced fit' mechanism rare among riboswitches. Finally, high-resolution optical tweezers are used to explore the link between the aptamer's secondary structure (the sequence of paired nucleotides) and its tertiary structure (three-dimensional folding) to illuminate the role of ligand binding in gene regulation, which depends on the equilibrium between competing secondary structures. Hybrid biophysical techniques like optical force/fluorescence microscopy are shown to be indispensable for addressing all the states in the reaction pathways of complex biomolecules like riboswitches and for discriminating between multiple levels of structure formation and interaction with the environment. Not only do the results presented here shed light on the RNA folding problem, particularly the role of tertiary structure in determining the minimum-energy configuration of an RNA sequence, but they could have implications for biomedical research, as the lysine riboswitch has already been shown to be a potential target for next-generation antibiotics.

I advanced the understanding of particle morphology and its implications for the behavior and effects of atmospheric aerosol particles. I have developed new experimental methods for the Aerosol Optical Tweezers (AOT) system and expanded the AOT’s application into studying realistic secondary organic aerosol (SOA) particle phases. The AOT is a highly accurate system developed to study individual particles in real-time for prolonged periods of time. While previous AOT studies have focused on binary or ternary chemical systems, I have investigated complex SOA, and how they interact with other chemical phases, and the surrounding gas-phase. This work has led to new insights into liquid-liquid phase separation and the resulting particle morphology, the surface tension, solubility, and volatility of SOA, and diffusion coefficients of SOA phases. I designed a new aerosol optical tweezers chamber for delivering a uniformly mixed aerosol flow to the trapped droplet’s position. I used this chamber to determine the phase-separation morphology and resulting properties of complex mixed droplets. A series of experiments using simple compounds are presented to establish my ability to use the cavity enhanced Raman spectra to distinguish between homogenous single-phase, and phase-separated core-shell or partially-engulfed morphologies. I have developed a new algorithm for the analysis of whispering gallery modes (WGMs) present in the cavity enhanced Raman spectra retrieved from droplets trapped in the AOT. My algorithm improves the computational scaling when analyzing core-shell droplets (i.e. phase-separated or biphasic droplets) in the AOT, making it computationally practical to analyze spectra collected over many hours at a few Hz. I then demonstrate for the first time the capture and analysis of SOA on a droplet suspended in an AOT. I examined three initial chemical systems of aqueous NaCl, aqueous glycerol, and squalane at ~ 75% relative humidity. For each system I added α-pinene SOA – generated directly in the AOT chamber – to the trapped droplet. The resulting morphology was always observed to be a core of the initial droplet surrounded by a shell of the added SOA. By combining my AOT observations of particle morphology with results from SOA smog chamber experiments, I conclude that the α-pinene SOA shell creates no major diffusion limitations for water, glycerol, and squalane under humid conditions. My AOT experiments highlight the prominence of phase-separated core-shell morphologies for secondary organic aerosols interacting with a range of other chemical phases. The unique analytical capabilities of the aerosol optical tweezers provide a new approach for advancing the understanding of the chemical and physical evolution of complex atmospheric particulate matter, and the important environmental impacts of aerosols on atmospheric chemistry, air quality, human health, and climate change.

Mild Traumatic Brain Injuries (mTBIs) are non-penetrating brain injuries that do not result in gross pathological lesions, yet they may cause a spectrum of cognitive and behavioral deficits. mTBI has been placed in the spotlight because of increased awareness of blast induced and sports-related concussions, but the underlying pathophysiological mechanisms are poorly understood. Several studies have implicated neuronal membrane poration and ion channel dysfunction as the primary mechanism of injury. We hypothesized that injury forces utilize mechanically-sensitive, transmembrane integrin proteins, which are coupled to the neuronal cytoskeleton (CSK) and distribute injury forces within the intracellular space, disrupting CSK organization and reducing intercellular neuronal functionality. To test this, magnetic beads were coated with adhesive protein, allowing them to bind to integrins in the neuronal membrane in vitro. To apply forces to the neurons via the bound beads, we built custom magnetic tweezers and demonstrated that focal adhesions (FACs) formed at the site of bead binding. We showed that the beads were coupled to the CSK via integrins by measuring the disparate adhesion of the soma and neurite to their underlying substrate. The soma also required more force to detach than neurites, correlating with the FAC density between each neuronal microcompartment and substrate. We then utilized the magnetic tweezers to test whether beads bound to integrins injured neurons more than beads that bound to neurons nonspecifically. Integrin-bound beads injured neurons more often and the injury was characterized by the formation of focal swellings along axons, reminiscent of Diffuse Axonal Injury. While integrin-bound beads initiated swellings throughout neurons, beads bound nonspecifically only caused local injury where beads were attached to neurons. To demonstrate the electrical dysfunction of integrin-mediated injury forces, we adapted Magnetic Twisting Cytometry to simultaneously apply injury forces to beads bound to multiple cells within neuronal networks in vitro. The formation of focal swellings resulted in reduced axonal electrical activity and decreased coordinated network activity. These data demonstrate that the mechanical insult associated with mTBI is propagated into neurons via integrins, initiating maladaptive CSK remodeling that is linked to impaired electrical function, providing novel insight into the underlying mechanisms of mTBI. / Engineering and Applied Sciences

L'ARN s'est longtemps vu attribuer un simple role de transmission entre l'ADN, garant de l'information genetique, et les proteines, assurant les fonctions et donc la survie cellulaire. Ce n'est qu'avec les decouvertes des ARNs de transfert dans les annees 70, puis des ribozymes dans les annees 80, qu'il a ete realise que l'ARN pouvait assurer ces deux roles : l'information genetique est stockee dans sa sequence lineaire, et l'adoption de structures tridimensionnelles complexes rend possible une activite catalytique. Depuis, de nouvelles fonctions de l'ARN n'ont cesse d'etre decouvertes, a tous les niveaux de regulation de l'expression genique entre autres. La majorite de ces fonctions repose sur la structuration tridimensionnelle d'ARNs simple brin.Dans ce travail, differents aspects de la structuration de l'ARN sont abordes, toujours en utilisant la technique de mesure de forces sur molecules uniques par piegeage optique. Dans un premier temps, une etude comparative d'une structure secondaire modele, le hairpin dans ses formes ARN et ADN, a ete realisee. La question de l'interaction d'une structure secondaire avec une proteine helicase (DbpA) a ensuite ete abordee. Enfin, dans le cadre plus general d'une etude sur l'assemblage du ribosome, nous avons debute le developpement d'une nouvelle methode d'analyse des structures secondaires. Cette methode repose sur le suretirement d'un hybride ARN ribosomique / ADN. / Traditionally, RNA has been considered as a mere intermediate between DNA, keeper of the genetic information, and proteins, which assume cells self-sustenance. With the discoveries of the transfert RNA in the 70s, and of the ribozymes in the 80s, RNA took on both roles: it can store information in its linear sequence, and tridimensional structuration enables catalytic functions. Since then, numerous roles devoted to RNA have been discovered, particularly for gene expression regulation. Most of these functions rely on tridimensional structuration of single stranded RNA. In this work, we used an optical tweezers setup to study several aspects of RNA structuration by single molecule force measurement. In a first part, we compared the dynamic behaviour of a model secondary structure made of either RNA or DNA, the hairpin. Then we considered the interaction of a secondary structure with a protein, the RNA helicase DbpA. Finally, within a wider study of ribosome assembly, we worked on the development of a new method to study tridimensional structuration. This method relies on the overstretching of a hybrid ribosomal RNA / DNA molecule.

Nous avons développé un dispositif combinant pinces optiques et imagerie par feuillet de lumière. Nous montrons que les interfaces cellulaires de l'épithélium précoce de l'embryon de Drosophile peuvent être piégées et manipulées directement avec des pinces optiques. La manipulation optique est réalisée à la fin de la cellularisation, processus par lequel des membranes cellulaires séparent les noyaux pour donner naissance à un épithélium ; à ce stade, les mouvements cellulaires sont minimes et les cellules ont des formes hexagonales similaires. En imposant un mouvement sinusoïdal au piège perpendiculairement à une interface, nous étudions la déflection de l'interface en fonction de la puissance laser, de l'amplitude du mouvement du piège et de la fréquence d'oscillation. En outre, des expériences de déflection-relaxation par déplacement instantané puis arrêt du piégeage, ont été réalisées, fournissant une alternative à l'analyse fréquentielle pour étudier les propriétés viscoélastiques de l'interface. Un modèle de type solide linéaire standard rend compte des observations et permet d'extraire les paramètres viscoélastiques de l'interface. Nous mettons également en évidence que la déflection imposée à une interface se propage aux interfaces voisines en s'affaiblissant exponentiellement sur une distance d'une à deux cellules. Cette technique étant établie, nous l'utilisons pour mesurer les tensions durant l'extension de la bandelette germinale. Les tensions sont anisotropes, les jonctions parallèles à la direction dorsoventrale ayant une tension trois fois plus élevée que celles perpendiculaires. Ce travail fournit des mesures absolues des tensions intercellulaire. / Here, an optical tweezers setup was developed on a pre-existing single-plane illumination (SPIM) setup. The cell-cell interface in embryonic epithelia could be trapped and manipulated directly with optical tweezers. The interaction of the interface with the trap was initially characterized at the end of cellularization where the tissue has minimal movements and actomyosin turnover. With a sinusoidal trap excursion, the interface amplitude was found to increase linearly with applied laser power as well as trap amplitude and time period. Furthermore, push and pull experiments on the interface responding to a stationary trap, provided another way to address the viscoelastic properties of the interface. The interface kinetics in stationary experiments could fit adequately to a passive viscoelastic model. This model also explained well the linear response to trap amplitude and time period, and formed the basis of estimating interface tension from its amplitude. Moreover, the propagation of the sinusoidal movement to neighbouring interfaces decayed rapidly with minimal phase lag in both experiments and the model. Having established a suitable regime of trapping conditions, where interface deflection is small and linear, the mechanical anisotropy of the epithelium was at the onset of gastrulation. The interface tension increased by 2-3 fold, exhibiting both apico-basal and dorso-ventral polarization of tension, concomitant with polarized accumulation of myosin. The role of myosin was established further through ROCK-inhibition. Perturbation of actin also decreased the interface tension. My work provides a crucial insight into the mechanical behaviour of dynamic epithelia.

Orientador: Carlos Lenz Cesar / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-08T01:06:24Z (GMT). No. of bitstreams: 1 Pozzo_LilianadeYsasa_M.pdf: 4076293 bytes, checksum: 5197fda41d6fa440de56b82b104a4bcd (MD5) Previous issue date: 2006 / Resumo: Microorganismos unicelulares, como os demais seres vivos, necessitam interagir com o ambiente e procurar ativamente por alimentos. Seus orgãos sensoriais, entretanto, se limitam a sensores hidrodinâmicos e receptores bioquímicos de membrana. A quimiotaxia estuda a resposta de organismos unicelulares frente a gradientes de concentração de substâncias atratoras ou repulsoras. Essa resposta é parte essencial do processo de infecção por parasitas no direcionamento e reconhecimento das células a serem infectadas. Dessa forma, um estudo quantitativo da quimiotaxia de parasitas requer o monitoramento tanto dos movimentos do parasita quanto das intensidades, direção e sentido das forças que exerce na presença de gradientes de concentração das substâncias atratoras e repulsoras. Forças de microorganismos com dimensões da ordem de 10 µm são da ordem de pico-Newtons. A pinça óptica é a microferramenta ideal para esse tipo de estudo pelas seguintes razões: (1) tem sensibilidade para medir forças desde 20 femto-Newtons até 200 pico-Newtons, da mesma ordem de grandezas das forças geradas pelos parasitas; (2) é uma técnica remota, sem necessidade de contacto e (3) não destrutiva, pois os lasers no infravermelho não geram calor suficiente para causar danos térmicos. Nesse trabalho mostramos como se pode utilizar o deslocamento da posição de equilíbrio de uma microesfera como transdutor de forças vetoriais, determinando quantitativamente intensidade, direção e sentido das forças na microesfera. Um detector de quadrante da luz do próprio laser da pinça óptica fornece o deslocamento da mesma em duas dimensões. Dessa forma foi possível monitorar a força exercida pelo parasita em tempo real em diferentes gradientes de concentração de várias substâncias. Também desenvolvemos uma câmera de gradiente que garantiu um gradiente unidimensional estacionário na qual se pode aprisionar os parasitas e realizar a medida vetorial da força em função do tempo. Demonstramos a capacidade do nosso sistema de realização de medidas de quimiotaxia utilizando o protozoário Leishmania amazonensis, responsável pela doença leishmaniose, na forma promastigota, na presença de gradientes de glucose. Nossos resultados mostram que além das forças serem fortemente direcionadas na direção do gradiente, que a intensidade das mesmas diminui nas direções contrária ou perpendicular ao gradiente. Com esse trabalho mostramos a construção de um sistema de medidas quantitativa capaz de estudar quantitativamente a quimiotaxia de qualquer parasita frente a qualquer gradiente de concentração de diferentes substâncias / Abstract: Unicellular microorganisms, like others live beings, need to interact with environment to find nourishment. They have only hydrodynamics sensors and biochemical receptors on membrane to accomplish this task. The response of the microorganism to concentration gradients of attractive or repulsive chemical substances is called chemotaxis. The investigation of chemotaxis is essential to understand infection processes and how parasites recognizes and directs itself to the cells to be infected. This way a quantitative study of parasites chemotaxis requires the observation of not only its movements, but also the strength, direction and sense of forces exerted in the presence of concentration gradient of attractive or repellent chemical substances. Microorganism¿s forces with dimensions of around 10µm are the order of pico-Newton. Optical tweezers are a suitable tool for this kind of investigation, we can justify this affirmation by the following reasons: (1) it has high sensibility to measure forces from 20 femto-Newton until 200 pico-Newton. These forces are in same order of the forces exerted by the parasites. (2) This technique does not need contact, therefore it is a remote technique; and (3) this tool does not destroy the parasites, because lasers on infrared band do not generate enough heat to cause thermal damage on them. In this work we used the displacements of a microsphere trapped in an Optical Tweezers as the force transducer to measure the direction and the strength of the propulsion forces of flagellum of the microorganism under several gradient conditions. A quadrant detector was utilized to sense the displacement of optical tweeter¿s laser in two dimensions. So we monitor the force exerted by the parasites in real time in several concentrations gradients of different substances. We also developed a system enable to create concentration gradient. This system guaranteed a stationary one-dimensional gradient, which the parasites were arrested and the vectorial measurements of force in time function were realized. In this study, we used the protozoa Leishmania amazonensis in its promastigote form. It is responsible by the disease called leishmaniose and it represents a serious problem of public health. We demonstrated that our system is able to perform chemotaxis measurements in gradients of glucose. Our results suggest that the direction and strength of force can be used to identify the movement of the parasite. We also notice differences in strength forces for both direction x and y for different glucose in several concentrations. Using that system we can investigate quantitatively taxias of any parasites in any concentration gradient with different chemical substance, temperature or other variables / Mestrado / Física / Mestre em Física