<P> A great number of cellular processes can lead to local changes in proton concentration and temperature. So, it is desirable to be able to measure pH and temperature with non-invasive and spatially resolved methods. In this thesis, I describe two unique methods to measure pH and temperature using the environmental sensitivity of the proton transfer of fluorescent molecules. The first method is based on the detection of the temperature and pH dependent parameters associated with the blinking of fluorophores by fluorescence correlation spectroscopy (FCS). Employing EGFP as the probe, I used this method to characterize temperature increase at a laser focus due to light absorption in a thin liquid sample. Using pyranine as a probe, I extended the applicability of this method to a range of pH including physiological pH. Also, I investigated in details the effect of buffer composition on the blinking of the fluorophores. Then, I concluded that one limitation of this method is its strong dependence on buffer conditions, which are not well characterized in vivo. The second approach is a pH measurement method based on ratiometric imaging. This method is not as dependent on buffer condition. We improved on current ratiometric imaging techniques by demonstrating the possibility of using two-photon excitation. This method was used to measure pH in pyranine loaded vesicles created during receptor-mediated endocytosis of µ-Opioid receptors expressed in HEK 293 cells. Preliminary results showed that the pH in the endocytic vesicles dropped to a value similar to those measurements in late endosomes roughly~ 10 minutes after triggering the endocytosis, and eventually, the pH reading reached a value similar to that of the pH oflysosomes. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

A Terapia Fotodinâmica (PDT) é a uma modalidade terapêutica promissora para o tratamento do câncer e outras doenças, que visa a destruição do tecido alterado. A PDT se caracteriza por um conjunto de processos físicos, químicos e biológicos que ocorrem após a administração de uma droga, retida preferencialmente nos tecidos tumorais, seguida pela irradiação com luz visível. Quando as células tumorais tratadas são irradiadas, o fotossensibilizador absorve luz e inicia uma reação em cadeia produzindo espécies reativas de oxigênio que destroem as células tumorais. Neste trabalho procuramos avaliar os principais aspectos da PDT para o desenvolvimento de uma dosimetria baseada em conceitos fundamentais tais como: a estabilidade do Photogem ®, a farmacocinética, a penetração da luz no tecido e os mecanismos de morte celular. Com esse propósito realizamos quatro experimentos distintos nos quais esses aspectos foram investigados. Realizamos experimentos in vitro para averiguar a estabilidade do Photogem ®. As soluções foram submetidas a condições extremas de irradiação, onde variamos comprimentos de onda e potência de irradiação. Analisamos também a variação de pH, concentração e solvente. A farmacocinética, a profundidade de penetração da luz em tecido biológico e os processos de morte celular foram realizados in vivo. Os resultados obtidos foram os seguintes: o fotossensibilizador é degradado ao variarmos o comprimento de onda e a potência de irradiação; ele não sofre degradação ao ser aquecido até 60°C. No estudo da farmacocinética verificamos que os tecidos estudados apresentaram tempos de acúmulo e eliminação diferente, com o fígado apresentando menores tempos. Na avaliação da penetração da luz verificamos que para tecidos menos pigmentados obtivemos maior penetração. Os nossos resultados estão em concordância com os encontrados na literatura. Finalmente para a respiração mitocondrial observamos que para maiores tempos de irradiação a razão de controle respiratório foi elevada, indicando modificações no ciclo respiratório. Os resultados obtidos nesse trabalho fornecem subsídios importantes para auxiliar a determinação de um protocolo controlado de dosimetria para PDT, aumentando a eficiência e conseqüentemente a taxa de sucesso da terapia. / The Photodynamic Therapy (PDT) is a promising therapeutical modality for the treatment of cancer and other diseases, seeking the altered tissue destruction. The PDT is characterized by a set of physical, chemical and biological processes, which occur after a drug injection and selective retention in tumoral tissues, followed by visible light irradiation. When the tumoral cells are irradiated, the photosensitiser absorbs light and begins a chain reaction with the formation of reactive species of oxygen leading to the destruction of the tumoral cells. In this study we evaluate important PDT aspects for the development of a dosimetry based in some fundamental concepts such as Photogem ® stability, pharmacokinetics, light penetration in tissue and all death mechanisms. Four experiments were done to investigate these aspects. In vitro studies were performed to evaluate the Photogem ® stability under different wavelength and light power parameters. It were also investigated the dependence under pH, concentration and solvent type variation. The pharmacokinetics, light penetration in biological tissue and cell death mechanisms were studied in vivo experiments. The results showed that the photosensitiser is degradeted depending on the was no degradation until 60 °C. In the pharmacokinetics study we verified that the investigated tissues present different accumulation and elimination times, the hepatic tissue showing the smallest times. In the light penetration evaluation we verified that the less pigmented ones showed the greater penetration in accordance with the literature results. Finally in mitochondrial study we observed that for longer irradiation times the respiratory cicle. Our results give important data to aid the establishment of a controlled PDT dosimetry protocol, improving the efficiency and consequently the success rate of this therapy.

The electrical activity of skeletal muscle¡ªthe electromyogram (EMG)¡ªis of value to many different application areas, including ergonomics, clinical biomechanics and prosthesis control. For many applications the EMG is related to muscular tension, joint torque and/or applied forces. In these cases, a goal is for an EMG-torque model to emulate the natural relationship between the central nervous system and peripheral joints and muscles. This thesis mainly describes an experimental study which relates the simultaneous biceps/triceps surface EMG of 12 subjects to elbow torque at seven joint angles (ranging from 45¡ÃƒÂ£to 135¡ÃƒÂ£) during constant-posture, quasi-constant-torque contractions. The contractions ranged between 50% maximum voluntary contractions (MVC) extension and 50% MVC flexion. Advanced EMG amplitude (EMG¦Ãƒâ€™) estimation processors were investigated, and three nonlinear EMG¦Ãƒâ€™-torque models were evaluated. Results show that advanced (i.e., whitened, multiple-channel) EMG¦Ãƒâ€™ processors lead to improved joint torque estimation, compared to unwhitened, single-channel EMG¦Ãƒâ€™ processors. Depending on the joint angle, use of the multiple-channel whitened EMG¦Ãƒâ€™ processor with higher polynomial degrees produced a median error that was 50%-66% that found when using the single-channel, unwhitened EMG¦Ãƒâ€™ processor with a polynomial degree of 1. The best angle-specific model achieved a minimum error of 3.39% MVCF90 (i.e., error referenced to MVC at 90¢X flexion), yet it does not allow interpolation across angles. The best model which parameterizes the angle dependence achieved an error of 3.55% MVCF90. This thesis also summarizes other collaborative research contributions performed as part of this thesis. (1) Decomposition of needle EMG data was performed as part of a study to characterize motor unit behavior in patients with amyotrophic lateral sclerosis (ALS) [with Spaulding Rehabilitation Hospital, Boston, MA]. (2) EMG-force modeling of force produced at the finger tips was studied with the purpose of assessing the ability to determine two or more independent, continuous degrees of freedom of control from the muscles of the forearm [with WPI and Sherbrooke University]. (3) Identification of a nonlinear, dynamic EMG-torque relationship about the elbow was studied [WPI]. (4) Signal whitening preprocessing for improved classification accuracies in myoelectric control of a prosthesis was studied [with WPI and the University of New Brunswick].

We used the combination of molecular dynamics and Monte Carlo method to investigate protein folding problems. The environments of proteins are very big, and often very time-consuming. If simulations are based on traditional methods of molecular simulations, it will cost very long time to accomplish the simulation. We use a special designed method, in which the molecular dynamics is used for determining the soft part of protein, and use Monte Carlo method to move and rotate the bonds of proteins. By removing a lot impossible movements in traditional Monte Carlo method, we shorten simulation time and simulate protein folding process effectively. In this work, we used GBSA solvent model, AMBER force field, and semi-local movements to accelerate the simulations. We obtained good result by this simulation method of a small peptide 1L2Y.

A Terapia Fotodinâmica (PDT) é a uma modalidade terapêutica promissora para o tratamento do câncer e outras doenças, que visa a destruição do tecido alterado. A PDT se caracteriza por um conjunto de processos físicos, químicos e biológicos que ocorrem após a administração de uma droga, retida preferencialmente nos tecidos tumorais, seguida pela irradiação com luz visível. Quando as células tumorais tratadas são irradiadas, o fotossensibilizador absorve luz e inicia uma reação em cadeia produzindo espécies reativas de oxigênio que destroem as células tumorais. Neste trabalho procuramos avaliar os principais aspectos da PDT para o desenvolvimento de uma dosimetria baseada em conceitos fundamentais tais como: a estabilidade do Photogem ®, a farmacocinética, a penetração da luz no tecido e os mecanismos de morte celular. Com esse propósito realizamos quatro experimentos distintos nos quais esses aspectos foram investigados. Realizamos experimentos in vitro para averiguar a estabilidade do Photogem ®. As soluções foram submetidas a condições extremas de irradiação, onde variamos comprimentos de onda e potência de irradiação. Analisamos também a variação de pH, concentração e solvente. A farmacocinética, a profundidade de penetração da luz em tecido biológico e os processos de morte celular foram realizados in vivo. Os resultados obtidos foram os seguintes: o fotossensibilizador é degradado ao variarmos o comprimento de onda e a potência de irradiação; ele não sofre degradação ao ser aquecido até 60°C. No estudo da farmacocinética verificamos que os tecidos estudados apresentaram tempos de acúmulo e eliminação diferente, com o fígado apresentando menores tempos. Na avaliação da penetração da luz verificamos que para tecidos menos pigmentados obtivemos maior penetração. Os nossos resultados estão em concordância com os encontrados na literatura. Finalmente para a respiração mitocondrial observamos que para maiores tempos de irradiação a razão de controle respiratório foi elevada, indicando modificações no ciclo respiratório. Os resultados obtidos nesse trabalho fornecem subsídios importantes para auxiliar a determinação de um protocolo controlado de dosimetria para PDT, aumentando a eficiência e conseqüentemente a taxa de sucesso da terapia. / The Photodynamic Therapy (PDT) is a promising therapeutical modality for the treatment of cancer and other diseases, seeking the altered tissue destruction. The PDT is characterized by a set of physical, chemical and biological processes, which occur after a drug injection and selective retention in tumoral tissues, followed by visible light irradiation. When the tumoral cells are irradiated, the photosensitiser absorbs light and begins a chain reaction with the formation of reactive species of oxygen leading to the destruction of the tumoral cells. In this study we evaluate important PDT aspects for the development of a dosimetry based in some fundamental concepts such as Photogem ® stability, pharmacokinetics, light penetration in tissue and all death mechanisms. Four experiments were done to investigate these aspects. In vitro studies were performed to evaluate the Photogem ® stability under different wavelength and light power parameters. It were also investigated the dependence under pH, concentration and solvent type variation. The pharmacokinetics, light penetration in biological tissue and cell death mechanisms were studied in vivo experiments. The results showed that the photosensitiser is degradeted depending on the was no degradation until 60 °C. In the pharmacokinetics study we verified that the investigated tissues present different accumulation and elimination times, the hepatic tissue showing the smallest times. In the light penetration evaluation we verified that the less pigmented ones showed the greater penetration in accordance with the literature results. Finally in mitochondrial study we observed that for longer irradiation times the respiratory cicle. Our results give important data to aid the establishment of a controlled PDT dosimetry protocol, improving the efficiency and consequently the success rate of this therapy.

No / Synthetic Biology aspires to design, compose and engineer biological systems that implement specified behaviour. When designing such systems, hypothesis testing via computational modelling and simulation is vital in order to reduce the need of costly wet lab experiments. As a case study, we discuss the use of computational modelling and stochastic simulation for engineered genetic circuits that implement Boolean AND and OR gates that have been reported in the literature. We present performance analysis results for nine different state-of-the-art stochastic simulation algorithms and analyse the dynamic behaviour of the proposed gates. Stochastic simulations verify the desired functioning of the proposed gate designs.

A modelagem farmacocinética permite prever a concentração de medicamentos em diferentes tecidos do organismo humano. O desenvolvimento de modelos matemáticos é importante para verificar a adequação de certos procedimentos realizados na administração de medicamentos. O objetivo deste trabalho é o desenvolvimento de um modelo farmacocinético capaz de prever a concentração plasmática de drogas no organismo para diversas formas de infusão. Foram utilizados dois tipos de abordagem. Inicialmente, na abordagem monocompartimental, considerou-se que a droga adentra ao organismo diretamente no compartimento sanguíneo, que representa todo o corpo humano. Já na abordagem bicompartimental foram considerados os seguintes compartimentos: um representando o meio pelo qual a droga é infundida no organismo (podendo ser via gastrointestinal, transdermal ou pulmonar) e outro representando o plasma sanguíneo. Em ambos os casos, foi considerada a hipótese de concentração homogênea da droga nos compartimentos em questão. O modelo foi estruturado na forma de diagramas de blocos e a solução foi feita com a utilização da Transformada de Laplace. Foi feita a validação dos modelos e verificou-se que os resultado gerados foram muito próximos dos resultados presentes na literatura. A utilização do modelo monocompartimental permitiu comparar os resultados da administração da mesma quantidade de droga por infusão constante e por infusão periódica. A análise dos resultados gerados pelo modelo mostrou que as concentrações atingidas pelos dois métodos não são as mesmas. O modelo bicompartimental permitiu simular administrações orais e transdermais, e inalação. Foi possível prever a concentração sanguínea após a interrupção da terapia com anti-concepcionais e anti-depressivos e foi verificado o tempo necessário para que esta concentração seja atingida novamente. Foram propostos métodos para que esta concentração fosse atingida em um menor período de tempo. Outra aplicação foi na comparação entre o tratamento com comprimidos inteiros e tomados pela metade em um intervalo menor de tempo. Verificou-se que a concentração atingida é diferente mesmo que a massa ingerida seja a mesma. O modelo também foi utilizado para calcular a concentração de nicotina após o consumo de cigarros e verificou-se que, o indivíduo que fuma a cada três horas não consegue eliminar totalmente a nicotina de seu organismo. Além disso, foi possível simular a sobredosagem de um anti-inflamatório e verificar o tempo em que a concentração fica acima do nível terapêutico. Foi proposto um método para obtenção do parâmetro farmacocinético relacionado à absorção, que pode ser obtido facilmente a partir de dados presentes nas bulas dos medicamentos. Este método é muito mais simples e preciso do que e proposto na literatura, que utiliza análise gráfica e dados clínicos que não são obtidos com tanta facilidade. / The pharmacokinetic modeling can predict the concentration of drug in different tissues of the human body. The development of mathematical models is an important tool to verify the appropriateness of certain procedures performed in medication administration. The objective of this work is to develop a pharmacokinetic model able to predict the plasma concentration of drug in the body after various forms of infusion. Two approaches were used. Initially, in the one-compartment approach it was considered that the drug enters the body directly into the blood compartment, which represents the entire human body. In the two-compartment approach it was considered the following compartments: one representing the means by which the drug is infused into the body (either via the gastrointestinal tract, lung, or transdermal) and one representing the blood plasma. In both cases, it was considered homogeneous concentration of the drug in the compartments. The model was built by using block diagrams and the solution was obtained using the Laplace Transform. The model was validated by comparing its results to literature data, with very good agreement. The model allowed comparing the one-compartment constant infusion of drug in the body with the periodic infusion. The analysis of the results generated by the model showed that the concentrations achieved by these methods are not the same. The two-compartment model allowed simulating oral and transdermal administration, and inhalation. It was possible to predict blood concentration after interruption of therapy with anti-depressants and anti-conceptional drugs. The model was able to verify the time it takes to reach the former level. Methods have been proposed to achieve the same concentration in a shorter period of time. Another application was the comparison of the treatment with whole tablets and taken by half in a smaller interval of time. It was found that the concentration achieved is different even though the same mass is ingested in both cases. The model was also used to calculate the concentration of nicotine after cigarette smoking and it was found that the individual who smokes every three hours, nicotine is not entirely eliminated from body. Furthermore, it was possible to simulate overdose of an anti-inflammatory and the period of time when the concentration is above the therapeutic level. It has been proposed a method to obtain pharmacokinetic parameter related to absorption, which can be easily obtained based on data present in the drug bull. This method is much simpler and more accurate than the method proposed in the, which uses graphical analysis and clinical data that are not so easy to be obtained.

Les travaux présentés dans ce manuscrit sont divisés en deux grandes parties, et abordent des applications biologiques relatives au cancer et plus particulièrement à leur traitement. La première partie est consacrée à une recherche technologique, dont l'objectif est le développement d'un dispositif innovant permettant de contrôler plus efficacement la phase cytotoxique de la thérapie photodynamique. Cette thérapie contre le cancer met en jeu trois éléments principaux : un agent photosensibilisant, de l'oxygène et de la lumière. La solution proposée repose sur une stratégie d'asservissement d'un indicateur thérapeutique observable durant le traitement : le photoblanchiment. Le système d'asservissement développé utilise un observateur d'état qui a nécessité de résoudre en pratique des problèmes d'identifiabilité et d'identification d'un processus non-linéaire. Il est implanté dans une plateforme pilote opérationnelle validée par des tests in vitro. Une demande de brevet pour le dispositif développé est en cours. La seconde partie de cette thèse s'inscrit dans le cadre d'une recherche appliquée, sur le thème de l'identification à temps continu de systèmes biologiques, à partir de séquences d'images au travers de trois cas d'études aux échelles cellulaire, tissulaire et animale. Une première étude est dédiée à la proposition d'un modèle à compartiments de la pharmacocinétique intratumorale de nanoparticules multifonctionnelles dans des cerveaux de rats, ainsi qu'à son identification à partir de séquences d'images IRM in vivo. La seconde traite de la modélisation, à partir de données d'imagerie expérimentale de fluorescence, de la fonctionnalité des jonctions communicantes intercellulaires. L'objectif est de discriminer deux types de cellules cancéreuses, grâce à leur dynamique de recouvrement de fluorescence. Enfin, un troisième cas d'étude aborde le problème de l'identification d'une cohorte de systèmes à partir de petits échantillons de données. Le contexte applicatif est l'étude de l'angiogenèse tumoral et de l'effet des traitements anti-cancer sur le développement du réseau vasculaire / The presented works are divided into two main parts and deal with biological applications to cancer, and more specifically to their treatments. The first part is dedicated to a technological research, in which a new device is designed and built to efficiently control the cytotoxic phase of photodynamic therapy. This anti-cancer therapy involves three main compounds: a photosensitizer agent, oxygen and light. The proposed solution relies on the control of an observable therapeutic indicator during the treatment: the photobleaching phenomenon. The developed control system uses a state observer which required to solve practical identifiability issues and the identification of a non-linear process. It has been implemented in a technical platform and validated during in in vitro tests. A patent application for this device is currently under review. The second section of this thesis deals with the applicability of continuous-time identification approaches to three biological systems from image sequences recorded at cellular, tissue and animal scales. A first study examines how continuous-time system identification may be used to determine a pharmacokinetic compartmental model of multifunctional nanoparticles within rat brain from in vivo MRI images. The second study deals with the empirical modeling of the junctional intercellular communication functionalities. The purpose is to discriminate two cancer cells types from their fluorescence recovery dynamics. Finally, a third study case addresses the issue of identifying a systems cohort from small amount of data. The applied context is the study of tumoral angiogenesis and the anti-cancer treatment effects on vascular network development

<p>The inverse problem of modeling biochemical processes mathematically from measured time course data falls into the category of system identification and parameter estimation. Analyzing the time course data would provide valuable insights into the model structure and dynamics of the biochemical system. Based on the types of biochemical reactions, such as metabolic networks and genetic networks, several modeling frameworks have been proposed, developed and proved effective, including the Michaelis-Menten equation, the Biochemical System Theory (BST), etc. One bottleneck in analyzing the obtained data is the estimation of parameter values within the system model.</p> <p>As most models for molecular biological systems are nonlinear with respect to both parameters and system state variables, estimation of parameters in these models from experimental measurement data is thus a nonlinear estimation problem. In principle, all algorithms for nonlinear optimization can be used to deal with this problem, for example, the Gauss-Newton iteration method and its variants. However, these methods do not take the special structures of biological system models into account. When the number of parameters to be determined increases, it will be challenging and computationally expensive to apply these conventional methods.</p> <p>In this research, several methods are proposed for estimating parameters in two classes of widely used biological system models: the S-system model and the linear fractional model (LFM), by utilizing their structure specialties. For the S-system, two estimation methods are designed. 1) Based on the two-term structure (production and degradation) of the model, an alternating iterative least squares method is proposed. 2) A separation nonlinear least squares method is proposed to deal with the partially linear structure of the model. For the LFM, two estimation methods are provided. 1) The separation nonlinear least squares method can also be adopted to treat the partially linear structure of the LFM, and moreover a modified iterative version is included. 2) A special strategy using the separation principle and the weighted least squares method is implemented to turn the cost function into a quadratic form and thus the estimates for parameters can be analytically solved. Simulation results have demonstrated the effectiveness of the proposed methods, which have shown better performance in terms of estimation accuracy and computation time, compared with those conventional nonlinear estimation methods.</p>