Made available in DSpace on 2014-06-12T16:30:16Z (GMT). No. of bitstreams: 2 arquivo2758_1.pdf: 877607 bytes, checksum: 307ea2159d2a3bd8df068f531d04a4d8 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Plantas com atividade antioxidante podem estar envolvidas no tratamento e/ou prevenção de diversas enfermidades, em especial as que envolvem processos inflamatórios. Foram selecionadas vinte espécies de plantas utilizadas para tratar inflamação pela comunidade de Carão, localizada em Altinho-PE, e dezenove espécies de forma aleatória. A coleta foi realizada na referida região no mês de setembro de 2009, de no mínimo 3 indivíduos de cada espécies, submetidas a extração por maceração com metanol 80% durante 6 dias e evaporado o solvente sob pressão reduzida. Buscando avaliar a correlação entre métodos utilizados para determinar a atividade antioxidante foram aplicados três: atividade sequestrante de radical livre DPPH (DPPH), ensaio da atividade quelante do íon ferroso (FIC) e poder antioxidante redutor férrico (FRAP). Avaliou-se também o poder antioxidante de espécies utilizadas para tratamento de inflamações comparadas às espécies selecionadas de forma aleatória. Observou-se que não houve correlação entre os métodos FIC e DPPH (rs = -0,3008 e p = 0,1975) e entre FIC e FRAP (rs = 0,3042 e p = 0,1921). Entretanto, foi possível observar correlação significativa entre FRAP e DPPH (rs = -0,9563 e p = < 0,0001). Através da análise de variância de Kruskal-Wallis os dois grupos de espécies estudadas foram estatisticamente iguais através dos métodos DPPH e FRAP e que as espécies aleatórias foram estatisticamente mais efetivas quando avaliado o poder quelante do íon ferroso. Contudo, quando utilizada a classificação de Melo et al (2010), observa-se que 45% das espécies antiinflamatórias apresentaram boa atividade seqüestradora de radicais livres comparadas às 36,84% das aleatórias. Conclui-se que os métodos utilizados avaliam a atividade antioxidante por meio de mecanismos distintos, não sendo recomendável o emprego de um único método para determinação da atividade antioxidante. Sendo o método DPPH mais indicado para avaliar a atividade antioxidante de espécies utilizadas para tratar inflamação. Dados sugerem que as espécies anttinflamatórias não agem quelando o íon ferroso, não sendo este método mais indicado para avaliação da atividade antioxidante de espécies antiinflamatórias, visto a quelação de metais de transição tem um papel secundário no mecanismo de inibição dos radicais livres

Magíster en Ciencias, mención Física / En esta tesis se diseñó e implementó una metodología experimental a escala microfluídica, enfocada al estudio de la difusión en redes cuasi-bidimensionales aleatorias de microcanales. Para ello, se adaptó la técnica de la recuperación de la fluorescencia después del fotoblanqueo (FRAP) con el objetivo de determinar el coeficiente de difusión efectivo adimensional $D/D_0$ de la fluoresceína en una solución acuosa dentro de estas redes; donde $D_0$ es el coeficiente de difusión molecular de esta especie. Se estudió el comportamiento de $D/D_0$ en función de dos parámetros adimensionales que describen las cualidades geométricas de estas redes: la homogeneidad $\gamma=R/\langle L \rangle$ y el aspecto reticular $\eta=\langle L \rangle/w$, donde $\langle L \rangle$ y $w$ son la longitud media y el ancho de los canales, y $R$ es el radio de la zona en que la difusión es medida. El parámetro $\gamma$ describe los efectos de los detalles geométricos sobre la difusión y $\eta$ cuantifica el volumen accesible al fluido. Adicionalmente, en estas geometrías estudiamos la respuesta de $D/D_0$ frente la acción de un flujo periódico de amplitud $x$ y promedio nulo. Determinamos un parámetro crítico $\gamma_c\sim2.7$ a partir del cual la red puede ser descrita como un medio efectivo. En esta escala, encontramos que $D/D_0\sim 0.7$ para un valor fijo de $\eta=3.64$. Además, medimos el efecto del aspecto reticular $\eta$ sobre la difusión; en este caso $D/D_0$ disminuye cuando $\eta$ aumenta, llegando rápidamente a un nivel de saturación $D/D_0\sim0.4-0.5$ para $\eta>2.5$. Por otro lado, para $\gamma=0.6$, lo que llamamos la micro-escala, se encontró que existe una dependencia del número de conexiones existentes en un nodo de la red sobre $D/D_0$. En el caso de la acción del flujo externo, observamos un importante incremento de $D/D^*$ en términos de la amplitud adimensional de oscilación de este flujo $x/\langle L \rangle$, siendo $D^*$ el coeficiente efectivo de difusión en ausencia de flujo. En la primera parte de esta tesis establecemos la base teórica correspondiente del proceso de difusión, incluyendo una descripción para medios no homogéneos. También discutimos los aspectos relevantes del FRAP, que es la metodología experimental que usamos en este trabajo. Más adelante, describimos los métodos experimentales desarrollados, donde reseñamos, entre otros detalles, la forma de obtener geometrías reticulares desordenadas mediante los mosaicos de Voronoi y el procedimiento de construcción de los microcanales que reproducen estas geometrías. Luego, mostramos en detalle los resultados experimentales, entre ellos, los ya mencionados. Finalmente incluimos las conclusiones del presente trabajo, presentando además las sugerencias para futuras investigaciones en este tema.

The Bcl-2 family of proteins strictly regulates the intrinsic pathway of apoptosis. Direct physical interactions between Bcl-2 proteins regulate mitochondrial outerpermeabilisation (MOMP), which occurs in response to various cell stresses andapoptotic stimuli. How changes in Bcl-2 protein activity regulate apoptosiscommitment is still unclear, especially with regard to how they interact with eachother within the context of the mitochondrial membrane. Recent studies haveshown that Bcl-2 proteins exist in a dynamic equilibrium between the mitochondriaand the cytosol. In this thesis, by using FRAP, I have measured changes in Bcl-XLand Mcl-1 dynamics in single cells. Surprisingly, individual cells within a populationshow widely differing Bcl-XL and Mcl-1 dynamics. There is a corelation betweenBcl-XL and Mcl-1 dynamics with BH3-only protein expression. Anti-apoptotic andpro-apoptotic Bcl-2 proteins stabilise each other on the OMM. Together, theseresults indicate that cells constantly fine tune mitochondrial priming and thatanalysing anti-apoptotic Bcl-2 proteins by FRAP allows this to be measured at asingle cell level in real time before MOMP.

No description available.

This thesis examines the influence of wheat Citrus with higher content of lutein in comparison with common wheat on antioxidant activity of the rat blood plasma and liver tissue. In the experiment, 32 male rats of Wistar strain were used. The influence of the feeding of yellow wheat varieties Citrus on antioxidant activity was measured in the blood and liver tissue. The experimental group (N = 16) was fed with dried granules of 100% wheat meal from Citrus. The control group (N = 16) was fed with similar feed prepared from two common wheat. Content of crude protein was the same in the both group. Body weight gain were followed in three-day intervals and feed consumption was followed daily. Antioxidant activity was measured by DPPH, FR, FRAP and ABTS methods. Values are expressed in gallic acid equivalent. In the blood significant differences (P < 0.05) of antioxidant activity was determined only by ABTS method between "Citrus" group (405.51 +- 7.64 mg /ml) in comparison to "Control" group (479.99 +- 22.54 mg/ml) in contrary to expectation this value of control group was of 18% higher. Values of other antioxidant methods were not significant. In the liver tissue only average value of ABTS method was little higher in Citrus group (409,439 +-13,626) in comparison with control group (389,493 +- 9,271 mg/ml). Similar situation was with DPPH method (20.711 +- 0.867 in Citrus group and 20.037 +- 1.109 mg/ml in the Control group) and difference was also not significant (P> 0.05).

After more than a century, there are no analytical solutions for the Navier-Stokes equations to describe complex fluid behavior, and we often resort to different computational methods to find solutions under specific conditions. In particular, to address many biological questions, we need to use techniques which are accurate at the mesoscale regime and computationally efficient, since atomistic simulations are still incredibly computationally costly, and continuum methods based on Navier-Stokes present challenges with complicated moving boundaries, in the presence of fluctuations. Here, we use a novel particle-based coarse-grained method, known as MPCD, to study ciliated swimmers. Using experimentally measured beating patterns, we show how we recapitulate the emergence of metachronal waves (MCW) on planar surfaces, and present new results on curved surfaces. To quantitatively study these waves, we also analyzed their effect on beating intervals, energy fluctuations, and fluid motion. We then extended our model to realistic cellular geometries, using experimentally obtained Basal Bodies locations.\par In the second part of our study, we focused on the intracellular fluid motion, neglecting hydrodynamic interactions. We developed the Digital Confocal Microscopy Suite (DCMS) that can run on multiple platforms using GPUs and can input realistic cell shapes and optical properties of the confocal microscope. It has this ability to simulate both (Fluorescence Recovery After Photobleaching) FRAP and Fluorescence Correlation Spectroscopy (FCS) experiments, as well as the capability to model photo-switching of fluorophores, acquisition photo-bleaching, and reaction-diffusion systems. With this platform, in collaboration with the Vidali Lab, we were able to elucidate the role of boundaries in interpreting FRAP experiments in \textit{moss} and estimate the binding rates of myosin XI.

After more than a century, there are no analytical solutions for the Navier-Stokes equations to describe complex fluid behavior, and we often resort to different computational methods to find solutions under specific conditions. In particular, to address many biological questions, we need to use techniques which are accurate at the mesoscale regime and computationally efficient, since atomistic simulations are still incredibly computationally costly, and continuum methods based on Navier-Stokes present challenges with complicated moving boundaries, in the presence of fluctuations. Here, we use a novel particle-based coarse-grained method, known as MPCD, to study ciliated swimmers. Using experimentally measured beating patterns, we show how we recapitulate the emergence of metachronal waves (MCW) on planar surfaces, and present new results on curved surfaces. To quantitatively study these waves, we also analyzed their effect on beating intervals, energy fluctuations, and fluid motion. We then extended our model to realistic cellular geometries, using experimentally obtained Basal Bodies locations.\par In the second part of our study, we focused on the intracellular fluid motion, neglecting hydrodynamic interactions. We developed the Digital Confocal Microscopy Suite (DCMS) that can run on multiple platforms using GPUs and can input realistic cell shapes and optical properties of the confocal microscope. It has this ability to simulate both (Fluorescence Recovery After Photobleaching) FRAP and Fluorescence Correlation Spectroscopy (FCS) experiments, as well as the capability to model photo-switching of fluorophores, acquisition photo-bleaching, and reaction-diffusion systems. With this platform, in collaboration with the Vidali Lab, we were able to elucidate the role of boundaries in interpreting FRAP experiments in \textit{moss} and estimate the binding rates of myosin XI.

L'endocytose dépendante de la clathrine (EDC), c’est-à-dire la formation de vésicules recouvertes de clathrine (VRC) à partir de la membrane plasmique, est un processus essentiel dans les cellules eucaryotes. Au cours de l’EDC, la GTPase dynamine est recrutée au cou de la VRC naissante où elle s'oligomérise en hélice. Les changements de conformation induits par l'hydrolyse du GTP catalysent la scission du cou vésiculaire. Ce processus a été étudié en détail par reconstitution in vitro sur des tubules membranaires, mais il doit être établi dans des cellules vivantes, où les interactions de la dynamine avec d'autres protéines comme l'amphiphysine sont critiques. L'imagerie TIRF (Total Internal Reflection Fluorescence) avec le protocole pH pulsé (ppH) sur cellules vivantes permet la détection de la formation de VRC avec une résolution spatiale (~100 nm) et temporelle (2 s) élevée. Ce protocole a révélé que la dynamine présente un recrutement biphasique aux puits recouverts de clathrine (PRC) en maturation avec un pic au moment de la scission mais les paramètres de son recrutement dans les cellules vivantes restent peu clairs. Pour déterminer ces paramètres, j’ai utilisé des techniques d’imagerie sur cellules vivantes pour étudier le recrutement de la dynamine à l’échelle globale et à l’échelle de la molécule unique lors de perturbations aiguës de sa fonction. Mes résultats de thèse ont montré que la dynamine est recrutée à la membrane plasmique, diffuse à l'extérieur des PRC et y est transitoirement piégée. De plus, j’ai déterminé avec des dynamines mutées (1) que le domaine PRD de la dynamine est crucial pour son recrutement aux PRC ; (2) que le domaine PH est important pour la scission vésiculaire mais par pour son recrutement aux PRC ou à la membrane plasmique. Enfin, j’ai observé que la dynamine s'échange en permanence avec un pool extra-PRC, ce qui permettrait son recrutement ultérieur par l'ajout de nouveaux sites de liaison et sa capacité à rétrécir le cou des vésicules suite à l’hydrolyse du GTP. En conclusion, ces données suggèrent qu’aux PRC, les molécules de dynamine (1) sont constamment échangées ; (2) diffusent à des taux similaires tout au long du processus de formation, maturation et scission des vésicules; et (3) l'activité GTPase de la dynamine contribue à la maturation et à la scission des VRC. / Clathrin-mediated endocytosis (CME), the formation of clathrin-coated vesicles (CCV) from the plasma membrane, is an essential process in eukaryotic cells. During CME, the GTPase dynamin is recruited to the neck of nascent CCV where it oligomerizes into helical filaments. Conformational changes induced by the hydrolysis of GTP catalyze the scission of the vesicle neck. This process has been studied in detail with in vitro reconstitution on membrane tubules but it needs to be established in living cells, where interactions between dynamin and other proteins such as amphiphysin are critical. Live cell total internal reflection fluorescence (TIRF) imaging with the pulsed pH (ppH) assay allows the detection of CCV formation with high spatial (~100 nm) and temporal (2 s) resolutions. It has revealed that dynamin is recruited to maturing clathrin-coated pits (CCP) in two phases with a peak at the time of scission but the parameters of its recruitment in living cells remain unclear. To determine these parameters, we have performed live cell imaging of dynamin recruitment at collective and single molecule levels during acute perturbations of its function. My PhD results showed that dynamin is recruited to the plasma membrane, diffuses outside of CCP and is trapped at CCP. Furthermore, we determined with mutated dynamins that (1) the PRD domain of dynamin is crucial for its recruitment at CCP; (2) the PH domain is important for vesicular scission but not for recruitment to CCP or to the plasma membrane. Finally, I observed that dynamin exchanges with an extra-CCP pool at all times: this would allow for its further recruitment by addition of new binding sites and its ability to narrow the vesicle neck after GTP hydrolysis. Altogether, these data suggest that in CCP dynamin molecules (1) are constantly exchanged; (2) diffuse at similar rates throughout the entire process of vesicle formation, from maturation until scission; and (3) that dynamin’s GTPase activity contributes to CCP maturation and scission.

Didžiausias fenolinių junginių kiekis bei didžiausios radikalų surišimo gebos ir redukcinio aktyvumo reikšmės nustatytos po 20 min ekstrakcijos. Didžiausi fenolinių junginių kiekiai nustatyti UAB „Švenčionių vaistažolių fabrikas“ (Herba Origani vulgaris 50g ir 2g N25) arbatose, mažiausias fenolinių junginių kiekis - “Emili” (Herba Origani vulgaris 1,5g N20) arbatoje. Didžiausia laisvųjų radikalų surišimo geba ir redukcinio aktyvumo reikšmės nustatytos UAB „Švenčionių vaistažolių fabrikas“ (Herba Origani vulgaris 50g) arbatoje. Mažiausia laisvųjų radikalų surišimo geba nustatyta Emili” (Herba Origani vulgaris 1,5g N20) arbatoje. Nustatyta stipri koreliacija tarp arbatų suminio fenolinių junginių kiekio ir antioksidantinio aktyvumo. Didžiausi koreliacijos koeficientai tarp suminio fenolinių junginių kiekio bei radikalinio ir redukcinio aktyvumo nustatyti UAB „Švenčionių vaistažolių fabrikas“ (Herba Origani vulgaris 50g) gamintojo arbatoje. / The biggest amount of phenolic compounds, free radical binding capacity and reducing activity were estimated after 20 minutes of extraction. The biggest amount of phenolic compounds was estimated in JSC “ Švenčionių vaistažolių fabrikas“ (Herba Origani vulgaris 50g and 2g N25) teas, while the smallest in “Emili” (Herba Origani vulgaris 1,5g N20) teas. The biggest free radical binding capacity and reducing activity were estimated in JSC “Švenčionių vaistažolių fabrikas“ (Herba Origani vulgaris 50g) tea. The smallest free radical binding capacity was estimated in “Emili” (Herba Origani vulgaris 1,5g N20) tea. A strong correlation between the total amount of phenolic compounds and antioxidant activity was established. The biggest coefficient between the total amount of phenolic compounds and radical, reducing activity was estimated in JSC “Švenčionių vaistažolių fabrikas“ tea.

La majorité des études sur la dynamique des facteurs de transcription en cellules vivantes s'accordent sur une dynamique rapide. Il existe cependant quelques exceptions, comme la dynamique du facteur de transcription HSF « Heat Shock Factor », sur les chromosomes polyténiques de drosophile. Notre projet a consisté à étudier la dynamique d'HSF1 dans des cellules humaines. L'exposition des cellules à un stress tel qu'un choc thermique induit une réponse ubiquitaire et transitoire, dont la fonction est de protéger les cellules contre les effets délétères du stress. Au cours d'un choc thermique, plusieurs phénomènes se produisent : i) un arrêt global de la transcription excepté pour certains gènes tels que ceux codant pour les protéines de choc thermique (HSPs), dont l'expression est sous le contrôle du facteur de transcription HSF1. ii) une activation d'HSF1 qui se relocalise de façon rapide et transitoire sur les corps nucléaires de stress (nSBs), où il induit la transcription des séquences satellite III. Les nSBs forment un site d'activité naturellement amplifié et visible en microscopie. Nous avons utilisé deux techniques complémentaires pour étudier la dynamique d'HSF1 en cellules vivantes : le recouvrement de fluorescence après photoblanchiment (FRAP) et la spectroscopie à corrélation de fluorescence multi-confocale (mFCS), qui permet l'analyse FCS en plusieurs points simultanément. En cellules HeLa, la protéine HSF1-eGFP présente une dynamique rapide qui est significativement ralentie suite à un choc thermique. En mFCS, nous avons obtenu des constantes de diffusion de 14 µm²/s avant choc thermique et de 10 µm²/s après choc thermique. En FRAP, le temps de demi-recouvrement est de 0,2 s avant choc thermique, 2,6 s après choc thermique dans le nucléoplasme et 65 s sur les corps nucléaires de stress. Le ralentissement de la dynamique d'HSF1 s'explique par deux phénomènes : i) la formation de complexes de haut poids moléculaire, ii) une augmentation des interactions avec la chromatine. Pour mieux caractériser le changement de dynamique d'HSF1 après choc thermique, plusieurs mutants ont été analysés. Le domaine de trimérisation est indispensable pour le changement de dynamique après choc thermique, alors que le domaine de liaison à l'ADN et le domaine de transactivation n'ont que peu d'effet sur le changement de dynamique. Il ne peut donc pas être expliqué uniquement par les interactions directes à la chromatine du domaine de liaison à l'ADN, ni même par les liaisons indirectes du domaine de transactivation via d'autres protéines. La protéine HSF1 pourrait interagir de façon aspécifique avec la chromatine lors de la recherche de site de liaison, ou d'autres protéines via d'autres domaines pourraient entrainer des interactions indirectes avec la chromatine. / The majority of studies made on transcription factors dynamics on living cells agree with a fast dynamics process. However, there is some exceptions such as the dynamics of the transcription factor HSF “Heat Shock Factor” on drosophila polytenic chromosome. My project is to study HSF1 dynamics in human living cells. Cells exposure to a stress such as heat shock induces a transient and ubiquitous response that function's to protect cells against the deleterious effect of stress. During the course of a heat shock, several phenomenons take place: i) a global arrest of transcription, with the exception of some genes, such as those coding for the heat shock proteins (hsp), which expression is under the control of HSF1. ii) Activation of HSF1 that relocalize in a fast and transient way to nuclear stress bodies (nSBs), where it induces satellite III transcription. nSBs act as a natural amplification gene array, visible on microscopy. We have used two complementary techniques to look at HSF1 dynamics in living cells: Fluorescence recovery after photobleaching (FRAP) and multiconfocal fluorescence correlation spectroscopy (mFCS) that allow FCS analysis at several position simultaneously. On HeLa cells, HSF1-eGFP protein has a fast dynamics which is significantly slowed down following heat shock. On mFCS, we obtained a diffusion constant of 14 µm²/s before heat shock, and 10 µm²/s after heat shock. On FRAP, the half recovery time is 0.2 s before heat shock, 2.6 s after heat shock in the nucleoplasm and 65 s in nuclear stress bodies. HSF1 dynamics slowing down may be explain by two phenomenons: i) formation of high molecular mass complexes, ii) rise of interaction of HSF1 with chromatin. To better characterize changes in HSF1 dynamics after heat shock, several mutants have been analyzed. The trimerization domain of HSF1 is essential for dynamics changes after heat shock, while DNA binding domain (DBD) and transactivation domain (TAD) have only little effects on dynamics changes. These changes cannot only be explained by direct interaction of DNA binding domain with chromatin, neither by indirect interaction of the transactivation domain with other protein partners. HSF1 could be able to interact non-specifically with chromatin during the search for specific binding sites. Also other proteins via other domains might induce indirect binding to chromatin.