971 resultados para Microscope confocal
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L'ubiquitination est une modification des protines conserve, consistant en l'addition de rsidus ubiquitine et rgulant le destin cellulaire des protines. La protine TRAF-interacting protein TRAIP (ou TRIP) est une ligase E3 qui catalyse l'tape finale de l'ubiquitination. TRAIP est conserv dans l'volution et est ncessaire au dveloppement des organismes puisque l'ablation de TRAIP conduit la mort embryonnaire aussi bien de la drosophile que de la souris. De plus, la rduction de l'expression de TRAIP dans des kratinocytes pidermiques humains rprime la prolifration cellulaire et induit un arrt du cycle cellulaire en phase Gl, soulignant le lien troit entre TRAIP et la prolifration cellulaire. Comme les mcanismes de rgulation de la prolifration jouent un rle majeur dans l'homostasie de la peau, il est important de caractriser la fonction de TRAIP dans ces mcanismes. En utilisant des approches in vitro, nous avons dtermin que la protine TRAIP est instable, modifie par l'addition d'ubiquitine et ayant une demi-vie d'environ 4 heures. Nos analyses ont galement rvl que l'expression de TRAIP est dpendante du cycle cellulaire, atteignant un pic d'expression en phase G2/M et que l'induction de son expression s'effectue principalement au cours de la transition Gl/S. Nous avons identifi le facteur de transcription E2F1 comme en tant le responsable, en rgulant directement le promoteur de TRAIP. Aussi, TRAIP endogne ou surexprime est surtout localise au niveau du nuclole, une organelle nuclaire qui est dsassemble pendant la division cellulaire. Pour examiner la localisation subcellulaire de TRAIP pendant la mitose, nous avons imag la protine TRAIP fusionne une protine fluorescente, l'intrieur de cellules vivantes nommes HeLa, l'aide d'un microscope confocal. Dans ces conditions, TRAIP est majoritairement localise autour des chromosomes en dbut de mitose, puis est arrange au niveau de l'ADN chromosomique en fin de mitose. La dtection de TRAIP endogne l'aide d'un anticorps spcifique a confirm cette localisation. Enfin, l'inactivation de TRAIP dans les cellules HeLa par interfrence ARN a inhib leur capacit s'arrter en milieu de mitose. Nos rsultats suggrent que le mcanisme sous-jacent peut tre li au point de contrle de l'assemblage du fuseau mitotique. - Ubiquitination of proteins is a post-translational modification which decides the cellular fate of the protein. The TRAF-interacting protein (TRAIP, TRIP) functions as an E3 ubiquitin ligase mediating addition of ubiquitin moieties to proteins. TRAIP interacts with the deubiquitinase CYLD, a tumor suppressor whose functional inactivation leads to skin appendage tumors. TRAIP is required for early embryonic development since removal of TRAIP either in Drosophila or mice by mutations or knockout is lethal due to aberrant regulation of cell proliferation and apoptosis. Furthermore, shRNA- mediated knock-down of TRAIP in human epidermal keratinocytes (HEK) repressed cell proliferation and induced a Gl/S phase block in the cell cycle. Additionally, TRAIP expression is strongly down- regulated during keratinocyte differentiation supporting the notion of a tight link between TRAIP and cell proliferation. We thus examined the biological functions of TRAIP in epithelial cell proliferation. Using an in vitro approach, we could determine that the TRAIP protein is unstable, modified by addition of ubiquitin moieties after translation and exhibits a half-life of 3.7+/-1-6 hours. Our analysis revealed that the TRAIP expression is modulated in a cell-cycle dependent manner, reaching a maximum expression level in G2/M phases. In addition, the expression of TRAIP was particularly activated during Gl/S phase transition and we could identify the transcription factor E2F1 as an activator of the TRAIP gene promoter. Both endogenous and over-expressed TRAIP mainly localized to the nucleolus, a nuclear organelle which is disassembled during cell division. To examine the subcellular localization of TRAIP during M phase, we performed confocal live-cell imaging of a functional fluorescent protein TRAIP-GFP in HeLa cells. TRAIP was distributed in the cytoplasm and accumulated around mitotic chromosomes in pro- and meta-phasic cells. TRAIP was then confined to chromosomal DNA location in anaphase and later phases of mitosis. Immune-detection of endogenous TRAIP protein confirmed its particular localization in mitosis. Finally, inactivating TRAIP expression in HeLa cells using RNA interference abrogated the cells ability to stop or delay mitosis progression. Our results suggested that TRAIP may involve the spindle assembly checkpoint.
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L'immunoglobuline A scrtoire (SlgA) est l'anticorps qui est dominant dans la rponse humorale des muqueuses. IgA est produit localement sous la forme de dimre ou polymre. Ces derniers sont ensuite relchs dans les scrtions muqueuses sous forme d'un complexe avec la pice scrtoire (SC). SlgA est capable d'identifier des antignes microbiens dans l'environnement des muqueuses et de prvenir leur diffusion en bloquant l'adhsion ou la surface des cellules pithliales. Au-del de sa fonction classique d'exclusion immune, SlgA peut aussi adhrer aux cellules M prsente au niveau des follicules associs l'pithlium dans des structures organises appeles plaques de Peyer (PPs) dans le systme gastrointestinal. L'interaction slective avec les SlgA amne cette dernire tre transporte travers les cellules M Ce procs facilite l'association de l'anticorps avec les cellules dendritiques (DCs) CDllc+CDllb+, localises dans la rgion sous-pithliale des PPs. L'entre de SlgA ou de microorganismes couverts par la SlgA via ce chemin est cruciale pour la modulation de la rponse immunitaire locale. Dans la premire partie du travail, nous avons tabli les conditions pour l'analyse de l'interaction entre SlgA et une lignes de DCs drive in vitro. Nous avons aussi montr que l'internalisation de la SlgA par les DCs est affect aprs traitement avec des inhibiteurs de l'endocytose ou par l'utilisation de comptiteurs molculaires. En utilisant le microscope confocal, on a observ qu'aprs internalisation la SlgA suit la voie endocytique, vers le compartiment lysozomal. Dans la deuxime partie du travail, une partie des rsultats a t confirme pour les DCs CDllc+CDllb+ des muqueuses. En outre, l'importance des sucres prsents sur la SlgA a t mis en vidence par la rduction de l'interaction avec les DCs des muqueuses aprs dglycosylation. On a montr pour la premire fois notre connaissance que Dectin-1 et SIGNR3 sont des rcepteurs potentiels pour la SlgA sur les DCs des muqueuses de la souris. De plus, les DCs de la souris prleves des PPs, des ganglions lymphatiques msentriques (MLNs) et de la rate ont t infects avec Shigella flexneri (Sf) seule ou en complexe avec SlgA. Les DCs infectes ont montr une augmentation de l'expression des marqueurs co-stimulateurs, une forte production des cytokines proinflammatoires et une induction de la prolifration des cellules T. Aprs l'association des Sf avec SlgA, les profils pro-inflammatoires des DCs ont diminus. En particulier, SlgA a un effet sur les cytokines scrtes et sur la prolifration des cellules T. Ces donnes dmontrent le rle de la SlgA dans la rponse immunitaire par les DCs des muqueuses.
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Introduction : La chronicit de la rhinosinusite, sa rsistance aux antibiotiques, et ses exacerbations aigus laissent croire que les biofilms sont impliqus dans la rhinosinusite chronique. Objectifs : Nous avons valu la capacit des bactries Pseudomonas aeruginosa, staphylocoques coagulase ngative et Staphylococcus aureus former des biofilms par un essai in vitro, et si cette capacit de formation a un lien avec lvolution de la maladie. Nous avons valu in vitro leffet de la moxifloxacine, un antibiotique utilis dans le traitement de la rhinosinusite chronique sur des biofilms matures de Staphylococcus aureus. Mthodes : Trent et une souches bactriennes ont t isoles de 19 patients atteints de rhinosinusite chronique et qui ont subit au moins une chirurgie endoscopique des sinus. Lvolution de la maladie a t note comme "bonne" ou "mauvaise" selon lvaluation du clinicien. La production de biofilm a t value grce la coloration au crystal violet. Nous avons valu la viabilit du biofilm aprs traitement avec la moxifloxacine. Ces rsultats ont t confirms en microscopie confocale balayage laser et par la coloration au LIVE/DEAD BacLight. Rsultat et Conclusion : Vingt deux des 31 souches ont produit un biofilm. La production dun biofilm plus importante chez Pseudomonas aeruginosa et Staphylococcus aureus tait associe une mauvaise volution. Ceci suggre un rle du biofilm dans la pathogense de la rhinosinusite chronique. Le traitement avec la moxifloxacine, une concentration de 1000X la concentration minimale inhibitrice rduit le nombre des bactries viables de 2 2.5 log. Ces concentrations (100 g/ml - 200 g/ml) sont faciles atteindre dans des solutions topiques. Les rsultats de notre tude suggrent que lutilisation de concentrations suprieure la concentration minimale inhibitrice sous forme topique peut ouvrir des voies de recherche sur de nouveaux traitements qui peuvent tre bnfiques pour les patients atteints de forme svre de rhinosinusite chronique surtout aprs une chirurgie endoscopique des sinus.
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3 vidos sont dans des fichiers complmentaires ce mmoire
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Les cellules sont capables de dtecter les distributions spatiales de protines et ainsi de migrer ou stendre dans la direction approprie. Une comprhension de la rponse cellulaire aux modifications de ces distributions spatiales de protines est essentielle pour lavancement des connaissances dans plusieurs domaines de recherches tels que le dveloppement, limmunologie ou loncologie. Un exemple particulirement complexe est le guidage daxones se droulant pendant le dveloppement du systme nerveux. Ce dernier ncessite la prsence de plusieurs distributions de molcules de guidages tant attractives ou rpulsives pour connecter correctement ce rseau complexe quest le systme nerveux. Puisque plusieurs indices de guidage collaborent, il est particulirement difficile didentifier la contribution individuelle ou la voie de signalisation qui est dclenche in vivo, il est donc ncessaire dutiliser des mthodes pour reproduire ces distributions de protines in vitro. Plusieurs mthodes existent pour produire des gradients de protines solubles ou lies aux substrats. Quelques mthodes pour produire des gradients solubles sont dj couramment utilises dans plusieurs laboratoires, mais elles limitent ltude aux distributions de protines qui sont normalement scrtes in vivo. Les mthodes permettant de produire des distributions lies au substrat sont particulirement complexes, ce qui restreint leur utilisation quelques laboratoires. Premirement, nous prsentons une mthode simple qui exploite le photoblanchiment de molcules fluorescentes pour crer des motifs de protines lies au substrat : Laser-assisted protein adsorption by photobleaching (LAPAP). Cette mthode permet de produire des motifs de protines complexes dune rsolution micromtrique et dune grande porte dynamique. Une caractrisation de la technique a t faite et en tant que preuve de fonctionnalit, des axones de neurones du ganglion spinal ont t guids sur des gradients dun peptide provenant de la laminine. Deuximement, LAPAP a t amlior de manire pouvoir fabriquer des motifs avec plusieurs composantes grce lutilisation de lasers a diffrentes longueurs donde et danticorps conjugus des fluorophores correspondants ces longueurs donde. De plus, pour acclrer et simplifier le processus de fabrication, nous avons dvelopp LAPAP a illumination champ large qui utilise un modulateur spatial de lumire, une diode lectroluminescente et un microscope standard pour imprimer directement un motif de protines. Cette mthode est particulirement simple comparativement la version originale de LAPAP puisquelle nimplique pas le contrle de la puissance laser et de platines motorises, mais seulement denvoyer limage du motif dsir au modulateur spatial. Finalement, nous avons utilis LAPAP pour dmontrer que notre technique peut tre utilise dans des analyses de haut contenu pour quantifier les changements morphologiques rsultant de la croissance neuronale sur des gradients de protines de guidage. Nous avons produit des milliers de gradients de laminin-1 ayant diffrentes pentes et analys les variations au niveau du guidage de neurites provenant dune ligne cellulaire neuronale (RGC-5). Un algorithme pour analyser les images des cellules sur les gradients a t dvelopp pour dtecter chaque cellule et quantifier la position du centrode du soma ainsi que les angles dinitiation, final et de braquage de chaque neurite. Ces donnes ont dmontr que les gradients de laminine influencent langle dinitiation des neurites des RGC-5, mais ninfluencent pas leur braquage. Nous croyons que les rsultats prsents dans cette thse faciliteront lutilisation de motifs de protines lies au substrat dans les laboratoires des sciences de la vie, puisque LAPAP peut tre effectu a laide dun microscope confocal ou dun microscope standard lgrement modifi. Cela pourrait contribuer laugmentation du nombre de laboratoires travaillant sur le guidage avec des gradients lis au substrat afin datteindre la masse critique ncessaire des perces majeures en neuroscience.
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Mmoire numris par la Direction des bibliothques de l'Universit de Montral.
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Mmoire numris par la Direction des bibliothques de l'Universit de Montral.
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Background and Objectives: This study evaluated the hybrid layer (HL) morphology created by three adhesive systems (AS) on dentin surfaces treated with Er:YAG laser using two irradiation parameters. Study Design: Occlusal flat dentin surfaces of 36 human third molars were assigned into nine groups (n = 4) according to the following ASs: one bottle etch&rinse Single Bond Plus (3M ESPE), two-step Clearfil Protect Bond (Kuraray), and all-in-one S3 Bond (Kuraray) self-etching, which were labeled with rhodamine B or fluorescein isothiocyanate dextran and were applied to dentin surfaces that were irradiated with Er:YAG laser at either 120 (38.7 J/cm(2)) or 200 mJ/pulse (64.5 J/cm(2)), or were applied to untreated dentin surfaces (control group). The ASs were light-activated following MI and the bonded surfaces were restored with resin composite Z250 (3M ESPE). After 24 hours of storage in vegetable oil, the restored teeth were vertically, serially sectioned into 1-mm thick slabs, which had the adhesive interfaces analyzed with confocal laser microscope (CLSM-LSM 510 Meta). CLSM images were recorded in the fluorescent mode from three different regions along each bonded interface. Results: Non-uniform HL was created on laser-irradiated dentin surfaces regardless of laser irradiation protocol for all AS, while regular and uniform HL was observed in the control groups. ""Stretch mark""-like red lines were found within the HL as a result of resin infiltration into dentin microfissures, which were predominantly observed in 200 mJ/pulse groups regardless of AS. Poor resin infiltration into peritubular dentin was observed in most regions of adhesive interfaces created by all ASs on laser-irradiated dentin, resulting in thin resin tags with neither funnel-shaped morphology nor lateral resin projections. Conclusion: Laser irradiation of dentin surfaces at 120 or 200 mJ/pulse resulted in morphological changes in HL and resin tags for all ASs evaluated in the study. Lasers Surg. Med. 42:662-670, 2010. (C) 2010 Wiley-Liss, Inc.
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The aim of this work is to measure the stress inside a hard micro object under extreme compression. To measure the internal stress, we compressed ruby spheres (a-Al2O3: Cr3+, 150 m diameter) between two sapphire plates. Ruby fluorescence spectrum shifts to longer wavelengths under compression and can be related to the internal stress by a conversion coefficient. A confocal laser scanning microscope was used to excite and collect fluorescence at desired local spots inside the ruby sphere with spatial resolution of about 1 m3. Under static external loads, the stress distribution within the center plane of the ruby sphere was measured directly for the first time. The result agreed to Hertzs law. The stress across the contact area showed a hemispherical profile. The measured contact radius was in accord with the calculation by Hertzs equation. Stress-load curves showed spike-like decrease after entering non-elastic phase, indicating the formation and coalescence of microcracks, which led to relaxing of stress. In the vicinity of the contact area luminescence spectra with multiple peaks were observed. This indicated the presence of domains of different stress, which were mechanically decoupled. Repeated loading cycles were applied to study the fatigue of ruby at the contact region. Progressive fatigue was observed when the load exceeded 1 N. As long as the load did not exceed 2 N stress-load curves were still continuous and could be described by Hertzs law with a reduced Youngs modulus. Once the load exceeded 2 N, periodical spike-like decreases of the stress could be observed, implying a memory effect under repeated loading cycles. Vibration loading with higher frequencies was applied by a piezo. Redistributions of intensity on the fluorescence spectra were observed and it was attributed to the repopulation of the micro domains of different elasticity. Two stages of under vibration loading were suggested. In the first stage continuous damage carried on until certain limit, by which the second stage, e.g. breakage, followed in a discontinuous manner.
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In this paper, we present an algorithm to create 3D segmentations of neuronal cells from stacks of previously segmented 2D images. The idea behind this proposal is to provide a general method to reconstruct 3D structures from 2D stacks, regardless of how these 2D stacks have been obtained. The algorithm not only reuses the information obtained in the 2D segmentation, but also attempts to correct some typical mistakes made by the 2D segmentation algorithms (for example, under segmentation of tightly-coupled clusters of cells). We have tested our algorithm in a real scenario?the segmentation of the neuronal nuclei in different layers of the rat cerebral cortex. Several representative images from different layers of the cerebral cortex have been considered and several 2D segmentation algorithms have been compared. Furthermore, the algorithm has also been compared with the traditional 3D Watershed algorithm and the results obtained here show better performance in terms of correctly identified neuronal nuclei.
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Both in industry and research, the quality control of micrometric manufactured parts is based on the measurement of parameters whose traceability is sometimes difficult to guarantee. In some of these parts, the confocal microscopy shows great aptitudes to characterize a measurand qualitatively and quantitatively. The confocal microscopy allows the acquisition of 2D and 3D images that are easily manipulated. Nowadays, this equipment is manufactured by many different brands, each of them claiming a resolution probably not in accord to their real performance. The Laser Center (Technical University of Madrid) has a confocal microscope to verify the dimensions of the micro mechanizing in their own research projects. The present study pretends to confirm that the magnitudes obtained are true and reliable. To achieve this, a methodology for confocal microscope calibration is proposed, as well as an experimental phase for dimensionally valuing the equipment by 4 different standard positions, with its seven magnifications and the six objective lenses that the equipment currently has, in the xy and z axis. From the results the uncertainty will be estimated along with an effect analysis of the different magnifications in each of the objective lenses.
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Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound. uorophores. We demonstrate how information about the orientation of. uorophores can be used to calculate the orientation factor k(2) required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.
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Subtractive imaging in confocal fluorescence light microscopy is based on the subtraction of a suitably weighted widefield image from a confocal image. An approximation to a widefield image can be obtained by detection with an opened confocal pinhole. The subtraction of images enhances the resolution in-plane as well as along the optic axis. Due to the linearity of the approach, the effect of subtractive imaging in Fourier-space corresponds to a reduction of low spatial frequency contributions leading to a relative enhancement of the high frequencies. Along the direction of the optic axis this also results in an improved sectioning. Image processing can achieve a similar effect. However, a 3D volume dataset must be acquired and processed, yielding a result essentially identical to subtractive imaging but superior in signal-to-noise ratio. The latter can be increased further with the technique of weighted averaging in Fourier-space. A comparison of 2D and 3D experimental data analysed with subtractive imaging, the equivalent Fourier-space processing of the confocal data only, and Fourier-space weighted averaging is presented. (C) 2003 Elsevier Ltd. All rights reserved.
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Aiming to detail data obtained through brightfield microscopy (BM) on reproductive, excretory and digestive system, specimens of Schistosoma mansoni eight weeks old, were recovered from SW mice, stained with Langeron's carmine and analyzed under a confocal laser scanning microscope CLSM 410 (Carl Zeiss). The reproductive system presented a single and lobate testis, with intercommunications between the lobes without efferent duct. Supernumerary testicular lobe was amorphous and isolated from the normal ones. Collecting tubules (excretory ducts), followed by the excretory bladder, opening to the external media through the excretory pore, were observed at the posterior extremity of the body. In the digestive tract, a cecal swelling was noted at the junction that originates the single cecum. It was concluded that through confocal laser scanning microscopy, new interpretations of morphological structures of S. mansoni worms could be achieved, modifying adopted and current descriptions. The gonad consists of a single lobed testis, similar to that observed in some trematode species. Moreover, the same specimens can be observed either by BM or CLSM, considering that the latter causes only focal and limited damage in tissue structures.
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In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.
