Le débit sanguin et sa régulation sont des indicateurs importants de la santé des tissus. Leur mesure a de nombreuses applications en recherche fondamentale et clinique. Certaines techniques optiques constituent un moyen intéressant pour la mesure du débit sanguin, car en général elles sont peu invasives et relativement abordables car elles utilisent des systèmes d'illumination continus. Pendant ma thèse, j'ai contribué au développement de techniques de suivi de la circulation sanguine dans des modèles animaux avec la construction d'un dispositif multimodal basé sur la fluxmétrie laser et sur l'imagerie des signaux optiques intrinsèques, capable de mesurer les paramètre hémodynamiques microvasculaire au niveau superficiel du cerveau. Ce dispositif, testé sur des modèles animaux d'accident vasculaire cérébral, est adaptable et peut être utilisé à d'autres fins. En parallèle, j'ai mis au point des nouvelles méthodes expérimentales et des protocoles de traitement d'images qui ont permis de réaliser des études longitudinales. En outre, ce dispositif a été utilisé dans une étude multidisciplinaire pour comprendre le rôle d'une protéine impliquée dans le cas de lésions de reperfusion après un accident vasculaire cérébral ischémique dans des modèles animaux. Ma contribution majeure réside dans le développement de l'imagerie de contraste de speckle spectroscopique et tomographique, nouvelle technique d'imagerie 3D non invasive pour la mesure du débit sanguin en profondeur. Dans l'ensemble, ces contributions permettront le développement de méthodes tomographiques non invasives rentables pour la mesure du débit sanguin chez l'homme. / Blood flow and its regulation are important for the health of tissues and its measurement has many applications in research and clinical environments. Optical techniques are often attractive for the non- or minimally-invasive, continuous and relatively inexpensive measurement of blood flow. This work contributes to the monitoring of blood flow in translational research with the construction of a multimodal device, based on laser speckle flowmetry and optical intrinsic signals, capable of measuring superficial microvascular cerebral blood flow, blood oxygenation and blood volume. This device was applied in animal models of ischemic stroke and is flexible to be modified and used for other purposes. In doing so, I have developed new experimental methods and image processing protocols that allowed us to perform longitudinal studies where the animal can be removed from the device several times. This device has also been used to elucidate the role of the Mannose-binding lectin protein in reperfusion injury after an ischemic stroke in animal models. This led to the main contribution of this work: the development of the speckle contrast optical spectroscopy and tomography, a new non-invasive, optical technique for deep blood flow measurement that paves the way for deeper and three dimensional imaging of blood flow. This new method was first developed from a theoretical perspective. Then it was validated in tissue simulating phantoms and demonstrated to be feasible in measurements on the human arm muscle. Overall, these contributions will allow the development of cost-effective, non-invasive tomographic methods for the measurement of blood flow even in humans.

No description available.

Retinoblastoma Binding Protein 6 (RBBP6) is a 200 KDa protein shown to play a role in 3'- polyadenylation of mRNA transcripts, as well as to function as an E3 ligase catalysing ubiquitination of cancer-associated proteins. RBBP6 has been previously reported to localise to nuclear speckles, which are thought to play a role in mRNA splicing, presumably as a result of its RS domain, which is known to target mRNA splicing factors to nuclear speckles. However recent studies in our laboratory have shown that isoform 3 of RBBP6, consisting mainly of the DWNN domain, also localises to speckles in resting cells, but more strongly in cells subjected to various stresses, suggesting that the DWNN domain may be the speckle-targeting domain. / Magister Scientiae - MSc (Biotechnology)

We present measurements of the evolutionary timescales of speckles around adaptive optics-corrected PSFs. We placed a SELEX SAPHIRA HgCdTe detector behind the SCExA0 instrument at Subaru Telescope. We analyzed the behavior of speckles at radial distances of 2-8 A/D away from the diffraction-limited PSF in H-band (-1.6 m) images collected at 1 kHz framerates. Speckles evolve with a variety of timescales, and these have not previously been studied at near-infrared wavelengths. Ultimately we would like to image reflected-light exoplanets, which necessitates a fast speckle control loop. Based on our measurements, we calculate the parameters of an optimized control loop that would enable such observations.

No description available.

No description available.

Trypanosoma cruzi é o agente causador da Doença de Chagas, que segundo a OMS, atinge oito milhões de pessoas principalmente na América Latina, causando danos à saúde pública, juntamente com um impacto econômico negativo. Durante o processo de infecção, uma variedade de eventos celulares ocorre apenas pelo simples contato do parasito com a célula hospedeira, levando a modificações no metabolismo celular e alterações morfológicas. O parasita é capaz de modular respostas celulares e imunológicas da célula hospedeira para sua própria sobrevivência. Além do que, pode alterar compartimentos celulares como o número e tamanho de nucléolos, sugerindo que a presença do parasita poderia estar interferindo na maquinaria nuclear. Porém, pouco se conhece sobre a organização nuclear da célula hospedeira quando infectada por Trypanosoma cruzi. O objetivo deste estudo foi de investigar pela primeira vez os compartimentos nucleares das células hospedeiras durante o curso da infecção por T. cruzi. Células LLC-MK2 foram infectadas com T. cruzi e reações de imunofluorescência indireta foram realizadas utilizando anticorpos e marcadores específicos para proteínas nucleares. As análises das imagens de microscopia confocal e quantificação das fluorescências pelo ImageJ mostraram padrões distintos nos compartimentos nucleares quando comparadas com células não infectadas. Corpos de Cajal e Speckles sofrem alterações quando a célula está infectada e isso depende do ciclo celular do parasita. Neste trabalho também foi investigado através de quantificação de imagem e immunoblotting o comportamento das Ribonucleoproteínas A1 e A2B1 durante a parasitemia. Estas análises demonstram que o T. cruzi pode modular a célula hospedeira quando infectada a favor de sua sobrevivência, promovendo alterações na dinâmica dos compartimentos nucleares durante o seu ciclo celular. Esse estudo inédito poderá auxiliar a compreender a biologia do parasita e sua interação com a célula hospedeira e desta maneira contribuir na busca de possíveis alvos terapêuticos / Trypanosoma cruzi is the causal agente of Chagas disease, that affects about eight million people mostly in Latin America according to the WHO, causing damage to public health and a negative economic impact. During infection, a variety of signaling processes occur after contact of the parasite to the host cell, what can lead to metabolic modifications as well morphological alterations in both cells. The parasite can modulate host cell cellular and immunological responses for its own survival. In addiction, the presence of T. cruzi can modify the nuclear compartments such as nucleoli, suggesting that the presence of the parasite could be interfering with the nuclear machinery. However, little is know about the nuclear organization when the host cell is infected with Trypanosoma cruzi. This study aimed to investigate for the first time the nuclear compartment of host cells infected by T. cruzi using specific antibodies and fluorescent markers for nuclear compartments, in order to investigate the morphological and functional changes in the nucleus of the host cell. Using LLC-MK2 cells infected with T. cruzi, we performed indirect immunofluorescence using distinct nuclear antibodies. Confocal microscopy analysis of infected cells showed pattern variations in the nuclear compartments when compared to uninfected cells. Cajal bodies and Speckles suffer alterations when the cell is infected and it is related to the parasite life cycle. In this work we also investigated by image quantification and immunoblotting the behavior of Ribonucleoproteins A1 and A2B1 during infection. These evidences support the idea that T. cruzi can modulate host cell response to ensure its own survival during the infection, promoting changes in the dynamics of the nuclear compartments. This unpublished data may help to understand the biology of the parasite and its interaction with the host cell and thus contributing to seek for potential therapeutic targets

<p>An arthroscope is a diagnostic instrument for visualisation of the interior of a joint. By adding a laser to an arthroscope and feeding the images to a computer, one gets an method to measure the structure of the cartilage covering the joint. This gives an added diagnostic value. The laser will create laser speckles and this report covers the basic theories behind this. The anatomy of the joints, the properties of cartilage and the background on the disease arthritis are also covered, as well as the field of surface topography and image processing.</p><p>Experiments were performed on three different materials - metals of different definite surface roughness, polymerised collagen and bovine articular cartilage.</p><p>The conclusion is that the technique would work, providing that some obstacles could be overcome. The technique itself is very precise and detects nanometric differences in the surface structure, making it extremely interesting for research purposes, such as follow-ups on treatments and studies of arthritis and cartilage repair.</p>

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein &alpha; (C/EBP&alpha;) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBP&alpha;. Additionally, the original clone found to interact with C/EBP&alpha; consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBP&alpha; mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBP&alpha;, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBP&alpha;-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBP&alpha;-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBP&alpha;. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBP&alpha; was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBP&alpha;, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBP&alpha;.

An arthroscope is a diagnostic instrument for visualisation of the interior of a joint. By adding a laser to an arthroscope and feeding the images to a computer, one gets an method to measure the structure of the cartilage covering the joint. This gives an added diagnostic value. The laser will create laser speckles and this report covers the basic theories behind this. The anatomy of the joints, the properties of cartilage and the background on the disease arthritis are also covered, as well as the field of surface topography and image processing. Experiments were performed on three different materials - metals of different definite surface roughness, polymerised collagen and bovine articular cartilage. The conclusion is that the technique would work, providing that some obstacles could be overcome. The technique itself is very precise and detects nanometric differences in the surface structure, making it extremely interesting for research purposes, such as follow-ups on treatments and studies of arthritis and cartilage repair.