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Molecular magnetic resonance imaging of vascular inflammation using microparticles of iron oxideAkhtar, Asim January 2010 (has links)
One approach that has demonstrated success in the field of molecular imaging utilizes microparticles of iron oxide (MPIO) conjugated to specific antibodies and/or peptides to provide contrast effects on MRI in relation to the molecular expression of a specified target. The experimental aims of this thesis were 1) to investigate the ability of VCAM-1 and P-selectin targeted MPIO to detect the expression of VCAM-1 and P-selectin on the activated endothelium in-vitro and in-vivo in mouse models of renal and cerebral ischemia reperfusion injury, and 2) develop a novel contrast agent for imaging αvβ3-integrin expression in angiogenesis using RGD peptide conjugated MPIO (RGD-MPIO) in-vitro. MPIO (1.0 µm) were conjugated to monoclonal antibodies against VCAM-1 (VCAM-MPIO) or P-selectin (PSEL-MPIO). In vitro, MPIO bound in a dose-dependent manner to tumor necrosis factor (TNF)-alpha stimulated sEND-1 endothelial cells when conjugated to VCAM-1 (R² = 0.88, P<0.01) and P-selectin antibodies (R² = 0.93, P<0.01), reflecting molecular VCAM-1 and P-selectin mRNA and protein expression. Mice subjected to unilateral, transient (30 minutes) renal ischemia and subsequent reperfusion received intravenous VCAM-MPIO and PSEL-MPIO (4.5 mg iron/kg body weight). In ischemic kidneys, MR related contrast effects of VCAM-MPIO were 4-fold higher than unclamped kidneys (P<0.01) and 1.5-fold higher than clamped kidneys of PSEL-MPIO injected mice (P<0.05). VCAM-MPIO binding was less evident in IRI kidneys pre-treated with VCAM-1 antibody (P<0.001). VCAM-1 mRNA expression and VCAM-MPIO contrast volume were highly correlated (R² = 0.901, P<0.01), indicating that quantification of contrast volume reflected renal VCAM-1 transcription. In mice subjected to cerebral ischemia, contrast volume was 11-fold greater in animals injected with VCAM-MPIO versus control IgG-MPIO (P<0.05). Finally, S-nitroso-N-acetylpenicillamine (SNAP) stimulated HUVEC-C cells, which express αvβ3-integrin, showed 44-fold greater RGD-MPIO binding than unstimulated cells (P<0.001) and 4-fold greater RGD-MPIO binding than SNAP stimulated cells blocked with soluble RGD peptide (P<0.001) in-vitro. This thesis demonstrated that targeted MPIO exhibited contrast effects that defined and quantified the molecular expression of specific targets through the use of high-resolution MRI in in-vitro and in-vivo models of vascular inflammation.
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Développement et caractérisation de nouveaux agents de contraste lipidiques ultrasensibles pour l'imagerie par résonnance magnétique destinés à l'imagerie moléculaire / Development and characterization of new contrast agents for lipid ultrasensitive magnetic resonance imaging for molecular imagingChahid, Bochra 20 December 2012 (has links)
L’effet des composés paramagnétiques sur le déplacement chimique des protons, c’est-à-dire sur leur fréquence de résonance propre, beaucoup utilisé en RMN conventionnelle, peut également être un outil de contraste en Imagerie par Résonance Magnétique (IRM) pour réaliser des images encodées en fréquence et donc sélectives selon la nature ou l’environnement de l’entité que l’on cherche à révéler. Cette approche fait intervenir le transfert d’aimantation par échange chimique de protons mobiles, en anglais « Chemical Exchange Saturation Transfer » (CEST). Le principe consiste à saturer sélectivement un signal donné de protons labiles appartenant à la structure-même de l’agent de contraste ou aux molécules d’eau qui lui sont transitoirement liées, à l’aide d’une impulsion radiofréquence bien choisie. L’image résulte alors de l’altération du signal des protons échangés. Le fonctionnement de la méthode repose sur l’existence effective de deux ensembles ou réservoirs de protons, celui correspondant aux protons associés à l’agent de contraste et celui représenté par le milieu environnant, autrement dit l’eau des tissus, ces deux réservoirs présentant une fréquence de résonance bien distincte. Les systèmes LipoCEST, liposomes encapsulant un complexe paramagnétique de lanthanide, permettent une telle différenciation de deux réservoirs de protons constitutifs d’une part de l’eau contenue dans la cavité interne des liposomes (dont la fréquence de résonance est modifiée par l’agent paramagnétique) et d’autre part de l’eau présente à l’extérieur de la structure. La sensibilité de tels systèmes est principalement due au grand nombre de protons contenus dans le réservoir interne. La nature de l’agent paramagnétique joue un rôle déterminant dans la sélectivité de l’effet CEST et la nature de la membrane des liposomes dont la perméabilité permet un échange plus ou moins rapide entre les deux réservoirs d’eau doit être sélectionnée de manière à conduire à une réponse CEST efficace.Les travaux réalisés au cours de cette thèse portent sur une telle mise au point avec pour objectif l’optimisation de systèmes destinés à une IRM-CEST après administration par voie intraveineuse. De ce fait, le diamètre des liposomes a été fixé inférieur à 200 nm et leur surface recouverte de chaînes de poly(éthylène glycol) pour assurer leur future stabilité dans le compartiment sanguin. Le choix des agents de déplacement chimique à centre lanthanide, principalement des complexes de thulium, a été fixé à partir de leurs propriétés structurales et magnétiques. La méthodologie spécialement développée pour encapsuler ces entités au sein de liposomes de compositions lipidiques induisant des perméabilités membranaires distinctes a permis la mise au point d’un nouvel agent de contraste LipoCEST. / The effect of paramagnetic compounds in the chemical shift of endogenous protons, i.e., their resonance frequency, widely used in conventional NMR, can also be a tool to modulate the contrast in magnetic resonance imaging (MRI) by achieving frequency-encoded images depending on the nature or the environment of the entity or tissue to be revealed. This approach involves the transfer of magnetization by chemical exchange of protons also referred to as "Chemical Exchange Saturation Transfer" (CEST). The principle consists in selectively saturate by applying a radio frequency pulse, the signal of labile protons transiently belonging to the structure of the contrast agent or to the water molecules which are associated transiently to it. The image then results from the alteration of the signal of the exchanged protons. The method is based on the actual existence of two sets of protons or pools with two distinct resonance frequencies, one corresponding to the protons associated with the contrast agent and the other represented by the surrounding bulk water.LipoCEST systems, liposomes encapsulating a paramagnetic lanthanide complex, allows such a differentiation of two proton pools constituted on one hand by the water molecules contained in the inner cavity of the liposomes (with a resonance frequency changed by the paramagnetic agent ) and on the other hand by the water present outside the vesicle structure. The sensitivity of such systems is mainly due to the large number of protons in the inner pool. The nature of the paramagnetic agent plays a role in the selectivity of the CEST effect while the nature of the liposome membrane and related permeability behavior controls the proton exchange kinetics between the two water pools. These two parameters must be selected and adjusted to provide effective CEST contrast.The work in this thesis aimed at such a development by optimizing liposome systems for MRI-CEST after intravenous administration. Therefore, the diameter of the liposomes was set below 200 nm and their surface covered by chains of poly (ethylene glycol) to ensure stability in the blood compartment. The choice of chemical shift agents based on lanthanide complexes, mainly thulium-based derivatives, was established from their structural and magnetic properties. The methodology specially developed to encapsulate these entities into vesicles of different membrane composition and permeability to water allowed to generate a new LipoCEST contrast agent.
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Aplicação de métodos de imagem molecular no estudo dos efeitos terapêuticos da galectina-3 em glioblastoma / Application of molecular imaging methods in the study of the therapeutic effects of galectin-3 in glioblastomaMitsuoka, Ronny Mikyo 05 August 2015 (has links)
O glioma de grau IV (geralmente chamado de Glioblastoma Multiforme - GBM) é o tumor mais agressivo e maligno do sistema nervoso central. A elevada mortalidade e baixa expectativa de vida proporcionado por esta doença, tem direcionado os esforços de muitos pesquisadores no desenvolvimento de novas formas de diagnóstico precoce, assim como a busca por terapias inovadoras. A galectina-3, uma proteína ligante de glicanas, é expressa diferencialmente em tecido normal vs. neoplásico e possui um papel importante na adesão, diferenciação, imunomodulação, apoptose, ciclo celular, assim como processos de transformação e progressão neoplásica. Diversos estudos têm demonstrado que a interferência nas funções exercidas pela galectina-3 pode representar uma estratégia promissora no tratamento de vários tipos de tumores, incluindo o glioblastoma. De fato, tem se verificado que a administração da forma truncada de galectina-3 em modelos experimentais murinos, possui um efeito antitumoral significativo quando administrada em conjunto com quimioterápicos. No entanto, ainda não se encontra esclarecido se a interferência com as funções da galectina-3 endógena são exercidas diretamente no microambiente tumoral ou de maneira sistêmica. Dessa forma, neste trabalho buscou-se um entendimento mais profundo e completo sobre o papel anti-tumoral de galectina-3 nativa e truncada em associação com o quimioterápico temozolamida num modelo de glioblastoma humano. Adicionalmente, as ferramentas de PET-SPECT, assim como imagem molecular ótica por fluorescência ou bioluminescênca foram utilizadas para se avaliar a biodistribuição da galectinas-3 em camundongos Balb/c nude inoculados com tumor. Inicialmente demonstrou-se que, nas células U87 de glioblastoma humano, a galectina-3 nativa e não a galectina-3 truncada apresenta efeito antitumoral in vitro quando associada com temozolamida com valores de IC50 de 0.008 mM e 1.893 mM respectivamente. Os testes in vivo foram dessa forma prosseguidos com a galectina-3 nativa. Através da técnica de imagem ótica por bioluminescência, observou-se que o tratamento simultâneo de galectina-3 com temozolamida levou a uma redução do crescimento do tumor gerado pelas células U87-Luc2 (U87 transfectadas com o gene reporter da luciferase) em camundongos Balb/c nude. Esta redução foi observada mesmo após a parada do tratamento (período de acompanhamento). Curiosamente, no tratamento com apenas galectina-3 observou-se uma redução do crescimento tumoral das células U87-luc2 cujo efeito foi anulado após a suspensão do tratamento. Através de análises por PET-SPECT avaliou-se a biodistribuição da galectina-3 marcada com 99mTc (99mTc-HYNIC/Gal-3) em camundongos Balb/c nude previamente inoculados com a linhagem U87. Demonstrou-se que esta proteína migra principalmente para os rins e, em menores quantidades para o baço, não ligando todavia no tumor. Devido à meia-vida do 99mTc-HYNIC/Gal-3 não permitir estudos prolongados, a galectina-3 conjugada com VivoTag 680XL foi utilizada para se avaliar a biodistribuição por 48h, 96h e 14 dias em camundongos Balb/c nude inoculados com a linhagem de glioblastoma U87. Além de se confirmar o padrão de distribuição acima descrito, observou-se que a galectina-3 não liga no tumor independentemente do modelo tumoral utilizado. Os resultados obtidos neste estudo demonstram que galectina-3 possui um efeito antiproliferativo quando administrada em conjunto com temozolamida num modelo de glioblastoma humano (células U87). Surpreendentemente, foi possivel observar pela primeira vez que o efeito antiproliferativo de galectina-3 não se deve à sua atuação direta no tumor. Nossos dados sugerem que, quando administrada in vivo, a galectina-3 atua em locais distintos do microambiente tumoral como os rins e baço, afetando portanto de maneira indireta o crescimento tumoral / Grade IV glioma or glioblastoma multiforme (GBM) is the most aggressive and malignant tumor of the central nervous system. The high mortality and low life expectancy provided by this disease have directed the efforts of many researchers to develop innovative therapeutic strategies and early diagnosis tools. Galectin-3 is a glycan-binding protein differentially expressed in normal and neoplastic tissue and has an important role in adhesion, differentiation, immune modulation, apoptosis, cell cycle, tumor transformation and neoplastic progression. Several studies have shown that interference with the functions performed by galectin-3 could represent a promising strategy in the treatment of several kinds of tumors, including glioblastoma. Indeed, it has been found that galectin-3 truncated form has a significant antitumor effect when associated with chemotherapy in murine experimental models. However, it\'s not clear yet whether the interference with galectin-3 endogenous functions is performed directly in the tumor microenvironment or systemically. Thus, this study aimed to understand the anti-tumor effect of truncated and native galectin-3 in combination with temozolomide chemotherapy in a human glioblastoma model. Additionally, PET, SPECT and bioluminescence tools were used to evaluate the biodistribution of galectin-3 in Balb / c nude mice inoculated with the U87 glioblastoma cell line. Here it was shown that in U87 cells, native galectin-3 and not the truncated form has anti-tumor effect in vitro when associated with temozolomide with IC50 values of 0.008 mM and 1.893 mM, respectively. Therefore the in vivo studies were pursued with native galectin-3. Using the optical bioluminescence technique, it was observed that the simultaneous treatment of galectin-3 with temozolomide led to a reduction of tumor growth generated by U87- Luc2 cells (U87 cells transfected with the luciferase reporter gene) in Balb / c nude. This reduction was observed even after stopping treatment (follow-up period). Interestingly, treatment of U87-Luc2 derived-tumor with only galectin-3 led to a reduction of tumor growth whose effect was abolished after discontinuation of treatment. The biodistribution of 99mTc labeled-galectin-3 (99mTc-HYNIC / Gal-3) was performed by molecular image tools (PET-SPECT scan) in mice BALB/c nude previously inoculated with the U87 line. It was shown that this protein migrates primarily to the kidneys and, in a smaller amount to the spleen, but doesn\'t bind the tumor. Because the half-life of 99m Tc-HYNIC / 3-Gal doesn\'t allow prolonged studies, galectin-3 conjugated with VivoTag 680XL was used to assess in vivo biodistribution at 48h, 96h and 14 days in Balb / c nude mice inoculated with the human glioblastoma cell line U87. Besides confirming the distribution pattern described above, it was found that galectin-3 doesn\'t bind to the tumor, regardless the tumor model. This study shows that galectin-3 has an antiproliferative effect when associated with temozolomide in the human glioblastoma model U87. Surprisingly, it was observed, that the in vivo antiproliferative effect of galectin-3 is not due to its direct binding to tumor cells. Our data suggest that when administered in vivo, galectin-3 acts in distinct locations of the tumor microenvironment such as the kidneys and spleen, thus indirectly affecting tumor growth
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Développements de stratégies de quantification et de dispositifs expérimentaux pour l'IRM moléculaire de biomarqueurs endovasculaires et intratissulaires de pathologies cérébrales / Development of quantification strategies and experimental devices for molecular MRI of endovascular and intratissular biomarkers in cerebral pathologiesMarty, Benjamin 29 May 2012 (has links)
Au cours de cette thèse, réalisée dans le cadre du projet Iseult/INUMAC, nous avons réalisé un travail de développements méthodologiques et technologiques, dans l'optique de permettre à l'IRM de devenir un outil quantitatif pour l'imagerie moléculaire de pathologies cérébrales sur des modèles rongeurs. Pour cela, nous avons développé une stratégie de quantification, utilisant des séquences de cartographie T1 et T2, pour acquérir des cartes de concentration en agents de contraste paramagnétiques et superparamagnétiques avec une excellente sensibilité, une résolution spatiale élevée, ainsi qu'une résolution temporelle compatible avec l'imagerie in vivo. La méthodologie générale que nous avons mise en place lors de ces travaux de thèse nous a permis d'aborder un certain nombre de problématiques propres à l'imagerie moléculaire de pathologies cérébrales par IRM. Dans un premier temps, nous nous sommes intéressés à l'imagerie d'un biomarqueur endovasculaire de l'angiogenèse tumorale sur un modèle de glioblastome cérébral induit chez des souris immuno-déprimées. Nous avons étudié la fixation d'une émulsion paramagnétique, fonctionnalisée par l'ajout de peptides RGD, sur l'intégrine alpha-nu-beta-3 surexprimée à la surface des cellules endothéliales de capillaires tumoraux. Nous nous sommes ensuite intéressés à la délivrance des agents de contraste aux tissus cérébraux. À l'aide d'un protocole optimisé d'ouverture de la barrière hématoencéphalique (BHE) par ultrasons focalisés sous IRM, nous avons étudié les caractéristiques de cette ouverture, ainsi que la dynamique de refermeture sur des modèles de rats et souris sains. Dans une autre étude, la mesure du coefficient de diffusion apparent d'agents de contraste dans les tissus cérébraux de rats sains nous a permis d'évaluer le temps nécessaire à ces agents de tailles différentes pour atteindre leurs cibles, une fois la BHE franchie. Ces caractéristiques représentent des informations capitales dans le cadre de la délivrance d'agents de contraste aux tissus cérébraux. Ils sont en effet susceptibles d'intéresser les industriels pharmaceutiques pour optimiser la conception d'agents diagnostiques et thérapeutiques dédiés aux pathologies cérébrales. / In this thesis, which was part of the Iseult/INUMAC project, we propose several methodological and technological developments aiming to allow MRI to become a quantitative tool for molecular imaging of brain pathologies. To do so, we developed a quantification strategy based on T1 and T2 mapping sequences in order to acquire quantitative concentration maps of paramagnetic and superparamagnetic contrast agents with excellent sensitivity, high spatial resolution and temporal resolution compatible with in vivo imaging. This general methodology allowed us to address several issues specific to molecular imaging of cerebral pathologies using MRI. First, we focused on the imaging of a vascular biomarker of tumor angiogenesis on a glioblastoma mouse model. We studied the binding of a paramagnetic emulsion, functionalized using RGD peptides, on alpha-nu-beta-3 integrin over-expressed at the surface of freshly formed endothelial cells. Then, we focused on the delivery of contrast agents to brain parenchyma. A system was developed and optimized to open transiently and non-invasively rodents blood brain barrier (BBB) using focalized ultrasound monitored by MRI. The BBB opening features and closure dynamics induced by this protocol were extensively characterized. In another study, we measured the apparent diffusion coefficient of contrast agents with different sizes in cerebral tissues of healthy rats. From these measures we could estimate the time necessary for these particles to reach their targets once the BBB is crossed. These parameters are highly valuable in the context of drug delivery to the brain. They might indeed be used by pharmaceutical industries to optimize the design of diagnostic and therapeutic agents dedicated to cerebral diseases.
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Dynamique des électrons corrélés en champ laser intense / Correlated electron dynamics in intense laser fieldPeters, Michel 21 June 2012 (has links)
L'avancement technologique des sources de rayonnement laser est tel qu'il permet désormais l'observation résolue en temps des phénomènes se déroulant dans les atomes et les molécules sous l'effet de champs intenses et de très courte durée. La complexité croissante de ces expériences et des processus auxquels elles s'intéressent suscite plusieurs questions à propos de la dynamique multiélectronique de ces systèmes. Par exemple, dans un travail récent portant sur la molécule de CO2, une technique d'imagerie moléculaire exploitant les interférences dans le signal d'émission des harmoniques élevées a été proposée. Ces interférences qui dépendent fortement de la géométrie moléculaire sont également influencées par divers effets multiélectroniques déterminant l'importance relative des différentes voies d'ionisation possibles de la molécule sondée rendant difficile leur interprétation. Il est donc très important de développer des modèles théoriques suffisamment précis pour pouvoir s'adresser à de telles interrogations. La compréhension du déroulement de ces processus permet d'avoir une meilleure emprise sur ceux-ci et de tirer une juste part de ce type d'expérience. Ainsi, nos développements méthodologiques récents s'inscrivent dans cette ambition de dévoiler la nature des effets multiélectroniques sur la dynamique des molécules polyélectroniques dirigées par un champ laser intense. Le développement complet de ce schéma général multi-configurationnel à champ auto-cohérent dépendant du temps (TDMCSCF) sera présenté et illustré avec quelques résultats préliminaires obtenus pour des systèmes simples. Finalement, on discutera de quelques applications utiles de l'étude de la dynamique électronique, telle que l'imagerie moléculaire dynamique. / Latest advances in laser sources are such that it is now possible to observe, in real-time, phenomena occurring in atoms and molecules under the effect of an intense and ultra-fast external field. The increasing complexity of these experiments and the processes under scrutiny give rise to a number of questions concerning the multielectron dynamics of such systems. For example, in a recent work on CO2 molecule, a molecular imaging technique exploiting interference structures in the high harmonic emission signal was proposed. Such interferences are directly correlated to the molecular geometry but are also influenced by diverse multielectron effects that govern the relative importance of possible ionization channels, making their interpretation difficult. The developement of accurate theoretical models capable of addressing such questionings thus takes a significant importance. The complete understanding of how these processes take place allow one to control them with better ease and to gain meaningful insights on these kinds of experiments. Therefore, our methodology developments are precisely intended to unveil the nature of multielectron effects on the dynamics of polyatomic molecules driven by intense laser fields. The complete development of a general Time-Dependent Multi-Configuration Self-Consistent Field (TDMCSCF) methodology \cite{Nguyen-Dang2007, Nguyen-Dang2009} will be presented and illustrated with preliminary results obtained for some simple molecular systems. Finally, some useful applications of the study of electron dynamics, such as dynamic molecular imaging, will be discussed.
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Aplicação de métodos de imagem molecular no estudo dos efeitos terapêuticos da galectina-3 em glioblastoma / Application of molecular imaging methods in the study of the therapeutic effects of galectin-3 in glioblastomaRonny Mikyo Mitsuoka 05 August 2015 (has links)
O glioma de grau IV (geralmente chamado de Glioblastoma Multiforme - GBM) é o tumor mais agressivo e maligno do sistema nervoso central. A elevada mortalidade e baixa expectativa de vida proporcionado por esta doença, tem direcionado os esforços de muitos pesquisadores no desenvolvimento de novas formas de diagnóstico precoce, assim como a busca por terapias inovadoras. A galectina-3, uma proteína ligante de glicanas, é expressa diferencialmente em tecido normal vs. neoplásico e possui um papel importante na adesão, diferenciação, imunomodulação, apoptose, ciclo celular, assim como processos de transformação e progressão neoplásica. Diversos estudos têm demonstrado que a interferência nas funções exercidas pela galectina-3 pode representar uma estratégia promissora no tratamento de vários tipos de tumores, incluindo o glioblastoma. De fato, tem se verificado que a administração da forma truncada de galectina-3 em modelos experimentais murinos, possui um efeito antitumoral significativo quando administrada em conjunto com quimioterápicos. No entanto, ainda não se encontra esclarecido se a interferência com as funções da galectina-3 endógena são exercidas diretamente no microambiente tumoral ou de maneira sistêmica. Dessa forma, neste trabalho buscou-se um entendimento mais profundo e completo sobre o papel anti-tumoral de galectina-3 nativa e truncada em associação com o quimioterápico temozolamida num modelo de glioblastoma humano. Adicionalmente, as ferramentas de PET-SPECT, assim como imagem molecular ótica por fluorescência ou bioluminescênca foram utilizadas para se avaliar a biodistribuição da galectinas-3 em camundongos Balb/c nude inoculados com tumor. Inicialmente demonstrou-se que, nas células U87 de glioblastoma humano, a galectina-3 nativa e não a galectina-3 truncada apresenta efeito antitumoral in vitro quando associada com temozolamida com valores de IC50 de 0.008 mM e 1.893 mM respectivamente. Os testes in vivo foram dessa forma prosseguidos com a galectina-3 nativa. Através da técnica de imagem ótica por bioluminescência, observou-se que o tratamento simultâneo de galectina-3 com temozolamida levou a uma redução do crescimento do tumor gerado pelas células U87-Luc2 (U87 transfectadas com o gene reporter da luciferase) em camundongos Balb/c nude. Esta redução foi observada mesmo após a parada do tratamento (período de acompanhamento). Curiosamente, no tratamento com apenas galectina-3 observou-se uma redução do crescimento tumoral das células U87-luc2 cujo efeito foi anulado após a suspensão do tratamento. Através de análises por PET-SPECT avaliou-se a biodistribuição da galectina-3 marcada com 99mTc (99mTc-HYNIC/Gal-3) em camundongos Balb/c nude previamente inoculados com a linhagem U87. Demonstrou-se que esta proteína migra principalmente para os rins e, em menores quantidades para o baço, não ligando todavia no tumor. Devido à meia-vida do 99mTc-HYNIC/Gal-3 não permitir estudos prolongados, a galectina-3 conjugada com VivoTag 680XL foi utilizada para se avaliar a biodistribuição por 48h, 96h e 14 dias em camundongos Balb/c nude inoculados com a linhagem de glioblastoma U87. Além de se confirmar o padrão de distribuição acima descrito, observou-se que a galectina-3 não liga no tumor independentemente do modelo tumoral utilizado. Os resultados obtidos neste estudo demonstram que galectina-3 possui um efeito antiproliferativo quando administrada em conjunto com temozolamida num modelo de glioblastoma humano (células U87). Surpreendentemente, foi possivel observar pela primeira vez que o efeito antiproliferativo de galectina-3 não se deve à sua atuação direta no tumor. Nossos dados sugerem que, quando administrada in vivo, a galectina-3 atua em locais distintos do microambiente tumoral como os rins e baço, afetando portanto de maneira indireta o crescimento tumoral / Grade IV glioma or glioblastoma multiforme (GBM) is the most aggressive and malignant tumor of the central nervous system. The high mortality and low life expectancy provided by this disease have directed the efforts of many researchers to develop innovative therapeutic strategies and early diagnosis tools. Galectin-3 is a glycan-binding protein differentially expressed in normal and neoplastic tissue and has an important role in adhesion, differentiation, immune modulation, apoptosis, cell cycle, tumor transformation and neoplastic progression. Several studies have shown that interference with the functions performed by galectin-3 could represent a promising strategy in the treatment of several kinds of tumors, including glioblastoma. Indeed, it has been found that galectin-3 truncated form has a significant antitumor effect when associated with chemotherapy in murine experimental models. However, it\'s not clear yet whether the interference with galectin-3 endogenous functions is performed directly in the tumor microenvironment or systemically. Thus, this study aimed to understand the anti-tumor effect of truncated and native galectin-3 in combination with temozolomide chemotherapy in a human glioblastoma model. Additionally, PET, SPECT and bioluminescence tools were used to evaluate the biodistribution of galectin-3 in Balb / c nude mice inoculated with the U87 glioblastoma cell line. Here it was shown that in U87 cells, native galectin-3 and not the truncated form has anti-tumor effect in vitro when associated with temozolomide with IC50 values of 0.008 mM and 1.893 mM, respectively. Therefore the in vivo studies were pursued with native galectin-3. Using the optical bioluminescence technique, it was observed that the simultaneous treatment of galectin-3 with temozolomide led to a reduction of tumor growth generated by U87- Luc2 cells (U87 cells transfected with the luciferase reporter gene) in Balb / c nude. This reduction was observed even after stopping treatment (follow-up period). Interestingly, treatment of U87-Luc2 derived-tumor with only galectin-3 led to a reduction of tumor growth whose effect was abolished after discontinuation of treatment. The biodistribution of 99mTc labeled-galectin-3 (99mTc-HYNIC / Gal-3) was performed by molecular image tools (PET-SPECT scan) in mice BALB/c nude previously inoculated with the U87 line. It was shown that this protein migrates primarily to the kidneys and, in a smaller amount to the spleen, but doesn\'t bind the tumor. Because the half-life of 99m Tc-HYNIC / 3-Gal doesn\'t allow prolonged studies, galectin-3 conjugated with VivoTag 680XL was used to assess in vivo biodistribution at 48h, 96h and 14 days in Balb / c nude mice inoculated with the human glioblastoma cell line U87. Besides confirming the distribution pattern described above, it was found that galectin-3 doesn\'t bind to the tumor, regardless the tumor model. This study shows that galectin-3 has an antiproliferative effect when associated with temozolomide in the human glioblastoma model U87. Surprisingly, it was observed, that the in vivo antiproliferative effect of galectin-3 is not due to its direct binding to tumor cells. Our data suggest that when administered in vivo, galectin-3 acts in distinct locations of the tumor microenvironment such as the kidneys and spleen, thus indirectly affecting tumor growth
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Adiabatic pulse preparation for imaging iron oxide nanoparticlesHarris, Steven Scott 26 January 2012 (has links)
Iron oxide nanoparticles are of great interest as contrast agents for research and potentially clinical molecular magnetic resonance imaging (MRI). Biochemically modifying the surface coatings of the particles with proteins and polysaccharides enhances their utility by improving cell receptor specificity, increasing uptake for cell labeling and adding therapeutic molecules. Together with the high contrast they produce in MR images, these characteristics promise an expanding role for iron oxide nanoparticles and molecular MR imaging for studying, diagnosing and treating diseases at the molecular level. However, these contrast agents produce areas of signal loss with traditional MRI sequences that are not specific to the nanoparticles and cannot easily quantify the contrast agent concentration. With the expanding role of iron oxide nanoparticles in molecular imaging, new methods are needed to produce a quantitative contrast that is specific to the iron oxide nanoparticle. This dissertation presents a new method for detecting and quantifying iron oxide nanoparticles using an adiabatic preparation pulse and the failure of the adiabatic condition for spins diffusing near the particles. In the first aim, the theoretical foundation of the work is presented, and a Monte Carlo simulation supporting the proposed mechanism of the contrast is described. Adiabatic pulse prepared imaging sequences are also developed for imaging at 3 Tesla and 9.4 Tesla to highlight the translational potential of the approach for clinical examinations and scientific research, and the linear correlation of the contrast with iron concentration ideal for quantification is presented. Further, the physical characteristics of the nanoparticles and the parameters of the MRI sequence are modified to characterize the approach. In the second aim, the contrast is characterized in more realistic phantoms and in vitro, and a method to more accurately quantify nanoparticle concentration in the presence of magnetization transfer is presented. Finally, accelerated imaging methods are implemented to acquire the adiabatic contrast in a time compatible with in vivo imaging, and the technique is evaluated in an in vivo model of quantitative iron oxide nanoparticle imaging. Together, these aims present a method using an adiabatic preparation pulse to generate an MR contrast based on the microscopic magnetic field gradients surrounding the iron oxide nanoparticles that is suitable for in vivo quantitative, molecular imaging.
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Optical and MR Molecular Imaging Probes and Peptide-based Cellular Delivery for RNA Detection in Living CellsNitin, Nitin 10 August 2005 (has links)
Detection, imaging and quantification of gene expression in living cells can provide essential information on basic biological issues and disease processes. To establish this technology, we need to develop molecular probes and cellular delivery methods to detect specific RNAs in live cells with potential for in vivo applications. In this thesis work, the major focus is placed on the development of molecular beacons and biochemical approaches (peptides etc.) to deliver such probes to different cellular compartments. These approaches are then employed to study the expression and localization of mRNAs, co-localization of mRNAs with cytoplasmic organelles and cytoskeleton, and co-localization of RNA molecules in the nuclei of living cells.
Further along this direction, we were interested in developing a better understanding of the functional states of mRNAs and the fluorescent signal observed in optical imaging experiments. To acheive this goal, we altered the translational process and studied its effect on the detection of mRNAs in living cells. The results of these studies indicate that the translational state of mRNAs favors the hybridization of molecular beacon with its target sequence. This study has also provided the evidence that molecular beacons are reversibly bound to target mRNAs and the repression of the translational process can prevent molecular beacon from binding to its target mRNA. Further, using these approaches in combination with FRAP based biophysical analysis, the dynamics of endogenous RNA in living cells are studied. These studies revealed the possible subcellular organization of RNA molecules and their dynamics in living cells. The results also demonstrated the role of cytoskeleton and ATP in the mobility of specific mRNAs in the cytoplasm.
In addition to optical probes, studies have been carried out to develop an MRI contrast agent using iron-oxide nanoparticles for deep tissue molecular imaging. Specifically, we have functionalized magnetic nanoparticles that are water-soluble, mono-dispersed, biocompatible, and easily adaptable for multifunctional bioconjugation of probes and ligands. We have successfully delivered magnetic nanoparticle bioconjugates into live cells and demonstrated their effect on relaxivity. We have further studied the role of coating thickness for optimization of contrast and further enhance the fundamental understanding of contrast mechanisms.
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Μεθοδολογίες μοριακής απεικόνισης με επισημασμένα νανοσωματίδια για τον ποσοτικό προσδιορισμό της χωροχρονικής κατανομής της αγγειογένεσης σε καρκινικούς όγκους / Molecular imaging methodologies with radiolabeled nanoparticles for the quantitative evaluation of angiogenesis spatial distribution in malignant tumorsΤσιάπα, Ειρήνη 29 April 2014 (has links)
Αντικείμενο της παρούσας διατριβής αποτελεί η μελέτη της απεικόνισης και ποσοτικοποίησής της με χρήση τεχνικών μοριακής απεικόνισης. Ένα νέο κυκλικό πεπτιδικό παράγωγο RGDfK (Arg-Gly-Asp-D‐Phe–Lys), το cRGDfK-Orn3-CGG, αξιολογήθηκε ως νέο μοριακό μέσο στόχευσης του καρκίνου μέσω της ειδικής στόχευσης των υποδοχέων ιντεγκρίνης ανβ3 που υπερεκφράζονται κατά την αγγειογένεση. Το νέο πεπτιδικό παράγωγο φέρει τον περιφερειακό υποκαταστάτη CGG (Cys-Gly-Gly), κατάλληλο για την επισήμανση με σύμπλοκα του πεντασθενούς 99mTc(V) καθώς και για την σύζευξη με νανοσωματίδια. Συγκεκριμένα, αναπτύχθηκαν σιδηρομαγνητικά νανοσωματίδια (10±2 nm) συζευγμένα με το νέο παράγωγο RGD, κατάλληλα τόσο για SPECT/MRI απεικόνιση όσο και για υπερθερμία. Ειδικότερα, αξιολογήθηκαν ως νέα μοριακά μέσα απεικόνισης: 99mΤc-RGD (cRGDfK-Orn3-CGG), 99mΤc-NPs και 99mΤc-NPs-RGD, αναφορικά με τα ραδιοχημικά, ραδιοβιολιγικά και in vivo απεικονιστικά χαρακτηριστικά τους. Τα επισημασμένα παράγωγα λήφθηκαν σε υψηλές αποδόσεις και παρουσίασαν ικανοποιητική σταθερότητα in vitro: α) με την πάροδο του χρόνου, β) σε παρουσία περίσσειας ανταγωνιστών για το 99mTc, γ) σε παρουσία ανθρωπίνου πλάσματος ή ορού. Η μελέτη της in vivo συμπεριφοράς, αλλά και η βιοκατανομή των νέων παραγώγων πραγματοποιήθηκε σε φυσιολογικούς μύες και σε παθολογικά πρότυπα καρκίνου τύπου γλοιοβλαστώματος U87MG. Η αξιολόγηση της χωροχρονικής κατανομής της αγγειογένεσης κατέδειξε μέγιστη στόχευση στις ιντεγκρίνες ανβ3 σε ποσοστό 11,60±3,05 % ID/g για το παράγωγο 99mTc-RGD και 9,01±0,19 ID/g για τα στοχευμένα νανοσωματίδια 99mTc-NPs-RGD. Το 99mTc-RGD σχεδιάστηκε κατάλληλα ώστε να αποβάλλεται από τον οργανισμό μέσω του ουροποιητικού συστήματος, με την προσθήκη του υδρόφιλου μορίου ορνιθίνης (Orn3) στη δομή του. Ενώ, τα 99mTc-NPs αποβάλλονται κυρίως μέσω του ηπατοχολικού συστήματος, τα στοχευμένα 99mTc-NPs-RGD παρουσιάζουν χαμηλότερη πρόσληψη στο ήπαρ και υψηλότερη πρόσληψη στους νεφρούς, η οποία μπορεί να αποδοθεί στην πρόσδεση του RGD παραγώγου στην επιφάνεια των NPs. Ικανοποιητικές απεικονίσεις των όγκων ελήφθησαν και με τα επισημασμένα παράγωγα 99mTc-RGD και 99mΤc-NPs. Τέλος, η in vivo αξιολόγηση της θερμικής απόκρισης των NPs ανέδειξε ικανοποιητικά αποτελέσματα, με αντικαρκινική δράση σε πειραματόζωο που φέρει U87MG όγκο. Τα παραπάνω αρχικά αποτελέσματα οδηγούν στο συμπέρασμα ότι τα στοχευμένα νανοσωματίδια είναι πολλά υποσχόμενα στο πεδίο της μοριακής απεικόνισης για τον ποσοτικό προσδιορισμό της χωροχρονικής κατανομής της αγγειογένεσης με στόχο τη διάγνωση αλλά και τη θεραπευτική προσέγγιση. / The aim of the present project is the in vivo evaluation of quantitative monitoring of angiogenesis making use of the molecular imaging methodology. A new cyclic RGDfK (Arg-Gly-Asp-D‐Phe–Lys) derivative, namely the cRGDfK-Orn3-CGG, was evaluated as eventually promising in early tumor detection through specifically targeting integrin ανβ3 receptors, overexpressed in angiogenesis. This new peptide, availing the 99mTc-chelating moiety CGG (Cys-Gly-Gly), is appropriately designed for 99mTc-labeling, as well as consequent conjugation onto nanoparticles. Specifically, RGD-conjugated iron oxide nanoparticles (10±2 nm) have been developed appropriately for SPECT/MRI imaging and hyperthermia treatment. Particularly, they were evaluated as tumor imaging agents: 99mΤc-RGD (cRGDfK-Orn3-CGG), 99mΤc-NPs and 99mΤc-NPs-RGD. The new derivatives were examined with regard to their radiochemical, radiobiological and imaging characteristics. It has been demonstrated that they were obtained in high radiochemical yield and presented high in vitro stability being examined: a) at different time-points, b) in the presence of an excess of antagonist moites for 99mTc, c) in human plasma or serum. The in vivo study and the biodistribution evaluation of radiolabeled products were assessed in normal mice and in pathological models (scid mice) bearing experimental U87MG glioblastoma tumors. Τhe quantitative evaluation of angiogenesis spatial distribution confirmed high specific binding of the 99mTc-RGD peptides to ανβ3 integrins, with significantly high tumor uptake 11.60±3.06 % ID/g, while targeting with 99mTc-NPs-RGD demonstrates high tumor uptake 9.01±0.19 ID/g. The 99mTc-RGD was appropriately designed to have urine excretion due to the ornithine (Orn3) linker, while the 99mTc-NPs exhibits hepatobiliary excretion, compared to 99mTc-NPs-RGD, which exhibit lower values of liver uptake with a significantly higher kidney uptake, which can be attributed to the attachment of the RGD derivative on the surface of NPs. Satisfactory tumor images were obtained with the radiolabeled derivatives 99mTc-RGD and 99mΤc-NPs. Finally, the in vivo heating efficiency experiment showed that hyperthermia induction with the aid of iron oxide NPs was feasible, resulting to anti-tumor effect in a U87MG tumor-bearing mouse. The above preliminary results indicate that targeted iron oxide NPs are promising candidates for the quantitative monitoring of angiogenesis for molecular imaging and potential cancer therapy.
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Raman-encoded nanoparticles for biomolecular detection and cancer diagnosticsAnsari, Dominic O. 28 October 2008 (has links)
Optical assays to detect cancer-associated molecular biomarkers in biological substrates are commonly performed with antibody-targeted organic dye contrast agents but the potential for precise quantification, long-term imaging, and multiplexed readouts is limited by chemical and optical instability, non-optimal spectral characteristics, and complicated synthetic chemistry of the dyes. This dissertation tested the hypothesis that a novel class of optical contrast agents termed polymer-protected Raman-encoded nanoparticle tags (PRENTs) provides practical advantages over existing optical technologies for molecular diagnostic applications. First, PRENTs were developed through a modular design utilizing gold-nanoparticle-Raman reporter complexes protected and functionalized by polyethylene glycol derivatives. PRENTs produced optical readouts through surface enhanced Raman scattering (SERS) that were brighter and more photostable than the fluorescence of semiconductor quantum dots under identical experimental conditions. Unique spectral signatures were produced with a broader class of Raman reporters than is possible with silica coated Raman tags. Spectral signatures and colloidal stability of PRENTs were unaffected by harsh chemical conditions that cause spectral changes and aggregation of dyes, quantum dots, and protein coated Raman tags. Antibody-targeted PRENTs specifically tagged cell surface cancer biomarkers on living cells at reasonable integration times. PRENTs were non-toxic to cells under conditions exceeding those required for sensitive molecular detection. Next, PRENTs were efficiently optimized for excitation with near-infrared light through inclusion of near-infrared chromophores as Raman reporters and exploitation of the size-dependent optical enhancement of gold nanoparticles. Third, the development of a slide-based Raman-linked immunosorbent assay using antibody-conjugated PRENTs enabled quantification of protein biomarkers with a dynamic range of 3 to 4 logs. In summary, this dissertation establishes PRENTs as novel optical tags with unique features useful for biomedical applications and provides insights for further assay development.
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