Gd-Kontrastmittel werden in der Medizin für die Magnetresonanztomographie (MRT) benötigt, um einen besseren Bildkontrast zu erzielen. Nach der Anwendung gelangen diese Arzneimittel über das Abwasser in die Klärwerke und von dort in die jeweiligen anliegenden Oberflächengewässer. Somit wird es notwendig, den Verbleib der Gd-MRT-Kontrastmittel in der Umwelt zu verfolgen und aufzuklären. In dieser Arbeit werden für die Analyse von Umweltproben Methoden zur Speziesanalytik von Gd-Kontrastmitteln entwickelt und optimiert. Hierbei wird u.a. die Zwitterionische Hydrophile Interaktionschromatographie (HILIC) verwendet, die mit einer Induktiv- gekoppelten Plasma – Massenspektrometrie (ICP-MS) gekoppelt ist. / Gd based contrast agents are used for medical magnetic resonance imaging (MRI) to enhance the contrast of the image. After application and excretion, the drugs pass through the wastewater treatment plant (WWTP) and make their way into the surrounding surface water bodies. This makes it necessary to monitor the fate of the Gd based contrast agents in the environment. In this work, methods for speciation analysis of Gd based contrast agents in environmental samples were developed and optimised. For this purpose i.a. zwitterionic hydrophilic interaction chromatography (HILIC) is used, which is hyphenated with inductively coupled plasma mass spectrometry (ICP-MS).

Gadoliumbasierte Kontrastmittel werden in der Magnetresonanztomographie verwendet und gelangen nach der Exkretion durch behandelte Patienten in Oberflächengewässer, da sie von Klärwerken nur unzureichend entfernt werden. Das Verhalten der Kontrastmittel in der Umwelt und deren Wechselwirkung mit Organismen wurde in dieser Arbeit aufgrund der Toxizität des freien Gd-Ions und der damit verbundenen möglichen Gefahren, wie der Anreicherung in der menschlichen Nahrungskette, näher untersucht. Die bildgebenden elementanalytischen Methoden Laserablation mit induktiv gekoppelter Plasmamassenspektrometrie (LA-ICP-MS) und Röntgenfluoreszenzanalyse mit Synchrotronstrahlung (SRXRF) wurden genutzt, um verschiedene biologische Systeme hinsichtlich ihrer Aufnahme, Verteilung und Abgabe von Gd-basierten Kontrastmitteln zu untersuchen. Zuvor wurden beide Methoden für diese Anwendung analytisch charakterisiert und verglichen. Die Nachweisgrenzen von Gadolinium wurden mittels LA-ICP-MS (0.78 pg) und SRXRF (89 pg) unter jeweils optimierten Bedingungen durch die Kalibrierung über Rückstände eingetrockneter Elementstandardtropfen bestimmt. Anhand von Fadenalgen konnte die Aufnahme und Abgabe der Gd-basierten Kontrastmittel gezeigt werden. Die Abhängigkeit von der Konzentration des Kontrastmittels in der Expositionslösung und die Unabhängigkeit der zeitlichen Aufnahme innerhalb eines Zeitraums von 1-7 Tagen wurden an Wasserlinsen studiert. Gadolinium konnte in einem Kresseblatt quantifiziert werden. Die Verifizierung der LA-ICP-MS-Ergebnisse erfolgte mit der SRXRF und der ICP-MS nach Aufschluss. Auch die Aufnahme und Abgabe von Gd in Wasserflöhen wurde beobachtet. Die Lokalisierung des Gds wurde mittels 3D µ-Computertomographie durch den Vergleich mit einem unbelasteten Wasserfloh aufgeklärt. In allen Untersuchungen wurde Gadolinium in den exponierten Modellorganismen nachgewiesen, woraus geschlussfolgert werden kann, dass die Kontrastmittel aus der Umgebung aufgenommen werden. / Gadolinium based contrast agents are used for magnetic resonance imaging. After their excretion by medicated patients they reach surface water passing waste water treat-ment plants where they are not removed sufficiently. The behavior of the contrast agents in the environment and the interaction with organisms was investigated in this work due to the toxicity of the free Gd3+ ion and the associated risks, such as accumulation in the human food chain. In this work, the two elemental analytical imaging methods laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron radiation X-ray fluorescence analysis (SRXRF) have been used to investigate the uptake, distribution, and excretion of Gd-based contrast agents by various biological systems. Both methods were analytically characterized and compared for this application. The detection limits of gadolinium were determined under optimized conditions by LA-ICP-MS and SRXRF. With calibration by remains of dried elemental standard droplets detection limits of 0.78 pg absolute amount of gadolinium (LA-ICP-MS), respectively 89 pg (SRXRF) were reached. Based on filamentous algae as water plants the uptake and the excretion of Gd-based contrast agents were revealed. The dependence on concentration of the contrast agent in the exposition solution and the independence of temporal uptake within one to seven days were studied for duckweed. By LA-ICP-MS gadolinium was quantified in a leaf of cress plant. The verification of the results was performed by SRXRF and ICP-MS after digestion. Furthermore, the uptake and distribution of Gd-based contrast agents in higher organisms (water flea) were observed. The exact location of gadolinium was resolved by three-dimensional μ-computed tomography by the comparison of an exposed with a Gd-free water flea. In all studies, gadolinium was detected in the investigated exposed model organisms. It can be concluded that the contrast agents were taken from the environment.

Cette étude concerne le développement de nanoparticules hybrides offrant de nouvelles stratégies pour la thérapie et le diagnostic médical. Elles sont constituées d’un cœur magnétique jouant le rôle d’agent de contraste pour l’IRM et d’inducteur de chaleur par hyperthermie, d’une couronne de polymère thermosensible permettant d’encapsuler des principes actifs et de peptides de reconnaissance biologique. Une grande partie de l’étude a consisté à étudier les processus d'adsorption de copolymères poly(éther)-b-poly(L-lysine) de composition variable sur les particules magnétiques et à comprendre le rôle de la conformation des chaînes polymère à la surface des particules sur la stabilité des colloïdes en milieu physiologique. Un agent antitumoral a été encapsulé puis libéré de façon contrôlée sous l’effet d’un champ magnétique alternatif en exploitant le caractère thermosensible des blocs polyéthers. Des séquences peptidiques ciblant les zones d’inflammation de la barrière hémato-encéphalique ont été greffées sur les copolymères. L’efficacité du ciblage a été validée par IRM et fluorescence sur un modèle animal démontrant ainsi la multifonctionnalité des nanoparticules. / This work deals with the development of hybrid nanoparticles that could offer new strategies for therapy and diagnostic. These are based on a magnetic core which can play the role of contrast agent for MRI as well as heat inductor in AC magnetic field. This inorganic core is surrounded by a thermo-responsive polymeric brush that controls the loading and the release of drugs, and can be functionalized by specific ligands ensuring the targeting specificity. A large part of this work consists in studying the adsorption mechanism of poly(ether)-b-poly(L-lysine) based block copolymers onto magnetic particle and to better understand the influence of the polymer chain conformation at particles surface on the colloidal stability under physiological conditions. An anticancer drug has been loaded and released in a controlled manner under alternative magnetic field by taking advantage from the thermosensitivity of the polyether block. Targeting peptides specific of inflammation sites at the blood brain barrier have been grafted onto copolymers. The targeting specificity has been demonstrated by MRI and fluorescence imaging in rats attesting the multifunctionality of such nanoparticles.

Made available in DSpace on 2015-05-14T13:21:13Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1246890 bytes, checksum: d8de4e92a3d9c573a9ad39ccb546aa71 (MD5) Previous issue date: 2008-11-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Lanthanide macrocyclic complexes, especially containing Gd(III) ions, are largely used as contrast agents in the medical diagnostic technique named Magnetic Resonance Imaging (MRI). Theoretical investigation of these compounds by molecular modeling methods is an emerging research area today. In this work we have performed a conformational study of the following supramolecular compounds: the macrocyclic ligands 1,4,7,10- tetraazacyclododecane (DOTA) and 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (H4DOTA) and the macrocyclic complexes [Gd(DOTA).H2O]- and Gd(PhenHDO3A).H2O, where PhenHDO3A is (rel-10- [(5R,6R)-5,6-dihydro-6-hydroxy-1,10-Phenanthroline-5-yl)-1,4,7,10- tetraazacyclododecane-1,4,7-triacetic acid). For this, we have developed and carried out a procedure that involves: (1) the sampling of the conformation space of these compounds through some short molecular dynamics at different temperatures and (2) full geometry optimization in the geometries obtained in the previous stage using semi-empirical AM1 method for the macrocyclic ligands and Sparkle/AM1 model for the macrocyclic lanthanide complexes. Our results revealed that the high flexibility of non-coordinated macrocyclic ligands DOTA and H4DOTA, helped us to test the ability of our methodology to sample different regions of conformation space of these compounds. For the [Gd(DOTA).H2O]- complex, we were capable to find the same conformational isomers which were reported in published works as well as to comprehend relevant details about the mobility of the coordinated water molecule. Furthermore, we also were capable to identify some intermediate local minima related to the dynamics of rotation of the coordinated acetate groups in the studied complexes as well as to the dynamics of ring inversion of the coordinated tetraazadodecane moiety in the Gd(PhenHDO3A).H2O. In conclusion, we possess now an interesting strategy to conduct the search for conformational isomers of these compounds. This knowledge is very important to be applied in the rational design of new molecules to act as contrast agent in MRI. / Complexos macrocíclicos de lantanídeos, em especial com o íon Gd(III), são cada vez mais utilizados como agentes de contrastes na técnica médica de diagnóstico, imagem por ressonância magnética nuclear (MRI). O estudo teórico de novos agentes de contrastes através de modelagem molecular é uma área de pesquisa em grande expansão. No nosso trabalho fizemos um estudo conformacional dos seguintes compostos supramoleculares: os ligantes macrocíclicos 1,4,7,10- tetraazaciclododecano (DOTA) e ácido 1,4,7,10-tetraazaciclododecano- 1,4,7,10-tetraacético (H4DOTA), o complexo macrocíclico [Gd(DOTA)H2O]- e o complexo macrocíclico Gd(PhenHDO3A).H2O, onde PhenHDO3A é (rel-10-[(5R,6R)-5,6-dihidro-6-hidroxi-1,10-fenantrolina-5- il)-1,4,7,10-tetraazaciclododecano-1,4,7-ácido triacético). Para isso, elaboramos e executamos um procedimento que envolve o mapeamento ou amostragem do espaço conformacional desses compostos através de dinâmicas moleculares curtas em diferentes temperaturas para em seguida, essas geometrias serem usadas como pontos de partida para otimização de geometria usando tanto o método semi-empírico AM1 quanto o modelo Sparkle/AM1. Como resultado, podemos citar que a elevada flexibilidade dos ligantes macrocíclicos não coordenados DOTA e H4DOTA, serviu para testar a capacidade da nossa metodologia de visitar regiões distintas do espaço conformacional. Para o complexo [Gd(DOTA)H2O]-, conseguimos encontrar os mesmos isômeros conformacionais que já são reportados em outros estudos, bem como revelar detalhes relativos à mobilidade da molécula de água coordenada. Além disso, conseguimos identificar mínimos locais intermediários que estão relacionados com a dinâmica de rotação dos grupos acetato em ambos os complexos e a dinâmica de inversão do anel tetraazadodecano no complexo Gd(PhenHDO3A).H2O. A relevância desse trabalho está apoiada no fato de que agora temos uma estratégia interessante para realizar a busca de isômeros conformacionais desses compostos, sendo essas informações, muito importantes quando se deseja projetar uma nova molécula para atuar como agente de contraste em MRI.

L'athérosclérose, à l’origine de la plupart des maladies cardiovasculaires telles que l'infarctus du myocarde ou l'AVC, est la principale cause de décès dans le monde. Les cliniciens ont donc besoin de techniques d'imagerie fiables pour identifier les patients «vulnérables» porteur d’athérome à haut risque d'occlusion thrombotique. Cette pathologie est une maladie inflammatoire qui implique beaucoup d'acteurs cellulaires et moléculaires, parmi lesquels les cellules endothéliales et immunitaires, les lipoprotéines, les cellules apoptotiques et les plaquettes. L'imagerie moléculaire visant à détecter ces acteurs avant la survenue d'événements cardiovasculaires dramatiques est en plein essor. Des anticorps humains (HuAbs) sélectionnés par phage-display pour reconnaître des biomarqueurs de la pathologies ont ici proposés comme ligands servant à fonctionnaliser des vecteurs pour l'imagerie IRM, de fluorescence ou TEP. Les HuAbs ont été modifiés, en introduisant soit des Cystéines soit un site de reconnaissance pour la Sortase, afin de développer un greffage site-spécifique. Les agents ciblant ont été validés in vitro et ex vivo sur des coupes d'athérome de modèles animaux. Des résultats prometteurs ontété obtenus en injectant dans des souris ApoE-/- l’anticorps antiplaquettaire TEG4, apportant ainsi de nouvelles connaissances sur la biologie de l'athérome et la preuve de concept d'une possible détection des plaques à haut risque riches en plaquettes. Des améliorations sont en cours pour développer des agents de contraste multi-fonctionnalisés avec des HuAbs et permettant de réaliser une imagerie moléculaire multimodale de l'athérosclérose facilement transposable en clinique. / Because cardiovascular diseases are the leading cause of death in the world, providing clinicians with reliable and straightforward imaging techniques to identify "vulnerable" patients from the general population appears like the Holy Grail of the cardiovascular field. Atherosclerosis, identified as the underlying condition for most acute cardiovascular events, is characterized by the constitution of a lipidrich atheroma plaque, driven both by excess cholesterol and inflammation, which eventual rupture triggers clotting into the blood flow. It involves a wealth of cellular and molecular actors, which are so many potential markers for molecular imaging, aiming at deciphering how to warn clinicians about the possible occurrence of myocardial infarction or stroke. Here, human antibodies (HuAbs) selected by phage-display for their recognition of over-expressed biomarkers of the pathology are proposed as targeting ligands. They were further engineered for site-specific grafting, either by introducing Cysteine or Sortase recognition tags, and used to target contrast agents for MRI, fluorescence, or PET imaging. In vitro and ex vivo validation studies were carried out on atheroma sections of animal models. In vivo studies in the ApoE-/- mouse model were realized with the anti-platelet TEG4 HuAb using MRI, which provided insights on the biological relevance and feasibility to detect platelets-rich, high-risk atheroma plaques. The development of contrast agents useful in multi-modality imaging, and multi-functionalized with HuAbs is underway. It should serve as an accurate molecular imaging method for atherosclerosis, further more easily translated into the clinical arena.

Les agents de contraste sont de petites bulles qui répondent non linéairement lorsqu’ils sont exposés à ultrasons. La réponse non-linéaire donne la possibilité d’images échographiques harmoniques qui a beaucoup d’avantages sur l’imagerie fondamentale. Toutefois, afin d’accroître l’échographie de contraste d’imagerie harmonique de performance nous devons d’abord comprendre la propagation non linéaire d’ultrasons. La non-linéarité du milieu déforme l’onde qui se propage, tels que les harmoniques commencent à se développer. La théorie qui a été prévue est la mise en œuvre, qui a permis une nouvelle méthode de modélisation de propagation des ultrasons non-linéaire. La connaissance acquise au cours de ce processus a été utilisée pour construire un deuxième signal à composantes multiples pour la réduction des harmoniques générées en raison des non-linéarités des tissus. En conséquence, la détection d’agents de contraste ultrasonore aux harmoniques a été augmentée. Une puissante technique d’imagerie échographique (Pulse inversion) a été renforcée avec le deuxième signal pour la réduction des harmoniques. Qu’est-ce qui a été appris pendant l’investigation : le pulse inversion technique a donné une nouvelle phase codée, appelée inversion de seconde harmonique. En outre, il a été noté que pour différents types de médias le niveau de distorsion de l’impulsion à ultrasons est différent. Cela dépend en grande partie du paramètre non linéaire B / A. Les travaux sur ce paramètre n’a pas été fini, mais il est quand même important de continuer dans cette direction puisque B / A imagerie avec des agents de contraste ultrasonore a beaucoup de potentiel. / Ultrasound contrast agents are small micro bubbles that respond nonlinearly when exposed to ultrasound wave. The nonlinear response gives possibility of harmonic ultrasound images which has many advantages over fundamental imaging. However, to increase ultrasound contrast harmonic imaging performance we must first understand nonlinear propagation of ultrasound wave. Nonlinear propagation distorts the propagating wave such that higher harmonics appear as the wave is propagating. The theory that was laid down, was allowed implementing a new method of modelling nonlinear ultrasound propagation. The knowledge obtained during this process was used to construct a multiple component second harmonic reduction signal for reduction of their harmonics generated due to the tissue nonlinearities. As a consequence detection of ultrasound contrast agents at higher harmonics was increased. Further more, a powerful ultrasound imaging technique called Pulse Inversion, was further enhanced with multiple component second harmonic reduction signal. What was learned during investigation of the Pulse Inversion, technique lead to a new phase coded ultrasound contrast harmonic method called second harmonic inversion;. Also it was noted that for different type of media the level of distortion of ultrasound pulse is different. It depends largely on the nonlinear parameter B / A. Although the work on this parameter has not been finished it is very important to continue in this direction since B / A imaging with ultrasound contrast agents has a lot of potential.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) techniques offer the opportunity to understand the physiological processes involved in the distribution of the contrast agent in vivo. This work utilises a liver specific contrast agent (gadoxetate) and demonstrates the potential use of these techniques to study transporter-mediated process in vivo. In vitro experiments investigated gadoxetate’s interaction with uptake and efflux transporters at the cellular level, ideally a prerequisite to understand the contribution of transporter proteins in in vivo pharmacokinetics. MRI was used to measure the accumulation of gadoxetate in fresh rat hepatocytes. Furthermore, LC-MS/MS methodology was optimised in conjunction with two in vitro systems to determine the role of transporters in gadoxetate uptake and efflux. HEK-OATP1B1 transfected cells were used to optimise the LC-MS/MS technique and Caco-2 cell monolayers were used to examine whether gadoxetate is a substrate of the efflux transporters, Pgp and BCRP. Subsequent studies demonstrated the use of DCE-MRI techniques to study transporter-mediated processes. Two pharmacokinetic models were proposed to quantify the uptake and efflux of gadoxetate in vivo. The suitability of the models in describing the liver concentration profiles of gadoxetate was assessed in pre-clinical and clinical reproducibility studies. Further pre-clinical experiments demonstrated the ability of the proposed DCE-MRI techniques to monitor changes in the uptake and efflux rate estimates of gadoxetate into hepatocytes, through co-administration of the transporter inhibitor, rifampicin, at two doses. The work presented demonstrates the potential use of DCE-MRI techniques as a diagnostic probe to assess transporter-mediated processes and drug-drug interactions (DDIs) in vivo.

Image registration is widely used in clinical practice. However image registration and its~evaluation is still challenging especially with regards to new possibilities of various modalities. One of these areas is contrast-enhanced ultrasound imaging. The time-dependent image contrast, low signal-to-noise ratio and specific speckle pattern make preprocessing and image registration difficult. In this thesis a method for registration of images in ultrasound contrast-enhanced sequences is proposed. The method is based on automatic fragmentation into image subsequences in which the images with similar characteristics are registered. The new evaluation method based on comparison of perfusion model is proposed. Registration and evaluation method was tested on a flow phantom and real patient data and compared with a standard methods proposed i literature. The second part of this thesis contains examples of application of image registration in~ophthalmology and proposition for its improvement. In this area the image registration methods are widely used, especially landmark based image registration method. In this thesis methods for landmark detection and its correspondence estimation are proposed.

This work deals with possibilities of ultrasound perfusion analysis for the absolute quantification of perfusion parameters. In the theoretical part of this work are discussed possibilities of using of the ultrasound contrast agents and approaches for the perfusion analysis. New methods for the perfusion analysis are suggested and tested in the practical part of this work. The methods are based on convolutional model in which the concentration of the contrast agent is modeled as aconvolution of the arterial input function and the tissue residual function. The feasibility of these methods for the absolute quantification of perfusion parameters is shown on data from phantom studies, simulations and also preclinical and clinical studies. The software for the whole process of the perfusion analysis was developed for using in hospitals.

This master thesis deals with the contrast agents and their application in the ultrasound perfusion analysis. It is focused on Bolus & Burst method which, as a combination of two approaches that have been used so far, allows an absolute quantification of perfusion parameters in the region of interest. Contrast agent concentration time sequence is modeled as a convolution of the parametrically defined arterial input function and the tissue residual funkction. Thesis discusses different mathematical models of these functions as well as the methods of the parameters estimation. The methods functionality is validated on simulated and also preclinical data.