Magnetic resonance imaging has developed into a powerful diagnostic technique characterized by a very high spatial resolution and an inherently relatively low sensitivity. In order to improve the contrast of MRI images, contrast agents are commonly injected into the patients before an examination. These substances are paramagnetic, superparamagnetic, or ferromagnetic compounds that shorten the relaxations times of the water hydrogen atoms. At present, most of the contrast enhanced clinical exams are performed with gadolinium complexes. They are particularly useful as their ability to change the relaxation rate (or relaxivity) can be very high. Several factors have a strong influence on the relaxivity of MRI contrast agents but the water exchange time τm and the rotational correlation time τr are particularly important for obtaining an increased relaxivity. These parameters can be adjusted by suitable chemical modifications of the Gd(III) complexes. For instance, decreasing the tumbling rate by linking a Gd(III) complex to a macromolecule leads to an increased relaxivity. This goal can be achieved through a covalent or a noncovalent linkage with synthetic polymers, particles or biomacromolecules. However, the covalent bonding has a detrimental effect on the clearance of the metal complexes thus exposing the patient to the toxicity of released Gd(III) ions and metabolites. This problem could be circumvented by using covalent links that are cleaved by endogenous biomolecules or after administration of exogenous compounds following the exam. In this context, our approach was to bind Gd(III) chelates to macromolecules through disulfide links as the latter are known to be reduced in vivo by thiols present in the body. Towards this aim, we have developed two bifunctional chelator agents bearing a methanethiosulfonate group (MTS) which reacts specifically with thiols, thus spontaneously establishing a disulfide bond between the Gd(III) chelate and the thiolated macromolecule. The first ligand that we have prepared (MTS-ADO3A) is a monoamide derivative of DOTA with an ethyl-MTS substituent. This compound is relatively easily synthesized but amide arms such as the one it features are known to have a detrimental effect on relaxivity through the lengthening of water exchange times. The conjugate obtained by binding Gd(III) chelates of this ligand to albumin or to polythiolated silica nanoparticles has been studied by nuclear magnetic relaxation dispersion (NMRD),17O NMR and luminescence analyses. These measurements confirm that the method is suitable to increase the relaxivity (20 mM-1s 1, 20 MHz, 25°C) but that this relaxivity increase (of 300%) is limited by a slow water exchange (660 ns). To overcome this limitation, a second ligand called MTS-CyDOTA has been synthesized. This ligand is a DOTA ligand grafted with a cyclohexyl ring featuring a MTS function. The synthesis is more demanding but faster water exchange times are expected because of a more sterically crowded coordination sphere. Moreover, this second ligand has a more rigid structure that could limit the independent rotation of the chelate from the macromolecule. As expected, the water exchange time of the Gd(III) chelate of this ligand (120 ns) is clearly lower than the one determined for Gd MTS-ADO3A. After binding to albumin or to silica nanoparticles a notable relaxivity increase was expected. Unfortunately, if the obtained relaxivity is higher (30 mM-1s 1, 20 MHz, 25°C), its not as high as it could have been expected in view of the size of the conjugate and of the water exchange time of the free chelate. Results obtained in this work suggest that fixation on silica nanoparticles or on albumin drastically decreases the water exchange rate which remains the limiting parameter. This effect has already been reported for Gd(III) chelates linked to albumin by non-covalent bonds and has been assigned to stable layers of water molecules on the macromolecule surface. Thanks to the high loading of the silica nanoparticles (10000 Gd(III) per particle), we have reached very high molecular relaxivities (>200000 mM-1s-1). Stability tests carried out on the disulfide links formed suggest that the small amount of free thiols in the circulation is not sufficient to cause a significant degradation of the disulfide bond in the conjugate within a reasonable length of time. An injection of glutathione would be necessary to achieve a complete degradation. To avoid the problem of water exchange lenghtening, we propose to increase the distance between Gd(III) chelates and macromolecules without loss of rigidity by developing double anchor chelates with substituents grafted on the side of the ring. Considerable synthetic efforts have devoted to the synthesis of such a system and are discussed in chapter VI. At present, this work is still in progress in the laboratory and recent results suggest that it should be possible to evaluate this double arms system in a near future. On the fringe of this synthesis, we present a relaxometric study on the interaction between HSA and a hydrophobic Gd(III) chelate obtained during the preparation of our double anchor chelate. Finally, a chapter of this work is devoted to the study of two compounds, phenEDTA and phenDTPA, which are ditopic chelates featuring a dihydro-1,10-phenanthroline unit that spontaneously self-assemble in the presence of a transition metal ion. The tris-complex generated by this process rotates more slowly in solution and thus presents an increased relaxivity (+130%). As part of this work, we have determined by potentiometric titration the acidity constants of phenEDTA and its stability constant with Gd(III). Moreover, the protonation scheme of this ligand has been studied by NMR titration. The particular behavior of Gd phenDTPA and Fe(Gd phenDTPA)3 in the presence of Zn(II) has also been studied by relaxometry, luminescence and EXAFS.

Citrate-coated gadolinium trifluoride (Cit-GdF3) and poly(acrylic acid)-coated nanoparticles (PAA-GdF3 NPs) were synthesized, the former reproduced from literature (though using more refined conditions), the latter through a new, two-step, ligand exchange method. Diamagnetic nanoparticle analogs (Cit-YF3 NPs) were prepared to investigate citrate interactions with the nanoparticle surface using NMR. Citrate was found to bind in numerous conformations, with a total of between 29 – 46 % bound at 0 ºC. Exchange studies revealed short residence lifetimes of one and twelve seconds respectively for bound and free forms of citrate (0 ºC), perhaps explaining the colloidal instability of these nanoparticles. PAA-GdF3 NPs were synthesized by first producing their Cit-GdF3 counterparts, and then exchanging citrate for PAA. The impetus behind this latter synthesis was the relative enhancement in stability and relaxivity attainable by these nanoparticles. The displacement of citrate by PAA was verified using diffusion NMR studies.

Citrate-coated gadolinium trifluoride (Cit-GdF3) and poly(acrylic acid)-coated nanoparticles (PAA-GdF3 NPs) were synthesized, the former reproduced from literature (though using more refined conditions), the latter through a new, two-step, ligand exchange method. Diamagnetic nanoparticle analogs (Cit-YF3 NPs) were prepared to investigate citrate interactions with the nanoparticle surface using NMR. Citrate was found to bind in numerous conformations, with a total of between 29 – 46 % bound at 0 ºC. Exchange studies revealed short residence lifetimes of one and twelve seconds respectively for bound and free forms of citrate (0 ºC), perhaps explaining the colloidal instability of these nanoparticles. PAA-GdF3 NPs were synthesized by first producing their Cit-GdF3 counterparts, and then exchanging citrate for PAA. The impetus behind this latter synthesis was the relative enhancement in stability and relaxivity attainable by these nanoparticles. The displacement of citrate by PAA was verified using diffusion NMR studies.

More than one-third of all Magnetic Resonance Imaging (MRI) scans employ image-enhancing contrast agents to increase the differential signal intensity between diseased and normal tissue. Because current clinical contrast agents exhibit low relaxivity (mM-1 s-1), low dose efficiency, and rapid secretion, we have designed a group of protein-based MRI contrast agents with multiple gadolinium binding sites. In this study, the developed purification method for Class ProCA-3 agents allows for a quick and cost-effective way to abstract up to 109 mg of pure, soluble protein from a 1L E. Coli cell pellet devoid of DNA or RNA “contamination” for extensive animal studies. Circular dichroism far-UV spectra ensure the metal stability of the agents, revealing maintenance of their native α-helical structure in the presence and absence of metal ions. Furthermore, substantial evidence supports the high dose efficiency of these agents, exhibiting up to five folds higher relaxivity than their analogous commercial competitors.

ABSTRACT Heaves is triggered by exposure to dust and its components, such as endotoxin, and is characterized by clinical signs such as coughing, decreased exercise tolerance, difficulty breathing and abnormal lung sounds which are due to bronchoconstriction and accumulation of neutrophils in the airways. Pulmonary intravascular macrophages (PIMs) are believed to increase horses sensitivity to endotoxemia-induced lung inflammation. The first objective of this study was to investigate a hitherto unknown role of PIMs in equine heaves. I used mouldy hay (MH) to induce heaves and gadolinium chloride (GC) to deplete PIMs in order to compare responses between non-treated and GC-treated heaves horses. A modified randomized crossover study (2X2 factorial) was conducted in which mares (N=9) were exposed to 4 different treatments: alfalfa cubes (Cb), alfalfa cubes + GC (Cb-GC), mouldy hay (MH) and MH + GC (MH-GC). Each treatment was followed by broncholaveolar lavage (BAL). MH was fed for 7 days to induce heaves followed by Cb for 21 days to achieve remission, whereas the treatments in which heaves was not induced (Cb; Cb-GC), the cubes were fed prior to the BAL and for 14 days after the BAL to allow recovery from the BAL procedure. BAL fluids were processed to investigate total cell, neutrophil and alveolar macrophage concentrations. In addition, TNFá protein levels as well as TNFá, IL-8, and TLR4 mRNA expression in BAL cells were assessed in order to infer on their activation state.<p> Data showed higher concentration of dust (3X), endotoxin (20X), and endotoxin per milligram of dust (7X) in MH compared to the Cb environment. Clinical scores and neutrophil concentrations in BAL were higher when mares received MH compared to MH and GC (MH-GC). Real time reverse transcriptase PCR revealed a significant lower expression of IL-8 and TLR4 mRNA in BAL cells from MH-GC mares compared to MH. TNFá mRNA expression as well as protein concentration were not affected by the different treatments. In vitro secondary LPS challenge significantly increased IL-8 mRNA expression in cells from MH treatment compared to without LPS, but not in the MH-GC treatment. TLR4 expression was not affected by the secondary challenge. Although secondary LPS challenge increased expression of TNFá mRNA and protein, the differences among treatment groups were not meaningful. In conclusion, PIM depletion attenuates clinical scores, migration of inflammatory cells into the alveolar space and expression of pro-inflammatory molecules in BAL cells of heaves horses.<p> The observations on the role of PIMs in heaves in horses prompted me to examine the occurrence of PIMs in human lungs. I found a trend for higher numbers of septal macrophages in autopsied lungs from human patients who died of non-pulmonary pathologies compared to those having either COPD or asthma. If these septal macrophages indeed represent the PIMs, this finding is contrary to existing belief that humans, unlike horses, do not have PIMs.

No description available.

En vue d'application à l'éclairage ou dans les dispositifs de visualisation à plama, on a étudié la luminescence de l'ion Eu2+ dans les borates BaLnB9O16 (Ln = La, Ce, Gd, Y) et de l'ion Eu3+ dans les diverses phases des systèmes Gd2O3-P2O5 et Y2O3-P2O5. Des informations sur les structures des réseaux-hôtes lorsqu'elles étaient inconnues, ont pu être obtenues par diffraction X et spectroscopie Raman. Pour Ln = Y, Gd, les borates BaLnB9O16:Eu2+ présentent une émission dans le bleu stable...

ABSTRACT Heaves is triggered by exposure to dust and its components, such as endotoxin, and is characterized by clinical signs such as coughing, decreased exercise tolerance, difficulty breathing and abnormal lung sounds which are due to bronchoconstriction and accumulation of neutrophils in the airways. Pulmonary intravascular macrophages (PIMs) are believed to increase horses sensitivity to endotoxemia-induced lung inflammation. The first objective of this study was to investigate a hitherto unknown role of PIMs in equine heaves. I used mouldy hay (MH) to induce heaves and gadolinium chloride (GC) to deplete PIMs in order to compare responses between non-treated and GC-treated heaves horses. A modified randomized crossover study (2X2 factorial) was conducted in which mares (N=9) were exposed to 4 different treatments: alfalfa cubes (Cb), alfalfa cubes + GC (Cb-GC), mouldy hay (MH) and MH + GC (MH-GC). Each treatment was followed by broncholaveolar lavage (BAL). MH was fed for 7 days to induce heaves followed by Cb for 21 days to achieve remission, whereas the treatments in which heaves was not induced (Cb; Cb-GC), the cubes were fed prior to the BAL and for 14 days after the BAL to allow recovery from the BAL procedure. BAL fluids were processed to investigate total cell, neutrophil and alveolar macrophage concentrations. In addition, TNFá protein levels as well as TNFá, IL-8, and TLR4 mRNA expression in BAL cells were assessed in order to infer on their activation state.<p> Data showed higher concentration of dust (3X), endotoxin (20X), and endotoxin per milligram of dust (7X) in MH compared to the Cb environment. Clinical scores and neutrophil concentrations in BAL were higher when mares received MH compared to MH and GC (MH-GC). Real time reverse transcriptase PCR revealed a significant lower expression of IL-8 and TLR4 mRNA in BAL cells from MH-GC mares compared to MH. TNFá mRNA expression as well as protein concentration were not affected by the different treatments. In vitro secondary LPS challenge significantly increased IL-8 mRNA expression in cells from MH treatment compared to without LPS, but not in the MH-GC treatment. TLR4 expression was not affected by the secondary challenge. Although secondary LPS challenge increased expression of TNFá mRNA and protein, the differences among treatment groups were not meaningful. In conclusion, PIM depletion attenuates clinical scores, migration of inflammatory cells into the alveolar space and expression of pro-inflammatory molecules in BAL cells of heaves horses.<p> The observations on the role of PIMs in heaves in horses prompted me to examine the occurrence of PIMs in human lungs. I found a trend for higher numbers of septal macrophages in autopsied lungs from human patients who died of non-pulmonary pathologies compared to those having either COPD or asthma. If these septal macrophages indeed represent the PIMs, this finding is contrary to existing belief that humans, unlike horses, do not have PIMs.

Afin d'accéder à des agents de contraste efficaces, il est essentiel d'optimiser simultanément les paramètres moléculaires influençant la relaxivité : nombre de molécules d'eau en première sphère de coordination, échange de l'eau, dynamique de rotation du complexe, relaxation électronique, distance Gd(III)-proton. Le but de ce travail est double. D'une part, il s'agit de concevoir et étudier des complexes possédant un nombre élevé de molécules d'eau coordinées et de comprendre l'influence de la sphère de coordination du métal sur la stabilité des complexes et la relaxation électronique. D'autre part, nous avons utilisé les ligands comme chromophores pour la mise au point de sondes luminescentes pour l'imagerie biomédicale.<br />Nous présentons la structure, la stabilité et la relaxivité de complexes de Gd(III) de deux séries de ligands tripodes dérivés du picolinate basés, soit sur le cycle 1,4,7-triazacyclononane, soit sur un pivot amine tertiaire. Ces complexes possèdent une relaxivité élevée dans l'eau et dans le sérum et peuvent former des interactions non-covalentes avec l'albumine sérique. L'interprétation de la relaxivité des protons de l'eau au moyen de nouvelles méthodes relaxométriques basées sur l'utilisation de solutés sondes nous a permis de montrer que la présence de groupement picolinate et du cycle 1,4,7-triazacyclononane pouvait conduire à des complexes de Gd(III) possédant des propriétés de relaxation électronique favorables.<br />Du fait de la présence de chromophores picolinate, les complexes d'Eu(III) et Tb(III) avec ces ligands donnent lieu à une luminescence intense dans le visible. D'autres complexes dérivés de l'unité 8 hydroxyquinoléine possèdent une luminescence élevée dans l'infrarouge et ont également été étudiés.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 229-230). Also available in print.