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Diagnóstico precoce e tratamento de melanoma, um tumor cutâneo com o pior prognóstico, é extremamente importante para um resultado clínico favorável. A biblioteca de peptídeos phage display é um recurso útil de triagem para identificar peptídeos bioativos que interagem com alvos em cânceres. O objetivo desse estudo foi a avaliação de dois traçadores de tecnécio-99m com sequências peptídicas RGD e NGR, conjugados com o quelante bifuncional MAG3. Os conjugados peptídicos (10 μL de uma solução μg/μL) foram marcados com tecnécio-99m usando tampão de tartarato de sódio. A avaliação radioquímica foi feita por ITLC e confirmada por CLAE. O coeficiente de partição foi determinado e ensaios de internalização foram realizados em duas linhagens celulares de melanoma (B16F10 e SKMEL28). A avaliação da biodistribuição dos traçadores foi realizada em animais sadios em diferentes tempos e também em camundongos portadores de células tumorais aos 120 min após a sua administração. Estudos de bloqueamento também foram conduzidos pela co-injeção de peptídeo frio. O desempenho dos conjugados peptídicos mostraram-se bastante parecidos em diversas avaliações. Eles foram radiomarcados com alta pureza radioquímica (>97%). Ambos são hidrofílicos, com excreção renal preferencial. A captação tumoral foi maior para células SKMEL28 do que para as células B16F10, especialmente para o 99mTc-MAG3-PEG8-c(RGDyK) (7,85±2,34 %DI/g) aos 120 min pós-injeção. O desempenho do 99mTc-MAG3-PEG8-c(RGDyk) foi superior que o do traçador com NGR, quanto à captação no melanoma humano podendo ser considerado como um promissor radiofármaco para diagnóstico de melanoma. / Early diagnosis and treatment of melanoma, a cutaneous tumor with a serious prognosis, is extremely important for optimal clinical outcome. Phage display peptide libraries are a useful screening resource for identifying bioactive peptides that interact with cancer targets. The aim of this study was the evaluation of two technetium-99m tracers for angiogenesis detection in melanoma model, using cyclic peguilated pentapeptide with RGD and NGR motifs conjugated with bifunctional chelator MAG3. The conjugated peptides (10 μL of a μg/μL solution) were labeled with technetium-99m using a sodium tartrate buffer. Radiochemical evaluation was done by ITLC and confirmed by HPLC. Partition coefficient was determined and internalization assays were performed in two melanoma cells (B16F10 and SKMEL28). Biodistribution evaluation of the tracers was done in healthy animals at different times and also in mice bearing the tumor cells at 120 min post injection. Blocking studies were also conducted by co-injection of cold peptides. The conjugated showed the same profile in many evaluations. They were radiolabeled with high radiochemical purity (>97%). Both were hydrophilic, with preferential renal excretion. Tumor uptake was higher for human melanoma cells than for murinic melanoma cells, specially for 99mTc-MAG3-PEG8-c(RGDyK) (7.85±2.34 %ID/g) at 120 min post injection. The performance of 99mTc-MAG3-PEG8-c(RGDyk) was much better than NGR tracer concerning human melanoma uptake and might be considered in future investigations focusing radiotracers for melanoma diagnosis.

Diagnóstico precoce e tratamento de melanoma, um tumor cutâneo com o pior prognóstico, é extremamente importante para um resultado clínico favorável. A biblioteca de peptídeos phage display é um recurso útil de triagem para identificar peptídeos bioativos que interagem com alvos em cânceres. O objetivo desse estudo foi a avaliação de dois traçadores de tecnécio-99m com sequências peptídicas RGD e NGR, conjugados com o quelante bifuncional MAG3. Os conjugados peptídicos (10 μL de uma solução μg/μL) foram marcados com tecnécio-99m usando tampão de tartarato de sódio. A avaliação radioquímica foi feita por ITLC e confirmada por CLAE. O coeficiente de partição foi determinado e ensaios de internalização foram realizados em duas linhagens celulares de melanoma (B16F10 e SKMEL28). A avaliação da biodistribuição dos traçadores foi realizada em animais sadios em diferentes tempos e também em camundongos portadores de células tumorais aos 120 min após a sua administração. Estudos de bloqueamento também foram conduzidos pela co-injeção de peptídeo frio. O desempenho dos conjugados peptídicos mostraram-se bastante parecidos em diversas avaliações. Eles foram radiomarcados com alta pureza radioquímica (>97%). Ambos são hidrofílicos, com excreção renal preferencial. A captação tumoral foi maior para células SKMEL28 do que para as células B16F10, especialmente para o 99mTc-MAG3-PEG8-c(RGDyK) (7,85±2,34 %DI/g) aos 120 min pós-injeção. O desempenho do 99mTc-MAG3-PEG8-c(RGDyk) foi superior que o do traçador com NGR, quanto à captação no melanoma humano podendo ser considerado como um promissor radiofármaco para diagnóstico de melanoma. / Early diagnosis and treatment of melanoma, a cutaneous tumor with a serious prognosis, is extremely important for optimal clinical outcome. Phage display peptide libraries are a useful screening resource for identifying bioactive peptides that interact with cancer targets. The aim of this study was the evaluation of two technetium-99m tracers for angiogenesis detection in melanoma model, using cyclic peguilated pentapeptide with RGD and NGR motifs conjugated with bifunctional chelator MAG3. The conjugated peptides (10 μL of a μg/μL solution) were labeled with technetium-99m using a sodium tartrate buffer. Radiochemical evaluation was done by ITLC and confirmed by HPLC. Partition coefficient was determined and internalization assays were performed in two melanoma cells (B16F10 and SKMEL28). Biodistribution evaluation of the tracers was done in healthy animals at different times and also in mice bearing the tumor cells at 120 min post injection. Blocking studies were also conducted by co-injection of cold peptides. The conjugated showed the same profile in many evaluations. They were radiolabeled with high radiochemical purity (>97%). Both were hydrophilic, with preferential renal excretion. Tumor uptake was higher for human melanoma cells than for murinic melanoma cells, specially for 99mTc-MAG3-PEG8-c(RGDyK) (7.85±2.34 %ID/g) at 120 min post injection. The performance of 99mTc-MAG3-PEG8-c(RGDyk) was much better than NGR tracer concerning human melanoma uptake and might be considered in future investigations focusing radiotracers for melanoma diagnosis.

Diamond is a promising material with unique chemical properties. In this thesis, nano-scale diamond quantum size effects were investigated using several chemical property indicators. The results show that the chemical properties are strongly dependent on size for film thicknesses smaller than 1 nm (1D), and for nanodiamond particle diameters less than 2 nm (3D). When the sizes exceed these ranges there are no longer any quantum effects. The influence of surface termination coverage on the surface chemical properties has been calculated for the 2×1 reconstructed diamond (100) surface and for the diamond (111) surface. The terminating species included COOH and NH2 groups, which both are beneficial for the immobilization of biomolecules. The results of the calculations show that it is energetically possible to terminate the diamond surfaces up to 100% with NH2, while it is only possible to cover the surfaces up to 50% with COOH species. The reason for the latter result is most probably the larger sterical hindrance amongst the adsorbates. Both types of termination species were shown to influence the diamond surface electronic properties (e.g., HOMO/LUMO levels). In order to extend the diamond utility for biomedical applications, especially implant design, interactions of various growth factors with the diamond surfaces were also simulated. For non-solvent diamond-biomolecule systems, the results show that adhesion affinities are strongly dependent on biomolecule molecular weights. When including a water based solvent in the systems, the results show good physisorption affinities between proteins and diamond. Proteins structures, before and after physisorption, were visualized, and further investigated with respect to electrostatic properties and functional groups. By comparing the biomolecular structural changes during the adhesion processes, it can be concluded that both the general structures, as well as the binding pocket structures, were kept intact after the adhesion to the diamond surfaces (regardless of the adhesion affinities). In addition, the surface electronic potential distributions were maintained, which indicate preserved biomolecule functionalities. / Vascubone

No description available.

No description available.

Hintergrund: RGD-Peptide hemmen kompetitiv die Adhäsionsmoleküle von Linsenepithelzellen (LEC). Ziel unserer Untersuchungen war es herauszufinden, ob diese Peptide in der Lage sind, auch nach Kurzzeitinkubation eine suffiziente Inhibition der Adhäsion bzw. eine Ablösung adhärenter Zellen und damit eine ausreichende Prävention des Nachstars zu bewirken. Außerdem wurde überprüft, ob das von uns verwendete RGD-Peptid eine Toxizität für die Hornhaut aufweist. Material und Methoden: Kulturen boviner und humaner LEC, boviner Hornhautendothelzellen, humane und bovine Linsenkapselexzidate und humane explantierte Hornhäute wurden verwendet. Wir untersuchten die Inhibition der Adhäsion und die Ablösung konfluenter LEC-Layer mittels des zyklischen RGD-Peptids cRGDDFV (Inkubationszeiten von 1 Stunde bzw. 5-7 Tagen und Konzentrationen von 10-4 M, 10-3 M und 2x10-3 M wurden angewandt). Ergebnisse: Wir fanden nach nur einstündiger Inkubation in der Kulturschale eine Adhäsionsinhibition von 48% für bovine LEC und von 100% für humane LEC. Die Differenz zwischen Kontrollpeptid und cRGDDFV war statistisch signifikant (p / Purpose: RGD-peptides competitively inihibit adhesion molecules of the lens epithelial cells (LEC). The purpose of our study was to investigate whether this peptide could be able to inhibit adhesion sufficiently after short term incubation resp. to detach adherent cells and so to prevent posterior capsule opacification (PCO). Also there was proofed if there is any toxicity for the cornea. Methods: Cultures of bovine and human LEC, bovine cornea endothelial cells, humane and bovine fragments of the lens capsule and explanted humane corneas were used. The inhibition of adhesion and the detachment of confluent LEC-layer by the cyclic RGD-peptide cRGDDFV were studied (incubation time was 1 hour resp. 5-7 days and concentrations of 10-4, 10-3 M and 2x10-3 M were used). Results: After one hour incubation time in a culture dish inhibition of adhesion was 48% for bovine LEC resp. 100% for humane LEC. There was a statistically significant difference between the control-peptide-group and cRGDDFV (p

L’objectif de cette thèse est de développer l’imagerie de transfert de saturation des agents de contraste lipoCEST pour la détection de l’angiogenèse dans un modèle souris de tumeur cérébrale U87. Un lipoCEST offrant un seuil de sensibilité in vitro de 100 pM est optimisé afin de répondre aux contraintes de l’imagerie CEST in vivo. Grâce à la mise en place d’un dispositif expérimental dédié à l’imagerie CEST, nous évaluons les performances des lipoCEST pour détecter de façon spécifique l’angiogenèse tumorale. Nous montrons pour la première fois qu’il est possible de détecter un lipoCEST in vivo dans un cerveau de souris suite à une injection intraveineuse. De plus, l’utilisation d’un lipoCEST fonctionnalisé avec un peptide RGD permet de cibler spécifiquement l’intégrine ανβ3 surexprimée lors de l’angiogenèse tumorale. L’association spécifique du RGD-lipoCEST est confirmée grâce à des données d’immunohistochimie et de microscopie de fluorescence. Enfin, dans le but de tendre vers un protocole d’imagerie moléculaire par IRM-CEST, nous mettons en place un outil de quantification des lipoCEST. Cet outil repose sur la modélisation des processus d’échange de protons in vivo. Grâce à la prise en compte des inhomogénéités de champs B0 et B1 qui peuvent se révélées être délétères pour le contraste CEST, nous démontrons que la précision de notre outil de quantification est de 300 pM in vitro. La quantification des données CEST acquises chez la souris U87 permet d’estimer à 1,8 nM la concentration maximale en RGD-lipoCEST liés à leur cible moléculaire. / The study aimed at developing saturation transfer imaging of lipoCEST contrast agents for the detection of angiogenesis in a U87 mouse brain tumor model. A lipoCEST with a sensitivity threshold of 100 pM in vitro was optimized in order to make it compatible with CEST imaging in vivo. Thanks to the development of an experimental setup dedicated to CEST imaging, we evaluated lipoCEST to detect specifically tumor angiogenesis. We demonstrated for the first time that lipoCEST visualization was feasible in vivo in a mouse brain after intravenous injection. Moreover, the integrin ανβ3 overexpressed during tumor angiogenesis can be specifically targeted using a functionalized lipoCEST with RGD peptide. The specific association between the RGD-lipoCEST and its target ανβ3 was confirmed by immunohistochemical data and fluorescence microscopy. Finally, in order to tend to a molecular imaging protocol by CEST-MRI, we developed a quantification tool of lipoCEST contrast agents. This tool is based on modeling of proton exchange processes in vivo. By taking into account both B0 and B1 fields inhomogeneities which can dramatically alter CEST contrast, we showed that the accuracy of our quantification tool was 300 pM in vitro. The tool was applied on in vivo data acquired on the U87 mouse model and the maximum concentration of RGD-lipoCEST linked to their molecular targets was evaluated to 1.8 nM.

Herein we report the synthesis of an RGD-ruthenium bipyridine [Ru(Bpy)2(BpyRGD)]2+ complex aimed at the detection of angiogenesis. Angiogenesis plays a critical role in many pathophysiological processes, such as tumor growth. The αv-integrins (αv[beta]3, αv[beta]5) are currently used as molecular targeting sites for anti-angiogenic therapies. The [Ru(Bpy)2(BpyRGD)]2+ complex is an organometallic luminescent probe, which enables noninvasive, in vitro imaging of αv[beta]3 expression. Peptides containing the arginine-glycine-aspartic acid (RGD) sequence have been shown to bind strongly to the αvb3 integrin. The RuBpy probes are soluble in water, display long lifetimes, and are photochemically stable. These properties enable the Ru(tris-bpy) complexes to be useful in numerous applications in biophysical and cell biology. The [Ru(Bpy)2(BpyRGD)]2+ complex was synthesized by combining the succinimidyl ester on the RuBpy complex with the lysine of the c(RGDfK) peptide. The results of the one-photon fluorescence bioimaging showed selective binding of the cyclic RGD to αv[beta]3 integrin, which supports previous literature. The high luminescence intensity, long lifetimes, and low cell toxicity levels of dye [Ru(Bpy)2(BpyRGD)]2+, illustrates the potential usage of this probe for future biological applications.

Calcific aortic valve disease (CAVD) is one of the most common causes of cardiovascular disease in North America. Mechanical factors have been closely linked to the pathogenesis of CAVD and may contribute to the disease by actively regulating the mechanobiology of valve interstitial cells (VICs). Mechanical forces affect VIC function through interactions between the VIC and the extracellular matrix (ECM). Studies have shown that the transfer of mechanical stimulus during cell-ECM interaction depends on the local material properties at hierarchical length scales encompassing tissue, cell and cytoskeleton. In this thesis, biomechanical tools were developed and applied to investigate hierarchical cell-ECM interactions, using VICs and valve tissue as a model system. Four topics of critical importance to understanding VIC-ECM interactions were studied: focal biomechanical material properties of aortic valve tissue; viscoelastic properties of VICs; transduction of mechanical deformation from the ECM to the cytoskeletal network; and the impact of altered cell-ECM interactions on VIC survival. To measure focal valve tissue properties, a micropipette aspiration (MA) method was implemented and validated. It was found that nonlinear elastic properties of the top layer of a multilayered biomaterial can be estimated by MA by using a pipette with a diameter smaller than the top layer thickness. Using this approach, it was shown that the effective stiffness of the fibrosa layer is greater than that of the ventricularis layer in intact aortic valve leaflets (p<0.01). To characterize the viscoelastic properties of VICs, an inverse FE method of single cell MA was developed and compared with the analytical half-space model. It was found that inherent differences in the half-space and FE models of single cell MA yield different cell viscoelastic material parameters. However, under particular experimental conditions, the parameters estimated by the half-space model are statistically indistinguishable from those predicted by the FE model. To study strain transduction from the ECM to cytoskeleton, an improved texture correlation algorithm and a uniaxial tension release device were developed. It was found that substrate strain fully transfers to the cytoskeletal network via focal adhesions in live VICs under large strain tension release. To study the effects of cell-ECM interactions on VIC survival, two mechanical stimulus systems that can simulate the separate effects of cell contraction and cell monolayer detachment were developed. It was found that cell sheet detachment and disrupted cell-ECM signaling is likely responsible for the apoptosis of VICs grown in culture on thin collagen matrices, leading to calcification. The studies presented in this thesis refine existing biomechanical tools and provide new experimental and analytical tools with which to study cell-ECM interactions. Their application resulted in an improved understanding of hierarchical valve biomechanics, mechanotransduction, and mechanobiology.