141 |
Quality control of PET camera for small animal imaging / Έλεγχος ποιότητας κάμερας PET για απεικόνιση μικρών ζώωνΕυθυμίου, Νικόλαος 06 February 2009 (has links)
- / The term Molecular Imaging (MI) can be broadly defined as the in vivo characterization and
measurement of biological processes at the cellular and molecular level. In contradistinction to
“classical” diagnostic imaging, it sets forth to probe the molecular abnormalities that are the basis of
disease rather than to image the end effects of these molecular alterations. The underlying biology
represents a new arena for many researchers. A number of technological challenges such as signal
amplification, data and image processing and efficient imaging strategies, provide a fast growing
scientific domain. Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI), Single
Photon Emission Tomography (SPECT) and optical imaging are the main tools of clinical molecular
imaging. Nuclear medicine techniques (SPECT and PET) are key players in MI. New
radiopharmaceutical products are currently being developed, in order to increase the specificity and
sensitivity of existing imaging techniques. Small animal imaging is the main tool for the evaluation of
those derivatives, especially in dynamic in vivo studies. In this, preclinical part, high resolution and
high sensitivity imaging equipment is necessary; as a result a number of such prototypes have been
developed worldwide and some of them are commercialized. However, there cost is usually not
affordable for small or medium size laboratories.
In this work a low cost dual head PET camera, suitable for high resolution small animal studies
has been developed. It is the result of the collaboration between Jefferson lab and Technological
Educational Institute of Athens (TEI) and is currently evaluated in Institute of Radioisotopes and
Radiodiagnostic Products (IRRP), in “Demokritos” Center.
The system has a field of view of 5x5cm and is based on 2 H8500 position sensitive
photomultiplier tubes (PSPMTs), coupled to two LSO crystals with 2.5x2.5mm pixel size. Then an
FPGA based data acquisition system and proper data reconstruction system collect events, sort
coincidences and produce images. The DAQ board consists of 16-channel DAQ modules installed on a
USB2 carrier. Each channel is an independent acquisition system consisting of traditional analog pulse
processing, FPGA analog control, and FPGA signal processing. After acquisition and processing,
channel data are assembled into event blocks for readout by the carrier board. Application specific tasks
are performed in JAVA using the Kmax interface. The GUI was designed to be easy-to-use. In order to
further analyze images and process the results proper algorithms were developed in MATLAB and
ImageJ. Systems evaluation has been carried out using FDG. Point sources have been used for systems
calibration. Capillaries with 1.1mm inner diameter were imaged and used for resolution calculation.
Finally a mouse injected with 100μCi of FDG was imaged. Spatial resolution has been measured using
thin capillaries (1.1mm inner diameter) and found equal to 3,5mm in planar mode. This lower limit is
determined by LSO pixels size (2.5×25mm2). Simulation studies have shown that resolution lower than
2mm will be achieved in tomographic mode. Mice injected with FDG are presented. Brain and heart
are clearly imaged.
Currently, a rotating base is constructed, in order to upgrade the system to a tomographic PET.
PET results will be presented as well. In addition, IRRP and TEI are working on new
radiopharmaceuticals based on Cu-64. It must be stated that PET market opened in Greece four years
ago; This system is the first working small PET prototype in Greece and it initiates national preclinical
PET research.
|
142 |
Radiotracer für die molekulare Bildgebung: Radiomarkierung von Inhibitoren der CDK4/6 mit den Radionukliden Iod-124 und Fluor-18Köhler, Lena 25 June 2010 (has links) (PDF)
Krebserkrankungen stellen in Deutschland die zweithäufigste Todesursache dar und die Anzahl der Neuerkrankungen nimmt stetig zu. Frühzeitige Diagnosen und Therapiemöglichkeiten sind daher dringend erforderlich.
Cyklinabhängige Proteinkinasen (Cdk) spielen eine entscheidende Rolle bei der Regulation des Zellzyklus. Viele Tumore zeigen eine deregulierte Cdk4‑Aktivität und/oder ‑Expression. Insgesamt zeigen ca. 80% aller Tumore eine Fehlregulation der für den Zellzyklus zentralen Cdk4/CykD1/INK4/pRb/E2F Signalkaskade. Somit besitzen Cdks ein enormes therapeutisches Potential im Kampf gegen Krebs. Die spezifische Inhibierung der Cdks verhindert die Zellproliferation und damit das Tumorwachstum. In den letzten Jahren wurden verschiedenste Strukturklassen vorgestellt, die als Cdk4-Inhibitor wirken.
Im Rahmen der Promotion sollen die Möglichkeiten einer funktionellen Tumordiagnose mittels cyklinabhängiger Kinasen untersucht werden. Die Entwicklung von radioaktiv markierten Inhibitoren der Cdk4/6 als Radiotracer und ihre radiopharmakologische Charakterisierung stellt dabei einen neuen Ansatz dar. Um die Rolle der Cdk4/6 im Zellzyklus von gesunden und deregulierten (z.B. Tumor-) Zellen aufzuklären, sollten mit Iod-124 und Fluor-18 markierte Inhibitoren eingesetzt werden, die hochselektiv diese Cdks blockieren.
Zunächst wurden verschiedene Inhibitoren der Cdk4/6 und deren Vorstufen für die Radiomarkierung dargestellt. Die bereits aus den Vorarbeiten von VanderWel et al., 2005 und Toogood et al., 2001 bekannten Syntheserouten mussten dazu optimiert werden und für neue Verbindungen, wie die fluorethylierten Substanzen, wurden neue Reaktionswege gefunden. Die dargestellten Referenzverbindungen CKIA-E wurden anschließend mittels Durchflusszytometrie an den Zelllinien HT-29 und FaDu auf ihre inhibitorischen Wirkung untersucht. Die Untersuchungen der Verbindungen CKIA/B/E zeigte, dass ein Zellzyklusarrest unter Einwirkung der Inhibitoren erreichbar ist. Die weiteren Untersuchungen zur Radiomarkierbarkeit sowie die radiopharmakologische Evaluation sollten daher an den Verbindungen CKIA, CKIB und CKIE stattfinden.
Die Darstellung der Verbindungen [124I]CKIA und [124I]CKIB erfolgte in zwei Schritten über die elektrophile Substitution durch regioselektive Destannylierung mit anschließender Entschützung der Seitenkette. Die Darstellung der fluorethylierten Verbindung erfolgte ebenfalls über eine Zweischrittsynthese beginnend mit der Synthese der prosthetischen Gruppe [18F]BFE aus der Tosylmarkierungsvorstufe. Die zur Markierung des sekundären Amins zur Auswahl stehenden prosthetischen Gruppen [18F]Fluorethyltosylat ([18F]FETos) und [18F]Bromfluorethan ([18F]BFE) wurden auf ihre Eignung untersucht, ebenso wie die Auswahl einer geeigneten Markierungsvorstufe für die Darstellung der prosthetischen Gruppe.
Die optimierten Syntheserouten ermöglichten die Isolierung von ausreichenden Mengen an Produktaktivität für die radiopharmakologischen Untersuchungen. Es fanden, neben der Bestimmung der spezifischen Aktivität und der Lipophilie der Verbindungen, Zellaufnahmeuntersuchungen und Bestimmungen zur Stabilität der Verbindungen in vitro, ex vivo und in vivo statt. Die radioiodierten Verbindungen konnten des Weiteren zur Untersuchungen der Bioverteilung in normalen männlichen Wistar-Ratten eingesetzt werden.
Für alle drei Verbindungen konnte eine sehr hohe in vitro-Stabilität festgestellt werden. Die Zellaufnahmeuntersuchungen zeigten vor allem für die Verbindungen [124I]CKIA und [124I]CKIB eine beträchtliche Zellaufnahme von über 1000% ID/mg Protein nach 2 h. Die Zellaufnahme der Verbindung CKIE ist geringer, sollte allerdings für eine in vivo-Anwendung ausreichend sein. Die Untersuchung der in vivo‑Stabilität der Verbindungen [124I]CKIA, [124I]CKIB und [18F]CKIE im Blut von Wistar Ratten ergab allerdings, dass alle Verbindungen schnell metabolisiert werden. Die Untersuchung der Bioverteilung der radioiodierten Verbindungen belegen eine in vivo Radiodeiodierung sowie eine hohe hepatobliliäre Auscheidungsrate.
Im Hinblick auf eine Anwendung als Radiotracer konnten im Rahmen dieser Arbeit neue Erkenntnisse gewonnen werden. Die dargestellten Inhibitoren sind in der Lage am Zellmodell den Zellzyklusarrest in der G1-Phase zu induzieren. Eine Radiomarkierung der ausgewählten Strukturen liefert das Produkt mit reproduzierbarer Ausbeute in hoher radiochemischer Reinheit und ausreichender spezifischer Aktivität, allerdings ist eine Herstellung der fluorethylierten Verbindung unter GMP-Bedingungen nur schwer realisierbar. Die radiomarkierten Verbindungen zeigen eine hohe in vitro-Stabilität und werden energieabhängig in die Zelle aufgenommen. Anhand der Stabilitätsuntersuchungen in vivo wurde gezeigt, dass alle drei Verbindungen in vivo instabil sind und sehr schnell hepatobiliär eliminiert.
|
143 |
Noninvasive assessment and quantification of tumour vascularisation using MRI and CT in a tumour model with modifiable angiogenesis – An animal experimental prospective cohort studyMirus, Matthew M., Tokalov, Sergey V., Wolf, Gerald, Heinold, Jerilyn, Prochnow, V., Abolmaali, Nasreddin 06 June 2018 (has links) (PDF)
Background
To investigate vascular-related pathophysiological characteristics of two human lung cancers with modifiable vascularisation using MRI and CT.
Methods
Tumour xenografts with modifiable vascularisation were established in 71 rats (approval by the Animal Care Committee was obtained) by subcutaneous transplantation of two human non-small-cell lung cancer (NSCLC) cells (A549, H1299) either alone or co-transplanted with vascular growth promoters. The vascularity of the tumours was assessed noninvasively by MRI diffusion-weighted-imaging (DWI), T2-weighted, and time-of-flight (TOF) sequences) as well as contrast-enhanced CT (CE-CT), using clinical scanners. As a reference standard, histological examinations (CD-31, fluorescent beads) were done after explantation.
Results
Microvessel density (MVD) was higher in co-transplanted tumours (171 ± 19 number/mm2) than in non-co-transplanted tumours (111 ± 11 number/mm2; p = 0.002). Co-transplanted tumours showed higher growth rates and larger tumour vessels at TOF-MRI as well as larger necrotic areas at CE-CT. In co-transplanted tumours, DWI revealed higher cellularity (lower minimal ADCdiff 166 ± 15 versus 346 ± 27 mm2/s × 10−6; p < 0.001), highly necrotic areas (higher maximal ADCdiff 1695 ± 65 versus 1320 ± 59 mm2/s × 10−6; p < 0.001), and better-perfused tumour stroma (higher ADCperf 723 ± 36 versus 636 ± 51 mm2/s × 10−6; p = 0.005). Significant correlations were found using qualitative and quantitative parameters: maximal ADCperf and MVD (r = 0.326); maximal ADCdiff and relative necrotic volume on CE-CT (r = 0.551); minimal ADCdiff and MVD (r = −0.395).
Conclusions
Pathophysiological differences related to vascular supply in two human lung cancer cell lines with modifiable vascularity are quantifiable with clinical imaging techniques. Imaging parameters of vascularisation correlated with the results of histology. DWI was able to characterise both the extent of necrosis and the level of perfusion.
|
144 |
O uso de microRNA para tratamento do câncer de próstata: estudos in vitro e in vivo / The use of microRNA for the treatment of prostate cancer: in vitro and in vivo studiesAlexandre Iscaife 10 June 2016 (has links)
Introdução: O câncer de próstata (CaP) é o tumor mais comum do homem nos países ocidentais e a segunda causa de óbito por câncer em homens nos EUA, Europa e Brasil. O câncer localizado tem sobrevida câncer especifica elevada quando tratado adequadamente, porém a doença metastática ainda apresenta tratamentos pouco eficientes com sobrevida global de 28%. Os microRNAs (miRNAs) são um grupo de moléculas pequenas de RNA que contém entre 19 a 25 nucleotídeos não codificantes de proteína, com ação fundamental na regulação da expressão gênica. Eles estão envolvidos em processos essenciais nas células normais e neoplásicas como ciclo celular, proliferação, apoptose, metabolismo energético, invasão e metastatização. Objetivos: Realizar estudos in vitro e in vivo usando miRNA em um modelo de câncer de próstata metastático inédito no nosso meio com intuito de analisar o seu potencial como agente terapêutico dessa neoplasia. Métodos: Nos estudos in vitro, três linhagens celulares foram utilizadas (PC3, DU145 e LNCaP). Essas linhagens foram transfectadas com os miRNAs 100, 145 e 373 e seus respectivos antiMiRs utilizando-se lipofectamina. Analisamos a expressão dos genes alvo mTOR, SMARCA5, KRAS, CMYC, MMP9, CD44 por PCR quantitativo em tempo real (qRT-PCR). Foram realizados também estudos de apoptose, ciclo celular e ploidia utilizando o citômetro de fluxo. Alterações no potencial de invasão foram avaliadas pela técnica do matrigel. O modelo in vivo pré-clínico foi desenvolvido pela injeção intra-cardíaca da linhagem PC-3M-Luc-C6 em camundongos NUDE com 9 semanas. O crescimento tumoral foi avaliado com o sistema de bioluminescência in vivo. Após o pleno estabelecimento das metástases no dia 21, os animais foram tratados com três injeções na veia da cauda contendo o miRNA conjugado com o atelocolágeno. Os animais foram sacrificados e no dia 48 para análise dos tecidos. Resultados: miR-100 aumenta a apoptose na LNCaP, e reduz a apoptose na DU145. Na linhagem DU145 o miR 100 inibiu a proliferação. Na análise da expressão gênica o miR-100 inibe SMARCA5 na DU145 e PC3 e mTOR na LNCaP, o anti-miR 100 estimula mTOR e SMARCA5 na LNCaP. O miR-145 promoveu aumento da apoptose em 24% na DU145. Na linhagem PC3 o miR-145 age inibindo a proliferação, com uma diferença absoluta de 18% em relação ao seu controle. O miR-145 inibe KRAS e CMYC nas três linhagens e o anti-miR-145 estimula CMYC na DU145 e RAS nas três linhagens O miR-373 reduziu a apoptose em 29% na DU145 e diminui a proliferação com uma diferença absoluta de 13% em relação ao controle. O miR- 373 estimula a MMP9 na DU145 e na LNCaP e inibe o CD44 na PC3. O antimiR- 373 inibe MMP9 na DU145 e LNCaP. Nos estudos in vivo de CaP metastático o miR-100 apresenta tendência a redução no crescimento tumoral (p=0,23) e o miR-145 reduz o crescimento de forma significativa no dia 34 (p=0,02). Após esses dias o tumor volta a crescer de forma agressiva. Os animais tratados com anti-miR-373 não apresentaram alterações em relação aos controles. Conclusões: O miR-100 é um miRNA contexto dependente, com papel supressor tumoral em linhagens de tumor de próstata agressivo e o miR- 373 age in vitro como oncomiR. O miR-145 age como supressor tumoral in vitro e em modelo animal de CaP metastático apresentando resposta terapêutica consistente, podendo ser utilizado no arsenal terapêutico contra essa neoplasia. Estudos futuros devem avaliar o uso dos miRNAs isoladamente ou de forma adjuvante no tratamento do CaP metastático / Introduction: Prostate cancer (PCa) is the most common neoplasia of man in Western countries and the second cause of death by cancer in men in the US, Europe and Brazil. The localized cancer has high cancer-specific survival when treated properly, however metastatic disease still presents low effective treatments with 28% of global survival. microRNAs (miRNAs) are a group of small RNA molecules containing from 19 to 25 nucleotides of noncoding protein with fundamental action in the regulation of gene expression. They are involved in key processes in normal and neoplastic cells as cell cycle, proliferation, apoptosis, energy metabolism, invasion and metastasis. Objectives: To carry out studies in vitro and in vivo using miRNA in a novel model of metastatic prostate cancer in our country in order to evaluate its potential as a therapeutic agent of this neoplasia. Methods: In the in vitro studies, three cell lines were used (PC3, DU145 and LNCaP). These cell lines were transfected with miRNAs 100, 145 and 373 and their antiMiRs using lipofectamine. We analyzed the gene expression of mTOR, SMARCA5, KRAS, CMYC, MMP9, CD44 by real-time polymerase chain reaction (qRT-PCR). We also performed studies of apoptosis, cell cycle and ploidy using flow cytometer. Changes in the invasion potential were evaluated by the technique of matrigel. The pre-clinical model in vivo was developed by intracardiac injection of PC-3MLuc-C6 cell line in NUDE mice with 9 weeks. Tumor growth was evaluated with an in vivo image system (IVIS). After the full establishment of metastases on day 21, the animals were treated with three injections into the tail vein containing the miRNA plus atelocollagen. The animals were sacrificed on day 48 for tissues analysis. Results: MiR-100 increases apoptosis in LNCaP and reduces apoptosis in DU145. The anti-miR-100 increased apoptosis in 14% in PC3. In cell line DU145, miR-100 inhibited proliferation. In the analysis of gene expression, the miR-100 inhibits SMARCA5 in DU145 and PC3 and mTOR in LNCaP, anti-miR-100 stimulates mTOR and SMARCA5 in LNCaP. The miR-145 promoted an increased in apoptosis by 24% in DU145. In PC3 cell line miR-145 acts by inhibiting the proliferation, with an absolute difference of 18% compared to control. MiR-145 inhibits KRAS and CMYC in the three cell lines and anti-miR-145 stimulates CMYC in DU145 and KRAS in the three cell lines. The miR-373 reduced apoptosis by 29% in DU145 and reduces proliferation with an absolute difference of 13% relative to control. MiR-373 stimulates MMP9 in DU145 and LNCaP cells and inhibits CD44 in PC3. The anti -miR-373 inhibits MMP9 in DU145 and LNCaP. In the in vivo studies of metastatic PCa, miR-100 shows a tendency to decrease tumor growth (p=0.23) and miR-145 reduces tumor growth on day 34 (p=0.02). After those days, the tumor grows back aggressively. Animals treated with anti-miR-373 showed no changes relative to controls. Conclusion: The miR-100 is a context-dependent miRNA, with tumor suppressor role in aggressive tumor cell lines. The miR-373 acts in vitro as oncomiR and miR-145 acts as a tumor suppressor in vitro and in an animal model with consistent therapeutic response and can be used in the therapeutic arsenal against this neoplasia. Future studies should evaluate the use of miRNAs alone or adjuvant in the treatment of metastatic prostate cancer
|
145 |
Aide au ciblage du microenvironnement tumoral par le développement d’un nano-système de détection et de traitement des tumeurs avec inhibition ciblée de l’héparanase / Tumor microenvironment targeting by the development of a nano-system for the detection and treatment of tumors by targeted inhibition of heparanaseAchour, Oussama 07 July 2014 (has links)
Le microenvironnement des cellules tumorales présente plusieurs particularités comme l'hypoxie, l'acidification du milieu extracellulaire et l'hypersécrétion d'enzymes hydrolytiques. Ces hydrolases, comme la cathepsine D et l'héparanase, interviennent dans les étapes de la progression tumorale et notamment l'angiogenèse. Cette thèse s'intègre dans un projet dont la finalité est de concevoir un nano-objet moléculaire enzymo-sensible qui réagirait d'une manière spécifique aux enzymes hypersécrétées dans le microenvironnement tumoral pour assurer de façon simultanée, une fonction de détection et de ciblage des tumeurs. La première partie de nos travaux a été consacrée à la conception et à la validation d'un lien peptidique intégrable dans l'objet moléculaire, sensible aux formes de la cathepsine D actives du microenvironnement tumoral de cancers mammaires. Cet objectif a été réalisé suite à l'étude cinétique de l'hydrolyse de 5 peptides par la cathepsine D mature et la pro-cathepsine D dans les conditions de pH du microenvironnement tumoral. Nous avons également étudié l'effet de l'hypoxie et de l'acidification du milieu extracellulaire sur la sécrétion des formes actives de la cathepsine D par la lignée tumorale de cancer mammaire MCF-7. Dans une deuxième partie, nous avons travaillé sur l'élaboration d'héparines de bas poids moléculaire pouvant assurer la fonction thérapeutique de l'objet moléculaire grâce à leur activité anti-angiogénique. Nous avons mis au point une méthode innovante pour la dépolymérisation de l'héparine qui consiste en une hydrolyse radicalaire par le péroxyde d'hydrogène assistée par les ultrasons. Cette technique permet la production d'oligosaccharides d'héparines caractérisées par des poids moléculaires et des degrés de sulfatation contrôlés. En fonction des conditions de dépolymérisation par cette technique, les héparines de bas poids moléculaires produites peuvent être utilisées comme anticoagulant ou anti-angiogénique. / Tumor microenvironment is characterized by several particularities such as hypoxia, extracellular media acidification and the hyper-secretion of hydrolytic enzymes. These hydrolases, such as cathepsin D and heparanase, are involved in many steps of tumor progression like angiogenesis. This thesis is a part of a project that aims to develop a "smart" molecular nano-object that specifically reacts to hyper-secreted enzymes in the tumor microenvironment for the simultaneous detection and targeting of tumor. The first part of our work concerned the design and the validation of a peptide that is sensitive to active forms of cathepsin D which is a protease, unregulated in many tumors microenvironment such as breast cancers. This objective has been achieved following the kinetic study of the hydrolysis of 5 peptides by mature cathepsin D and procathepsin D in the pH conditions of the tumor microenvironment. On the other hand, we studied the effect of hypoxia and the acidification of the extracellular medium on the secretion of active forms of cathepsin D by the breast cancer cell line MCF-7. In a second part, we worked on the development of low molecular weight heparins that may provide therapeutic function of the molecular object through their anti-angiogenic activity. We have developed an innovative method for the depolymerization of heparin that consists on a radical hydrogen peroxide hydrolysis assisted by ultrasound. This technique allows the production of heparins oligosaccharides characterized by controlled molecular weight and degree of sulfatation. Depending on the depolymerization conditions by this technique, the produced low molecular weight heparins can be used as an anticoagulant or anti-angiogenic.
|
146 |
Synthèse d'agents chélatants bifonctionnels macrocycliques pour le marquage de molécules biologiques par des métaux : application en imagerie médicale / Synthesis of bifunctional chelating agents based on macrocyclic polyanines for medical imaging applicationsBernhard, Claire 27 May 2011 (has links)
L’imagerie moléculaire est devenue incontournable pour le diagnostic et le traitement de cancers. Cette discipline regroupe un ensemble de techniques telles que la tomodensitométrie (CT), l’Imagerie par Résonance Magnétique (IRM), l’imagerie optique ou encore l’imagerie nucléaire (tomographie par émission de positons TEP, tomographie d’émission monophotonique TEMP). Chacune de ces techniques possède ses propres avantages et inconvénients et ne peut apporter à elle seule des informations anatomiques et fonctionnelles suffisantes. Les travaux actuels sont portés sur la conception de systèmes dits multimodaux afin de combiner les avantages de différentes techniques, voire de bénéficier d’un effet synergique. De par leur sensibilité comparable et leur complémentarité, coupler l’imagerie nucléaire à l’imagerie optique devient alors avantageux. La conception des systèmes monomoléculaires (MOMIA) contenant deux fonctions détectables par imagerie nucléaire (complexe de radiométaux) et imagerie optique (sonde fluorescente) nécessite en amont la mise au point d’outils de synthèses performants. La première partie de ce travail de thèse est consacrée à la synthèse d’agents chélatants bifonctionnels à base de polyamines macrocycliques, destinés à une utilisation en imagerie médicale. Ces agents doivent présenter d’excellentes propriétés de coordination vis-à-vis du métal visé, et posséder une fonction de greffage pour assurer le couplage avec une biomolécule vectrice. L’accès à de tels systèmes a nécessité le développement d’outils de synthèse efficaces de précurseurs macrocycliques dérivés du cyclène et du 13aneN4. L’introduction sélective de diverses fonctions de greffage visant principalement les résidus de type lysine a permis la préparation de plusieurs familles de composés, dont certains ont pu être « bioconjugués» à des peptides ou anticorps au sein du laboratoire ou dans le cadre de diverses collaborations. Plus particulièrement, la facilité d’utilisation du système « DOTAGA anhydride » a permis l’introduction aisée d’unités DOTA sur des nanoparticules ou des anticorps monoclonaux. Egalement, l’introduction d’une fonction alcyne a permis l’accès à de nouvelles briques moléculaires préparées par « click chemistry ». Dans une seconde partie sont présentés les travaux relatifs à la synthèse d’agents bimodaux originaux. Pour accéder à de tels systèmes, l’introduction d’un fluorophore de la famille des bodipys a été envisagée. L’absence de travaux antérieurs relatifs au couplage d’une polyamine cyclique et une entité bodipy a nécessité la préparation préalable d’un système modèle « DOTA bodipy », permettant de s’assurer par des études photophysiques que la présence des complexes métalliques macrocycliques ne va pas, ou peu, interférer avec les propriétés de fluorescence du bodipy. L’utilisation d’un espaceur « acide aminé » a alors permis d’accéder à de nouveaux bodipys porteurs de deux groupes fonctionnels en position méso. La fonctionnalisation a posteriori de ces briques de construction a permis l’introduction en dernier lieu d’unités macrocycliques N- et/ou C- fonctionnalisés. La préparation de système émettant dans le proche I.R. a été également envisagée. / Molecular imaging became a major tool for the diagnosis and the treatment of cancers. This research field includes different techniques, such as Tomography (CT), Magnetic Resonance Imaging (MRI), Optical Imaging or nuclear Imaging (PET Positron Emission Tomography, SPECT Single Photon Emission Computed Tomography). Each imaging modality has its own strengths and weaknesses, and thus, combining different and complementary systems can overcome inherent limitations associated with any one individual techniques and improve the accuracy of disease diagnosis and enhancing patient management. In particular dual-modality Optical/Nuclear imaging may find important preclinical and clinical applications. One possible approach seeks to fuse the two imaging systems into one molecule (MonOmolecular Multimodality Imaging Agent [MOMIA]) in order to ensure the same biodistribution of the two probes. Our strategy consists in combining a DOTA-like compound allowing complexation of radiometal for nuclear imaging (SPECT or PET) with a bodipy moiety, valuable probe those fluorescent properties can be finely adjusted. The first part of this work is dedicated to the synthesis of bifunctional chelating agents based on macrocyclic polyamines for medical imaging application. These compounds must show excellent coordination properties towards the aimed radiometal and possess a grafting function to allow the coupling with a biomolecule. Powerful and general routes for the synthesis of a wide range of N- and C-functionalized macrocycles derived from cyclen and 13aneN4 are described, which enable to access to a wide range of new BFCs by introduction of different functional groups reactive towards primary amines, such as carboxylic acid, isothiocyanate or anhydride function. Some compounds were conjugated to different biomolecules, such as peptides or antibodies. Morever, the introduction of an alkyne function yields a novel family of bifunctional agents allowing chemoselective attachment to functionalized biomolecules or to modified amino acids using « click chemistry ». In a second part, we focused on the introduction of a bodipy moeity to obtain new bimodal agents for dual Optical/Nuclear imaging. Interestingly, the attachment of the polyaminocarboxylate (DOTA derivative) to the bodipy makes it soluble in water and complexation of different metal cations of interest in the macrocyclic cavity does not significantly alter the luminescence properties of the whole system. In addition, the functionalization of the meso position by using an appropriate linker between the bodipy and DOTA-like units, i.e. a 4-nitrophenylalanine derivative, could provide a new bimodal tag for labeling antibodies or peptides. Optimisation of the second generation bodipy-DOTA, i.e. derivatization reaction to reach the near-IR range or introduction of C-functionalised macrocycles was also investigated.
|
147 |
Development of Overhauser-enhanced magnetic resonance imaging in vivo : application to molecular imaging of proteolysis. / Développement de l'imagerie par résonance magnétique rehaussée par l'effet Overhauser in vivo : application à l'imagerie moléculaire de la protéolyse.Koonjoo, Neha 08 October 2015 (has links)
Ce travail fait l’objet d’une avancée scientifique dans le développement de la technique d’IRM rehaussée par l’effet Overhauser dans la souris à 0,2 T. Cette dernière repose sur le transfert de polarisation des spins électroniques saturés d’un radical libre vers les spins des protons (généralement de l’eau) voisins pour rehausser le signal RMN du proton. Notre équipe a développé cette technique pour détecter une activité protéolytique au travers de deux stratégies. La première partie de la thèse a été de détecter pour la première fois une activité protéolytique in situ dans des souris saines et in vitro sur cellules vivantes. L’efficacité du rehaussement par effet Overhauser repose sur le temps de corrélation des spins des électrons non-appariés. Un radical nitroxyde greffé à l’élastine a été utilisé comme substrat. La protéolyse de ce dernier par des élastases pancréatiques a conduit l’observation en 3D d’un rehaussement du signal RMN de plus de 10 fois dans le tube digestif de souris vivantes. De plus, des développements méthodologiques, tels que l’implémentation de la séquence TrueFISP, le sous-échantillonnage par la méthode “Keyhole”, et la reconstruction des données en 3D ont été faits. La deuxième stratégie repose sur des molécules de nitroxyde ayant l’unique propriété de pouvoir décaler leurs pics de résonance après hydrolyse. Un nitroxyde phosphorylé en position Béta pouvant être détecté à deux fréquences spécifiques différentes avant et après hydrolyse d’un groupement chimique a été synthétisé par des chimistes à Marseille. L’hydrolyse de cette macromolécule a été observée in vivo dans l’estomac de souris saines avec des rehaussements de plus de 400% et imagée en 3D avec une bonne résolution spatio-temporelle. Ainsi, une prochaine étape serait de poursuivre ce travail sur un modèle pathologique et développer cette technique à un champ magnétique plus bas. / This work relates the continuity and advances in the implementation of the Overhauser-enhanced Magnetic Resonance Imaging technique on a 0.2 T scanner. Briefly, OMRI technique is based on polarization transfer of saturated electronic spins from free nitroxide radicals to proton spins of surrounding water molecules in the aim to drastically enhance proton NMR signal. To this technique, our research team has merged specific strategies for proteolytic activity detection. The first strategy relies on a 3D visualization of proteolytic activity happening in intact living cells or in vivo in healthy mice. With an Overhauser switch based upon changes in molecular tumbling time, high Overhauser enhancements of 10-fold were observed in the intestinal tract of mice after that elastolytic activity of our probe: the nitroxide-labeled elastin macromolecule took place. In addition, MRI developments - TrueFISP sequence implementation, undersampling Keyhole method and data reconstruction were carried out for imaging these rapid biological processes. A second exquisite strategy is also described using nitroxides with shifting resonant peaks. Here, a Beta-phosphorylated nitroxide molecule was specifically detected at two distinct frequencies: one for its substrate and the other for its product once hydrolysis took place. This hydrolysis was imaged in 3D in the stomach of living mice with Overhauser enhancements of more than 400% and with a good spatiotemporal resolution. The perspectives of this work lie on a future detection of a pathological proteolytic activity in vivo and eventually and development of very low magnetic field OMRI.
|
148 |
Noninvasive assessment and quantification of tumour vascularisation using MRI and CT in a tumour model with modifiable angiogenesis – An animal experimental prospective cohort studyMirus, Matthew M., Tokalov, Sergey V., Wolf, Gerald, Heinold, Jerilyn, Prochnow, V., Abolmaali, Nasreddin 06 June 2018 (has links)
Background
To investigate vascular-related pathophysiological characteristics of two human lung cancers with modifiable vascularisation using MRI and CT.
Methods
Tumour xenografts with modifiable vascularisation were established in 71 rats (approval by the Animal Care Committee was obtained) by subcutaneous transplantation of two human non-small-cell lung cancer (NSCLC) cells (A549, H1299) either alone or co-transplanted with vascular growth promoters. The vascularity of the tumours was assessed noninvasively by MRI diffusion-weighted-imaging (DWI), T2-weighted, and time-of-flight (TOF) sequences) as well as contrast-enhanced CT (CE-CT), using clinical scanners. As a reference standard, histological examinations (CD-31, fluorescent beads) were done after explantation.
Results
Microvessel density (MVD) was higher in co-transplanted tumours (171 ± 19 number/mm2) than in non-co-transplanted tumours (111 ± 11 number/mm2; p = 0.002). Co-transplanted tumours showed higher growth rates and larger tumour vessels at TOF-MRI as well as larger necrotic areas at CE-CT. In co-transplanted tumours, DWI revealed higher cellularity (lower minimal ADCdiff 166 ± 15 versus 346 ± 27 mm2/s × 10−6; p < 0.001), highly necrotic areas (higher maximal ADCdiff 1695 ± 65 versus 1320 ± 59 mm2/s × 10−6; p < 0.001), and better-perfused tumour stroma (higher ADCperf 723 ± 36 versus 636 ± 51 mm2/s × 10−6; p = 0.005). Significant correlations were found using qualitative and quantitative parameters: maximal ADCperf and MVD (r = 0.326); maximal ADCdiff and relative necrotic volume on CE-CT (r = 0.551); minimal ADCdiff and MVD (r = −0.395).
Conclusions
Pathophysiological differences related to vascular supply in two human lung cancer cell lines with modifiable vascularity are quantifiable with clinical imaging techniques. Imaging parameters of vascularisation correlated with the results of histology. DWI was able to characterise both the extent of necrosis and the level of perfusion.
|
149 |
Synthèse de sondes chémiluminescentes et profluorescentes pour des applications en imagerie in vivo / Synthesis of chemiluminescent and profluorogenic probes for in vivo imagingGrandclaude, Virgile 23 September 2011 (has links)
L’imagerie moléculaire optique joue maintenant un rôle essentiel dans le diagnostic pré-clinique et le développement de médicaments. En effet, c’est un outil précieux dans la détection et le suivi de cellules vivantes que ce soit en utilisant de simples agents de marquage ou des sondes plus développées, dites « intelligentes » et activées uniquement par une interaction spécifique avec le bio-analyte ciblé. Ce travail de thèse a consisté à développer des outils synthétiques innovants afin d’optimiser les paramètres physico-chimiques et les propriétés optiques des sondes luminescentes. Ceci dans le but de répondre à la problématique complexe de l’imagerie dans le contexte in vivo. Nous avons notamment travaillé sur des aspects de pro-fluorescence et de chémiluminescence. De nouveaux pro-fluorophores à phénol basés sur une architecture originale de type bis-coumarinique ont été développés. De plus, nous avons mis en place une méthode d’hydrosolubilisation généralisable aux fluorophores à phénol de type coumarine et xanthène. Nos recherches en chémiluminescence ont permis la synthèse de nouveaux chémiluminophores couplés à des fluorophores organiques afin d‘augmenter l’efficacité d’émission de chémiluminescence dans le rouge. Enfin, nos travaux ont permis de mettre en place les premières « cassettes » chémiluminescentes basées sur une architecture de type 1,2-dioxétane. / Optical molecular imaging is now playing a pivotal role both in pre-clinical diagnosis and drug development. Indeed, this is a valuable tool for the real time detection and monitoring of living cells either through the use of structurally simple labels or more recently by means of sophisticated fluorescent probes, called “smart” probes and only activatable upon specific interaction with the targeted bio-analyte. The aim of this PhD work was the design of new synthetic tools aimed at optimizing physico-chemical and optical properties of fluorescent probes intended for challenging in vivo imaging applications. We have focused on the pro-fluorescence and chemiluminescence approaches. New phenol-based pro-fluorophores have been developed by using an original bis-coumarinic scaffold. In the context of the chemistry of fluorophores, we have also investigated a general method for the water-solubilisation of phenol-based fluorophore belonging to the coumarin and xanthene families. Our research in chemiluminescence has led the synthesis of new chemiluminophores covalently linked to fluorescent organic dyes aimed at increasing the emission efficiency in the red region of such chemiluminophores. Thus, the first chemiluminescent “energy transfer cassettes” based on a 1,2-dioxetane scaffold have been obtained.
|
150 |
Cyclopeptides containing the DEKS motif as conformationally restricted collagen telopeptide analogues: synthesis and conformational analysisWodtke, Robert, Ruiz-Gómez, Gloria, Kuchar, Manuela, Pisabarro, M. Teresa, Novotná, Pavlina, Urbanová, Marie, Steinbach, Jörg, Pietzsch, Jens, Löser, Reik 07 January 2020 (has links)
The collagen telopeptides play an important role for lysyl oxidase-mediated crosslinking, a process which is deregulated during tumour progression. The DEKS motif which is located within the N-terminal telopeptide of the α1 chain of type I collagen has been suggested to adopt a βI-turn conformation upon docking to its triple-helical receptor domain, which seems to be critical for lysyl oxidase-catalysed deamination and subsequent crosslinking by Schiff-base formation. Herein, the design and synthesis of cyclic peptides which constrain the DEKS sequence in a β-turn conformation will be described. Lysine-side chain attachment to 2-chlorotrityl chloride-modified polystyrene resin followed by microwave-assisted solid-phase peptide synthesis and on-resin cyclisation allowed for an efficient access to head-to-tail cyclised DEKS-derived cyclic penta- and hexapeptides. An Nε-(4-fluorobenzoyl)lysine residue was included in the cyclopeptides to allow their potential radiolabelling with fluorine-18 for PET imaging of lysyl oxidase. Conformational analysis by ¹H NMR and chiroptical (electronic and vibrational CD) spectroscopy together with MD simulations demonstrated that the concomitant incorporation of a D-proline and an additional lysine for potential radiolabel attachment accounts for a reliable induction of the desired βI-turn structure in the DEKS motif in both DMSO and water as solvents. The stabilised conformation of the cyclohexapeptide is further reflected by its resistance to trypsin-mediated degradation. In addition, the deaminated analogue containing allysine in place of lysine has been synthesised via the corresponding ε-hydroxynorleucine containing cyclohexapeptide. Both ε-hydroxynorleucine and allysine containing cyclic hexapeptides have been subjected to conformational analysis in the same manner as the lysine-based parent structure. Thus, both a conformationally restricted lysyl oxidase substrate and product have been synthetically accessed, which will enable their potential use for molecular imaging of these important enzymes.
|
Page generated in 0.0333 seconds