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No / AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.

Il est avéré que les signaux de pléthysmographie par inductance comportent des composantes cardiaques et respiratoires pouvant présenter un intérêt pour un suivi physiologique. Cette technique est largement utilisée chez l’homme et chez les mammifères de taille moyenne, mais n’a jamais été mise en œuvre chez les petits rongeurs de laboratoire comme le rat. Cette thèse vise à apporter la preuve analytique et expérimentale (TRL3) d’une application cardiaque de la pléthysmographie cardiorespiratoire par inductance (PCRI) fondée sur le concept amont de « bague aortique virtuelle » (BAOV). La BAOV permet de mettre en œuvre une mesure externe des débits aortiques « instantanés » à l’aide de la PCRI en lieu et place d’un instrument positionné autour du vaisseau lui-même.La thèse a débuté par une phase de spécification et de conception guidée par l’interdépendance entre la physiologie et les contraintes instrumentales. Les performances métrologiques à atteindre sont dictées par un saut d’échelle entre l’homme et le rat. Le développement d’une chaine de mesure optimisée a permis de repousser les limites des systèmes existants en rendant possible la mesure de variations de volumes de quelques microlitres. En parallèle, l’identification de critères de validation, de méthodes de référence et la mise au point de protocoles expérimentaux ont conduit à la définition d’une stratégie de validation de l’instrument de mesure développé et du concept de BAOV.À l’issue de ces trois années, un système de PCRI à ultra-haute résolution a été mis au point. Après calibration, l’exactitude sur les mesures de variations de section est de 5 % sur un banc de test micrométrique. L’interchangeabilité de la mesure des variations de volume du thorax sur la gamme physiologique a été évaluée par rapport à une mesure pneumotachographique sur 9 animaux anesthésiés. Les limites d’agrément obtenues sont inférieures à 20 %. L’induction d’un challenge hémodynamique sur 11 animaux anesthésiés dont le débit aortique est mesuré en parallèle avec la PCRI et une bague de débit ultrasonique placée au niveau sous-diaphragmatique démontre l’équivalence entre les deux systèmes. Par ailleurs, la grande similitude entre les signaux de débits des deux méthodes valide le concept de bague aortique virtuelle proposé / It is recognized that inductive plethysmographic signals contain cardiac and respiratory components, which can be of interest for physiological monitoring. This technology is widely used in humans and medium mammals, but it has never been implemented in small laboratory rodents. This PhD aims to provide the analytic and experimental proof (TRL3) of a cardiac application of the cardio-respiratory inductive plethysmography (CRIP), based on the upstream concept of an “virtual aortic probe” (VAP). The VAP allows to realize an extern measure of “instantaneous” aortic flows thanks to CRIP instead of an instrument located directly around the vessel.The PhD starts with a phase of specification and conception driven by the interdependency between physiology and instrumental constraints. The expected metrological performances are established by a scale jumping between man and rat. The development of an optimized acquisition line has enabled to stretch the limits of existing systems; it allows to measure volume variations of a few microliters. At the same time, validation criteria and reference methods have been identified and experimental protocols have been specified in order to define the validation strategy of the developed instrument and VAP concept.At the end of these 3 years, an ultra-high resolution CRIP system has been developed. After calibration, the accuracy on the section variation measurements is 5% on a micrometric test-bench. The interchangeability of the thorax volume variation measure on a physiological range has been evaluated by comparison with a pneumotachographic measure on 9 anesthetized animals and the limits of agreement are lower than 20%. A hemodynamic challenge has been induced on 11 anesthetized animals, and the aortic flow has been simultaneously measured by CRIP and with an ultrasonic flow probe at under diaphragm level. This demonstrates the equivalence between both systems. And the high similarity between flow signals from both methods validates the proposed concept of virtual aortic probe

This study aimed to investigate the pharmacology of selected pyrrolobenzodiazepine (PBD) compounds shown to have cytotoxic activity with predicted DNA sequence selectivity. Research focused upon the PBD dimer, SJG-136, selected for clinical trials, and the novel PBD monomer compounds D709119, MMY-SJG and SJG-303. SJG-136, a novel sequence-selective DNA minor groove cross-linking agent, was shown to have potent tumour cell type selective cytotoxicity in in vitro assays. Pharmacokinetic studies in mice via both the i.p. and i.v. route (dosed at the maximum tolerated dose (MTD)) showed that SJG-136 reaches concentrations in plasma well in excess of the in vitro IC50 values for 1 h exposure, and was detected in tumour and brain samples also above the in vitro IC50 values. Furthermore, SJG-136 showed linear pharmacokinetics over a 3-fold drug dose range. Metabolism studies showed SJG-136 is readily metabolised in vitro by hepatic microsomes, predominantly to a monodemethylated metabolite; this metabolite could be detected in vivo. Analytical method development work was also conducted for the imminent Phase I clinical trial of SJG-136 resulting in a sensitive and selective bio-analytical detection protocol. Comet analysis showed that SJG-136 dosed at the MTD and ⅓MTD causes significant interstrand DNA cross-linking in lymphocytes in vivo. In vitro studies demonstrated that SJG-136 localises within the cell nucleus, and acts to disrupt cell division via a G2/M block in the cell cycle at realistic concentrations and exposure times that are achievable in vivo. In vivo pharmacokinetic studies of D709119 showed the compound is easily detectable in mouse plasma following i.p. dosing at the MTD, but could not be detected in either tumour or brain samples. In vitro cytotoxicity studies revealed D709119 to have potent activity across a selection of tumour cell lines. SJG-136, D709119, MMY-SJG, SJG-303 and DC-81 demonstrated a non-enzyme-catalysed reactivity with the biologically relevant thiol, reduced glutathione (GSH). Studies demonstrated that reactivity of the PBD compounds toward GSH was dependent on GSH concentrations. At levels of GSH found in plasma, the PBD compounds showed considerably lower reactivity with GSH than at intracellular GSH levels. SJG-136 and D709119 also showed favourable pharmacokinetic profiles in mice, and warrant further study for anti-tumour activity in vivo and progression to use in patients.

Made available in DSpace on 2014-07-29T16:11:52Z (GMT). No. of bitstreams: 1 Dissertacao Flaubertt 1iniciais.pdf: 86457 bytes, checksum: 527b584507a695284399e78f10a55608 (MD5) Previous issue date: 2008-03-12 / O uso de plantas medicinais tem sido muito significativo nos últimos anos, sendo incentivado pela Organização Mundial de Saúde (OMS). Synadenium umbellatum Pax, Euphorbiacea (vulgo cola-nota, cancerola, milagrosa) tem o látex usado empiricamente como anti-tumoral e antiinflamatório. Por existir espécies tóxicas nesta família e visando à segurança no uso de extratos vegetais, tal estudo avaliou a toxicidade pré-clinica do látex e do extrato etanólico das folhas (EEF) de S. umbellatum, por via oral, em ratas Wistar. O estudo seguiu diretrizes da OECD (Organisation for Economic Cooperation and Development) para teste de toxicidade aguda (Guideline 423) e de toxicidade subaguda (Guideline 407). Na toxicidade aguda do látex e do EEF, não se observou, na dose de 2000 mg/kg, alterações fisiológicas e comportamentais, e nem a morte dos animais. No entanto, o látex ocasionou congestão e infiltrado leucocitário nos rins, fígado e pulmões, efeitos não observados com o EEF. Na toxicidade subaguda, doses de 50, 100 e 200 mg/kg de EEF não produziram alterações dose-dependentes significativas nos parâmetros laboratoriais e nem alterações fisiológicas, macroscópicas e histopatológicas dos órgãos. O EEF da S. umbellatum é praticamente atóxico em exposição aguda, já o látex pode ocasionar alterações histopatológicas no fígado e pulmões. O uso crônico da planta S. umbellatium merece mais estudos.

Les modifications post-traductionnelles de la tubuline sont des cibles attrayantes pour la thérapie du cancer. TTLL12 est impliqué dans la détyrosination de la tubuline, la triméthylation de l’histone H4K20 et le cancer de la prostate. La thèse porte sur les effets de la surexpression de TTLL12 sur ces modifications à différents stades du cycle cellulaire et sur la sensibilité à des agents ciblant les microtubules. Les résultats montrent que TTLL12 affecte ces modifications indépendant du cycle cellulaire et réduit la sensibilité des cellules à paclitaxel. XRP44X est un nouvel inhibiteur de la signalisation Ras-ERK-Elk3. Ses propriétés antitumorigène ont été montré in vitro et dans certaines études in vivo. Le projet de thèse était une continuation des études pré-cliniques sur XRP44X dans des modèles de cancer de la prostate de souris. Les résultats montrent que XRP44X est un inhibiteur efficace de la tumorigenèse et des métastases, ce qui peut être dû à son effet sur Elk3. / Tubulin posttranslational modifications are an attractive target for cancer therapy. TTLL12 isinvolved in tubulin detyrosination, histone H4K20 trimethylation and prostate cancer. The thesis addresses the effects of TTLL12 overexpression on these tubulin and histone modifications at different stages of the cell cycle and on sensitivity to microtubule-targeting agents. The results show that TTLL12 over expression affects tubulin detyrosination and H4K20 trimethylation independently of cell cycle phase and reduces cell sensitivity totaxanes.XRP44X is a novel inhibitor of Ras-ERK1/2-Elk3 signalling and tubulin-binding agent. Itsantitumorigenic properties had been shown in vitro and in initial in vivo studies. The thesis project was a continuation of pre-clinical studies on XRP44X in mouse prostate cancer models. The results show that XRP44X is an effective inhibitor of tumorigenesis and metastasis in prostate cancer, which may be due to its effect on Elk3.

L’IRM est de plus en plus utilisée pour diagnostiquer et évaluer un très grand nombre de pathologies. Cette technique présente cependant deux inconvénients majeurs. En effet, les examens restent encore très longs (notamment en imagerie 3D) et la quantification est très difficile par rapport à d’autres modalités comme la tomographie par émission de positons. L’objectif de ce travail de thèse a été de diminuer significativement les temps d’acquisition nécessaires pour l’imagerie volumique et de développer des techniques quantitatives robustes, permettant d’effectuer des suivis longitudinaux.Pour cela, des méthodes innovantes ont été développées à très haut champ magnétique (7T) et validées sur des modèles murins sains et pathologiques. Trois développements majeurs sont ressortis de cette thèse. Tout d’abord, une mesure rapide des temps de relaxation longitudinale (T1) a été développée.Cette méthode basée sur une approche Look-Locker a été couplée avec un échantillonnage en empilement de spirales et a permis d’obtenir au niveau cardiaque des cartes T1 en 3D sur des souris saines et des modèles d’infarctus du myocarde en moins de 15 minutes. Ensuite, une approche dite« spiral-in » a été couplée avec une méthode de multi-échos de spin afin d’accélérer la mesure des temps de relaxation transversale (T2). Cette méthode a permis d’obtenir des cartes T2 en 3D sur des cerveaux de souris saines et métastatiques en moins de 20 minutes. Enfin, une approche hybride couplant les avantages de l’acquisition spiralée et ceux de l’échantillonnage radial a été développée.Cette méthode a été couplée avec une technique de Golden-Angle pour échantillonner aléatoirement l’espace de Fourier et a permis pour la première fois de visualiser une angiographie 3D d’un foie de souris en respiration libre en moins de 12 minutes. Toutes les méthodes développées dans ce travail ont été validées au niveau de leur robustesse et démontrent que l’IRM peut être une technique à la fois rapide et quantitative. Ces développements pourront être transférés vers la clinique dans de futurs travaux. / MRI is more and more used to diagnose and assess a wide range of pathologies. However, this technique is still limited by two disadvantages. Indeed, the acquisition times are too long(especially in 3D) and the quantification is still difficult compared to other techniques like positron emission tomography. The aim of this PhD project was to significantly reduce acquisition times required for 3D imaging and to develop robust quantitative techniques allowing longitudinal studies.To these ends, innovative methods have been developed at very high magnetic field (7T) and validated on healthy and diseased mouse models. Three major developments arose from this work. Firstly, a fast measurement of the longitudinal relaxation time (T1) has been developed. This method based on a Look-Locker approach was coupled with a sampling using stack-of-spirals and allowed to get T1 mapsin 3D in healthy and myocardial infarction models in less than 15 minutes. Then, a "spiral-in" approach was coupled with a multi spin echoes acquisition to accelerate the measurement of the transverse relaxation time (T2). This method allowed to get T2 maps in 3D of healthy and metastatic mouse brains in less than 20 minutes. Finally, a hybrid approach combining the advantages of the spiral acquisition with those of the radial sampling has been developed. This method has been coupled with a Golden-Angle technique for randomly sampling the k-space and allowed for the first time to display a 3Dangiography of a mouse liver in free breathing in less than 12 minutes. All the protocols developed inthis PhD project were validated in terms of robustness and showed that MRI can be a technique both rapid and quantitative. These developments will be transferred to the clinic in future works.

This study aimed to investigate the pharmacology of selected pyrrolobenzodiazepine (PBD) compounds shown to have cytotoxic activity with predicted DNA sequence selectivity. Research focused upon the PBD dimer, SJG-136, selected for clinical trials, and the novel PBD monomer compounds D709119, MMY-SJG and SJG-303. SJG-136, a novel sequence-selective DNA minor groove cross-linking agent, was shown to have potent tumour cell type selective cytotoxicity in in vitro assays. Pharmacokinetic studies in mice via both the i.p. and i.v. route (dosed at the maximum tolerated dose (MTD)) showed that SJG-136 reaches concentrations in plasma well in excess of the in vitro IC50 values for 1 h exposure, and was detected in tumour and brain samples also above the in vitro IC50 values. Furthermore, SJG-136 showed linear pharmacokinetics over a 3-fold drug dose range. Metabolism studies showed SJG-136 is readily metabolised in vitro by hepatic microsomes, predominantly to a monodemethylated metabolite; this metabolite could be detected in vivo. Analytical method development work was also conducted for the imminent Phase I clinical trial of SJG-136 resulting in a sensitive and selective bio-analytical detection protocol. Comet analysis showed that SJG-136 dosed at the MTD and ⅓MTD causes significant interstrand DNA cross-linking in lymphocytes in vivo. In vitro studies demonstrated that SJG-136 localises within the cell nucleus, and acts to disrupt cell division via a G2/M block in the cell cycle at realistic concentrations and exposure times that are achievable in vivo. In vivo pharmacokinetic studies of D709119 showed the compound is easily detectable in mouse plasma following i.p. dosing at the MTD, but could not be detected in either tumour or brain samples. In vitro cytotoxicity studies revealed D709119 to have potent activity across a selection of tumour cell lines. SJG-136, D709119, MMY-SJG, SJG-303 and DC-81 demonstrated a non-enzyme-catalysed reactivity with the biologically relevant thiol, reduced glutathione (GSH). Studies demonstrated that reactivity of the PBD compounds toward GSH was dependent on GSH concentrations. At levels of GSH found in plasma, the PBD compounds showed considerably lower reactivity with GSH than at intracellular GSH levels. SJG-136 and D709119 also showed favourable pharmacokinetic profiles in mice, and warrant further study for anti-tumour activity in vivo and progression to use in patients.

There are an estimated 2 billion individuals infected with Mtb, and 37.7 million people living with HIV (PLWH) worldwide. HIV/Mtb co-infection increases the risk of developing active tuberculosis by over 20-fold, and 210,000 of 1.5 million deaths from TB were among co-infected PLWH in 2020. Therefore, development of effective TB vaccination, particularly within the vulnerable PLWH population, is an urgent global issue. With limited in vivo models to study co-infection, humanized NRG (huNRG) mice and humanized DRAG-A2 mice (a next-generation of huNRG mice expressing HLA class I and II transgenes with improved human immune reconstitution, huDRAG-A2) are promising tools for HIV and TB reserach as they develop robust human immune cell populations and recapitulate many aspects of HIV or TB clinical disease. HIV/Mtb co-infection was investigated using huNRG and hu-DRAG-A2 mice in separate experiments where intravaginal (with DMPA pre-treatment) or intraperitoneal HIV-1 infection was administered, respectively, and intranasal infection of Mtb was administered 3.5 weeks later. Both huNRG and huDRAG-A2 mice recapitulated hallmark features of HIV/Mtb co-infection such as severe granuloma pathology, hCD4+ T cell depletion in lung and spleen tissue, and human like lung pathology such as Mtb-infected foamy macrophages in the granuloma. Co-infected huDRAG-A2 mice also displayed significantly higher bacterial burden in the lungs, increased extrapulmonary dissemination into spleen and liver, and significantly lower hCD4+ T cells in the peripheral blood post-Mtb infection when compared to the Mtb-only infected group. To investigate TB vaccine immunogenicity, huNRG and huDRAG-A2 mice were immunized with a novel trivalent vaccine, AdCh68MV. Upon intranasal immunization, both models showed trends of developing higher Mtb antigen-specific hCD4+ T cell responses in the lung and spleen. Overall, this project sets the initial stages of a pre-clinical HIV/Mtb co-infection model in huNRG and huDRAG-A2 mice appropriate for immune investigations, therapeutic and vaccination development. / Thesis / Master of Science in Medical Sciences (MSMS) / There are over 2 billion individuals infected with TB and 37.7 million people living with HIV (PLWH) worldwide. When someone is co-infected with both diseases, the risk of death is greatly increased. Research in co-infection and developing effective TB vaccination for PLWH are urgent global issues. Animal studies are currently limited because studying HIV requires human immune cells. Our lab has established humanized mice (hu-mice) that develop many different human immune cells and are useful for HIV/Mtb co-infection research. When hu-mice were co-infected, they showed more dying lung tissue, immune cell loss, and bacteria in the lungs. Hu-mice were also used to study human immune responses to a novel TB vaccine delivered to the lungs. Trends of higher immune responses towards TB were observed in the lung and spleen of immunized hu-mice. Overall, this project shows the utility of hu-mice as pre-clinical models of HIV/Mtb co-infection and Mtb vaccine studies.

The blood oxygen level dependent functional MRI (BOLD-fMRI) signal is an indirect measure of the neuronal activity that most BOLD studies are interested in. This thesis uses generative embedding algorithms to investigate some of the challenges and opportunities that this presents for BOLD imaging. It is standard practice to analyse BOLD signals using general linear models (GLMs) that assume fixed neurovascular coupling. However, this assumption may cause false positive or negative neural activations to be detected if the biological manifestations of brain diseases, disorders and pharmaceutical drugs (termed "neuromodulations") alter this coupling. Generative embedding can help overcome this problem by identifying when a neuromodulation confounds the standard GLM. When applied to anaesthetic neuromodulations found in preclinical imaging data, Fentanyl has the smallest confounding effect and Pentobarbital has the largest, causing extremely significant neural activations to go undetected. Half of the anaesthetics tested caused overestimation of the neuronal activity but the other half caused underestimation. The variability in biological action between anaesthetic modulations in identical brain regions of genetically similar animals highlights the complexity required to comprehensively account for factors confounding neurovascular coupling in GLMs generally. Generative embedding has the potential to augment established algorithms used to compensate for these variations in GLMs without complicating the standard (ANOVA) way of reporting BOLD results. Neuromodulation of neurovascular coupling can also present opportunities, such as improved diagnosis, monitoring and understanding of brain diseases accompanied by neurovascular uncoupling. Information theory is used to show that the discriminabilities of neurodegenerative-diseased and healthy generative posterior parameter spaces make generative embedding a viable tool for these commercial applications, boasting sensitivity to neurovascular coupling nonlinearities and biological interpretability. The value of hybrid neuroimaging systems over separate neuroimaging technologies is found to be greatest for early-stage neurodegenerative disease.