Les glioblastomes sont des tumeurs gliales de hautgrade qui restent incurables de nos jours. Le traitement recommandé (résection chirurgicale suivie d’un traitement de radiothérapie externe associé à la chimiothérapie) conduit à une augmentation de la médiane de survie des patients de quelques mois. De nouvelles stratégies notamment dans le champ des nanomédecines véhiculant un radioélément (émetteur ou ) ont été évaluées en clinique. La première partie,après avoir dressé l’état des lieux des technologies utilisées dans ce domaine, rend compte des critères importants à prendre en compte que sont le choix du radioélément, les modalités d’administration et les vecteurs utilisés. Elle se conclut par une présentation des études précliniques en cours dont l’utilisation de nanovecteurs encapsulant un radioisotope : lesLNC188Re-SSS. La deuxième partie illustre l’application thérapeutique sur un modèle murin de xénogreffe et montre une éradication de la tumeur initiale suite à un protocole personnalisé d’injection fractionnée par convection-enhanced delivery. Elle décrit également la distribution des LNC ainsi que les effets directs des radiations sur les cellules tumorales (cellules géantes atypiques, supposées polyploïdes), accompagnés par un infiltrat inflammatoire (immunité innée). Une évaluation complémentaire sur modèle murin GL261 a été réalisée et constitue la troisième partie de cette thèse. Le transfert de ces résultats à l’application clinique pourrait être facilité par le recours à un modèle canin de gliome spontané homologue à celui de l’homme, dont ce travail prépare l’utilisation, dans le but de valider les procédures d’injection intracérébrales automatisées. / High grade glial brain tumors are defined as glioblastomas. Nowadays, they are incurable. The current therapeutic purposal (surgical resection, external radiotherapy and chemotherapy) doesn’t extend the patients median survival time up to a few months. Newstrategies as nanomedicines loaded with a radionuclide( or  emitter) have been evaluated in clinical trials. As tate of the art of this domain’s technologies is described in a first part which analyses the important criteria to take account in vectorized radiotherapy like the radionuclides, the route of administration and the vectors used. Then, it is concluded with a presentation of preclinical on going studies as the use of nanovectors loaded with a radioisotope : the LNC188Re-SSS. The second part illustrates the therapeutical strategy application on a xenograft mice model. The data showan eradication of the tumor mass of treated mice withthe personalized convection-enhanced delivery offractionated radiotherapy. Furthermore, it describes the LNC distribution and the direct radiation effects on tumor cells (atypical giant cells, polyploïdy) supported by an inflammatory infiltration (innate immune effectors). An evaluation on the GL261 mice model has been realized and concerns the third part of this thesis. In perspectives, the transfer of these data to clinical trials could be facilitated thanks to the dog spontaneous glioma model. This tumor share characteristics with the human neoplasma. Finally, this work will aim to validate the automated intracranial injection procedures.

In this thesis, we describe several imaging techniques specifically designed and developed for the assessment of pulmonary structure, function and pathology. We then describe the application of this technology within appropriate biological systems, including the identification, tracking and assessment of lung tumors in a mouse model of lung cancer. The design and development of a Large Image Microscope Array (LIMA), an integrated whole organ serial sectioning and imaging system, is described with emphasis on whole lung tissue. This system provides a means for acquiring 3D pathology of fixed whole lung specimens with no infiltrative embedment medium using a purpose-built vibratome and imaging system. This system enables spatial correspondence between histology and non-invasive imaging modalities such as Computed Tomography (CT), Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET), providing precise correlation of the underlying 'ground truth' pathology back to the in vivo imaging data. The LIMA system is evaluated using fixed lung specimens from sheep and mice, resulting in large, high-quality pathology datasets that are accurately registered to their respective CT and H&E histology. The implementation of an in vivo micro-CT imaging system in the context of pulmonary imaging is described. Several techniques are initially developed to reduce artifacts commonly associated with commercial micro-CT systems, including geometric gantry calibration, ring artifact reduction and beam hardening correction. A computer controlled Intermittent Iso-pressure Breath Hold (IIBH) ventilation system is then developed for reduction of respiratory motion artifacts in live, breathing mice. A study validating the repeatability of extracting valuable pulmonary metrics using this technique against standard respiratory gating techniques is then presented. The development of an ex vivo laser scanning confocal microscopy (LSCM) and an in vivo catheter based confocal microscopy (CBCM) pulmonary imaging technique is described. Direct high-resolution imaging of sub-pleural alveoli is presented and an alveolar mechanic study is undertaken. Through direct quantitative assessment of alveoli during inflation and deflation, recruitment and de-recruitment of alveoli is quantitatively measured. Based on the empirical data obtained in this study, a new theory on alveolar mechanics is proposed. Finally, a longitudinal mouse lung cancer study utilizing the imaging techniques described and developed throughout this thesis is presented. Lung tumors are identified, tracked and analyzed over a 6-month period using a combination of micro-CT, micro-PET, micro-MRI, LSCM, CBCM, LIMA and H&E histology imaging. The growth rate of individual tumors is measured using the micro-CT data and traced back to the histology using the LIMA system. A significant difference in tumor growth rates within mice is observed, including slow growing, regressive, disappearing and aggressive tumors, while no difference between the phenotype of tumors was found from the H&E histology. Micro-PET and micro-MRI imaging was conducted at the 6-month time point and revealed the limitation of these systems for detection of small lesions ( < 2mm) in this mouse model of lung cancer. The CBCM imaging provided the first high-resolution live pathology of this mouse model of lung cancer and revealed distinct differences between normal, suspicious and tumor regions. In addition, a difference was found between control A/J mice parenchyma and Urethane A/J mice ‘normal’ parenchyma, suggesting a 'field effect' as a result of the Urethane administration and/or tumor burden. In conclusion, a comprehensive murine lung cancer imaging study was undertaken, and new information regarding the progression of tumors over time has been revealed.

La SRM proton constitue un outil non invasif unique pour l'exploration biochimique quantitative des tissus vivants. Les deux études présentées dans cette thèse visent à maîtriser chacune des phases impliquées depuis l’acquisition des données jusqu’à l’estimation fiable et précise des profils métaboliques des tissus explorés. Des protocoles expérimentaux d’acquisition des signaux de spectrométrie de résonance magnétique proton in vivo à temps d’écho courts ont été définis puis optimisés pour une application pré-clinique (souris) sur un imageur 4.7T et pour une étude en environnement clinique menée à 1.5T. La première étude a permis de mesurer longitudinalement les altérations des métabolites cérébraux (N-acétyl-aspartate, choline, créatine, taurine) chez un modèle murin de neuro-inflammation sur un imageur 4.7T, et la seconde étude avait pour objectif la mesure de la quantité totale et la composition lipidique hépatique en environnement clinique à 1.5T chez des sujets stéatosiques. Des méthodes d’estimation des contributions métaboliques et lipidiques adaptées aux propriétés physiques de signaux ont été validées pour chacune de ces applications. Ces méthodes sont fondées sur des algorithmes de moindres carrés non linéaires. Des stratégies multi-tirages des valeurs initiales et des contraintes ont été favorablement validées. Les atouts et les originalités de ce projet reposent sur les développements synergiques des protocoles d’acquisitions et des méthodes de traitement du signal associées. Ces développements ont pour vocation d’enrichir la palette des informations biochimiques collectées pour l’aide au pronostic et diagnostic médical / The proton MRS is a unique non-invasive method to quantitative biochemical exploration of living tissues. The studies presented in this thesis aim to handle each one of the involved steps, from data acquisition to reliable and precise metabolic profile estimation of explored tissues. Protocols for experimental acquisition of in vivo, short echo-time magnetic resonance signals were defined, and optimized for a pre-clinical application (mice) on a 4.7T scanner and for a clinical study at 1.5T. The first study allowed yo measuring cerebral metabolite (N-acetyl-aspartate, choline, creatine, taurine) alterations along time in a murine model of neuro-inflammation on a 4.7T scanner and the second study aimed to measure the total quantity and the composition of liver fat in patients with hepatic steatosis in a clinical environment at 1.5T. Signal processing methods for metabolite and fat contribution estimates, coping with physical signal properties were validated for both studies. These methods are based on non-linear least squares algorithms. Multiple starting values and constraints strategies were successfully validated. The assets and the originality of this project are based on the synergic developments of acquisition protocols and the associated signal processing methods. These developments were designed to enrich the list of the biochemical information non-invasively measured, in order to help medical prognostic and diagnostic

Un enjeu majeur en cancérologie est de réduire le risque de développement métastatique et de rechute. La transition épithélio-mésenchymateuse (EMT), processus physiologique au cours de l'embryogenèse, est un mécanisme central de la carcinogenèse, contribuant de façon précoce à la transformation et la dissémination des cellules tumorales via l'inhibition de la surveillance cellulaire (apoptose et senescence) et l'acquisition de capacités migratoires et invasives. Une autre caractéristique des cancers est la capacité d'échapper à la réponse immunitaire, puissante barrière anti-tumorale. Mais les cellules tumorales entretiennent des relations complexes avec le système immunitaire. Alors que la propension de l'inflammation et des cellules immunitaires innées à favoriser le développement tumoral et l'échappement immunitaire, via l'induction de l'EMT et le maintien d'un microenvironnement immuno-suppresseur, a été bien étudiée, le rôle éventuel de la réponse immunitaire adaptative dans la promotion de l'EMT est quant à lui peu connu. Grâce au développement d'un modèle murin de lignée tumorale mammaire plastique surexprimant l'oncogène Her2/Neu, ce travail démontre in vivo la capacité des cellules tumorales à subir l'EMT, induite par la réponse immunitaire médiée par les lymphocytes T. La déplétion spécifique des lymphocytes T (LT) CD4 restaure le phénotype épithélial de la tumeur, indiquant que les LT CD4 médient une réponse immunitaire induisant l'EMT. En retour, l'EMT confère aux cellules tumorales la capacité de modeler l'immunité comme le recrutement de neutrophiles. Ce travail apporte un nouvel éclairage sur les interactions entre cellules tumorales et système immunitaire / Current clinical challenge in many carcinomas is to reduce the risk of metastasis development and cancer recurrence. Epithelial-mesenchymal transition (EMT), a physiological process during embryogenesis, is a central mechanism in oncogenesis. EMT induction contributes to early transformation and dissemination through inhibition of cellular surveillance (apoptosis and senescence) and increased migrative and invasive behavior. Another necessary hallmark of cancer is the ability of tumor cells to evade immune surveillance, a powerful barrier against tumor progression. But cancer cells enjoy intricate relations with the immune system. Whereas inclination of inflammation and innate immune cells to favor tumor development and immune escape, via EMT induction and immunosuppressive microenvironment maintenance, has been well investigated, the role of adaptive immune response in EMT promotion is understudied. Based on the development of a plastic murine mammary tumor cell line model overexpressing Her2/Neu oncogene, this study demonstrate in vivo that tumor cells keep an epithelial phenotype in adaptive immunodeficient mice but undergo EMT under the pressure of T-cell mediated immune response, characterized by loss of epithelial EpCAM marker and acquisition of mesenchymal features and EMT transcriptomic signature. CD4 T cell depletion but not CD8 restores the epithelial phenotype of tumors, suggesting that CD4 T cells mediate an immune response that could lead ton EMT induction. In return, EMT confers the ability of tumor cells to shape immunity like intra-tumor neutrophil infiltration. This work shed a new light on interactions between tumor cells and immune system

Cancer is responsible for upward of 13% of human deaths. Contemporary chemotherapy of disseminated cancer is often thwarted by dose limiting systemic toxicity and by multi-drug resistance (MDR). Riminophenazines are a novel class of potential anticancer agents that possess a potent multi-mechanistic antineoplastic action. Apart from their broad action against intrinsic, non-classical resistance, Riminophenazines inhibit the action of Pgp and hypothetically all ABC transporters demonstrating their great utility against classical MDR. Considering that combination chemotherapy is the norm, the vision directing R&D efforts was that Riminophenazines could be used with benefit within many standard chemotherapeutic regimes. The strategic intent of this project was to attain improved therapeutic benefit for patients through gains in both pharmaco dynamic and pharmacokinetic specificity for cancer cells over what is currently available. Tactically, this was driven through the use of synergistic Fixed-Ratio Drug Combinations (FRDC) encapsulated within tumour-targeting Nanoparticulate Drug Delivery Systems (NDDS). Long-term aims of this R&D project were to: 1) Screen FRDC of clofazimine (B663) and the lead derivative (B4125) with etoposide, paclitaxel and vinblastine for synergistic drug interactions in vitro. 2) Design, assemble and characterize a novel nanoparticulate, synergistic, anticancer co-formulation. 3) Evaluate the in vivo safety and efficacy of the developed product/s in accordance with international regulatory guidelines. Using the median effect and combination index equations, impressive in vitro synergistic drug interactions (CI<1) were shown for various FRDC of the three standard chemotherapeutics tested (etoposide, paclitaxel and vinblastine) in combination with either B663 or B4125 against MDR neoplastic cell cultures. Considering in vitro results and with the view to advance quickly to clinical studies, the already approved clofazimine (B663) was elected as the combination partner for paclitaxel (PTX). Considering the potency and wide action of PTX, a novel coformulation (designed to circumvent drug resistance) has the potential to greatly impact upon virtually all cancer types, particularly if selectively delivered through innovative delivery systems and loco-regional administration. A passively tumour targeting, micellular NDDS system called Riminocelles™ that encapsulates a synergistic FRDC of B663 and PTX has been designed, assembled using thin film hydration methods and characterized in terms of drug loading, particle size, zeta potential, CMC and drug retention under sink conditions. An acute toxicity and a GLP repeat dose toxicity study confirmed Riminocelles to be well tolerated and safe at clinically relevant dosages whilst Taxol® (QDx7) produced statistically significant (P<0.05) weight loss within 14 days. The same study demonstrated statistically significant (P<0.05) tumour growth delays superior to that of Taxol at an equivalent PTX dosage of 10 mg/kg. Importantly, all components (amphiphiles and drugs) used in assembly of Riminocelles are already individually approved for medicinal use - this promotes accelerated development towards advanced clinical trials and successful registration. Although these results are very promising (outperforming Taxol), this system was however found in a pharmacokinetic study to suffer from in vivo thermodynamic instability due to the high concentration (abundance) of albumin present in plasma. For this reason, in vivo longevity within circulation, permitting passive tumour accumulation was not fully realized. A second NDDS called the RiminoPLUS™ imaging system was additionally developed. This lipopolymeric nanoemulsion system has successfully entrapped Lipiodol® Ultra fluid (an oil based contrast agent) within the hydrophobic core of a monodisperse particle population with a size of roughly 100 nm and a stability of one week. This formulation is therefore thought capable of CT imaging of tumour tissue and drug targeting after either intravenous or loco-regional injection. In vivo proof of the imaging concept is warranted. The results of this study serve to highlight the great potential of in vitro optimized synergistic FRDC against drug resistant cancers. Lipopolymeric micelles are an effective way to formulate multiple hydrophobic drugs for intravenous administration and present a means by which cancer can be readily targeted; provided that the delivery system possess the prerequisite in vivo stability and surface attributes. Further experiments exploring synergistic drug and biological combinations as well as “intelligent” NDDS actively guided through specific molecular recognition are called for. / Thesis (PhD)--University of Pretoria, 2012. / Pharmacology / unrestricted

Background: The focus of this study was to investigate how producing human bone marrow (hBM) derived mesenchymal stromal cell (MSC) extracellular vehicles (EVs) in a high purity isolation system would affect their established characterization criteria and address the validity of these methods of EV production. Additionally, we set out to functionally characterize the hBM-MSC-EVs for their identified immunomodulatory ability while also assessing the presence of novel MSC-EV marker NRP1 identified by our group to further affirm its validity as a functional MSC-EV identity marker. Methods: Each hBM-MSC-EV donor was cultured in a hollow-fiber bioreactor system in non-stimulating serum/xeno-free conditions for 25 days to produce EVs persistently under quiescent conditions to characterize the hBM-MSC-EVs in their native form. EVs were isolated by traditional PEG-based precipitation for preliminary characterization to monitor bioreactor production wherein they were characterized using multimodal tangential flow filtration coupled with fast protein liquid chromatograph (FPLC) size exclusion/high-affinity purifications to obtain the final highly purified EV sample. Additionally, functional analysis of their immunomodulatory ability, EVs and MSCs were incubated with activated peripheral blood mononuclear cells (PBMCs) as an in-vitro model to evaluate their potency. Results: The hBM-MSC-EVs produced from the bioreactor system showed consistent characterization in accordance with the MISEV2018 establish criteria. We were also able to demonstrate their functional ability by observing statistically significantly immunomodulatory ability of activated PBMCs equivalent to native MSC ability. We were also able to validate the present of NRP1 on all hBM-MSC-EV samples produced confirming its validity as a MSC-EV marker. Conclusion: The significance of the results obtained from this study confirms the production of MSC-EV using a bioreactor and high purity isolation for obtaining consistent MSC-EVs for downstream investigation. Additionally, we were able to demonstrate the significance of MSC-EVs on MSC signaling for immunomodulation by showing equivalent functional potency when tested in-vitro. These results contribute to further understanding the biological attributes of MSC-EVs and contribute to the validation of currently established characterization guidelines.

No description available.

The thesis outlines possible medical applications of soft matter assemblies as nanotechnology based systems as well as their potential in the emerging field of nanomedicine. Nanomedicine can be defined as the investigation area encompassing the design of diagnostics and therapeutics at the nanoscale, including nanobots, nanobiosensors, nanoparticles and other nanodevices, for the remediation, prevention and diagnosis of a variety of illnesses. The ultimate goal of nanomedicine is to improve patient quality-of-life. Because nanomedicine includes the rational design of an enormous number of nanotechnology-based products focused on miscellaneous diseases, a variety of nanomaterials can be employed. Therefore, the thesis is driven by a focus on recent advances in the manufacture of soft matter-based nanomedicines specifically designed to improve cancer diagnostics and chemotherapy efficacy. It will in particular highlight liposomes, polymer-drug conjugates, drug- loaded block copolymer micelles and biodegradable polymeric nanoparticles, emphasizing the current investigations and potential novel approaches towards overcoming the remaining challenges in the field as well as a brief overview of formulations that are in clinical trials and marketed products. Based on vehicle-related and...

L'ablation par radiofréquence (ARF) de tumeurs abdominales est rendue difficile par l’influence des vaisseaux sanguins et les variations de la conductivité thermique, compliquant la planification spécifique à un patient donné. En fournissant des outils prédictifs, les modèles biophysiques pourraient aider les cliniciens à planifier et guider efficacement la procédure. Nous introduisons un modèle mathématique détaillé des mécanismes impliqués dans l’ARF des tumeurs du foie comme la diffusion de la chaleur et la nécrose cellulaire. Il simule l’étendue de l’ablation à partir d’images médicales, d’après lesquelles des modèles personnalisés du foie, des vaisseaux visibles et des tumeurs sont segmentés. Dans cette thèse, une nouvelle approche pour résoudre ces équations basée sur la méthode de Lattice Boltzmann est introduite. Le modèle est d’abord évalué sur des données de patients qui ont subi une ARF de tumeurs du foie. Ensuite, un protocole expérimental combinant des images multi-modales, anatomiques et fonctionnelles pré- et post-opératoires, ainsi que le suivi de la température et de la puissance délivrée pendant l'intervention est présenté. Il permet une validation totale du modèle qui considère des données les plus complètes possibles. Enfin, nous estimons automatiquement des paramètres personnalisés pour mieux prédire l'étendu de l’ablation. Cette stratégie a été validée sur 7 ablations dans 3 cas cliniques. A partir de l'étude préclinique, la personnalisation est améliorée en comparant les simulations avec les mesures faites durant la procédure. Ces contributions ont abouti à des résultats prometteurs, et ouvrent de nouvelles perspectives pour planifier et guider l’ARF. / The outcome of radiofrequency ablation (RFA) of abdominal tumors is challenged by the presence of blood vessels and time-varying thermal conductivity, which make patient-specific planning extremely difficult. By providing predictive tools, biophysical models may help clinicians to plan and guide the procedure for an effective treatment. We introduce a detailed computational model of the biophysical mechanisms involved in RFA of hepatic tumors such as heat diffusion and cellular necrosis. It simulates the extent of ablated tissue based on medical images, from which patient-specific models of the liver, visible vessels and tumors are segmented. In this thesis, a new approach for solving these partial differential equations based on the Lattice Boltzmann Method is introduced. The model is first evaluated against clinical data of patients who underwent RFA of liver tumors. Then, a comprehensive pre-clinical experiment that combines multi-modal, pre- and post-operative anatomical and functional images, as well as the interventional monitoring of the temperature and delivered power is presented. This enables an end-to-end validation framework that considers the most comprehensive data set for model validation. Then, we automatically estimate patient-specific parameters to better predict the ablated tissue. This personalization strategy has been validated on 7 ablations from 3 clinical cases. From the pre-clinical study, we can go further in the personalization by comparing the simulated temperature and delivered power with the actual measurements during the procedure. These contributions have led to promising results, and open new perspectives in RFA guidance and planning.