Blood perfusion is the local, non-directional blood flow through tissue. It is measured as the volumetric flow rate of blood through a given volume of tissue. One method that has been developed for measuring blood perfusion is a probe that measures the temperature response of the tissue when a thermal event is applied. From the temperature response, the blood perfusion and contact resistance can be estimated by comparing the experimental response to a predicted response, and employing Gaussian minimization techniques to estimate the blood perfusion and contact resistance. The objective of this research was to assess the measurement repeatability and sensitivity of the blood perfusion probe by testing the probe on phantom tissue, such that the effects of physiologic or pathologic conditions on the blood perfusion could be eliminated. Another objective was to conduct a preliminary in vivo study using rats for the purpose of establishing proper experimental protocols for future testing of the blood perfusion probe. A phantom tissue test stand comprised of porous material and water to simulate tissue and blood, respectively, was constructed for the phantom study. Inlet flow rates into the porous media ranging between 0 cc/min and 30 cc/min were tested. To test the measurement repeatability 7 flow rates (0, 5, 10, 15, 20, 25 and 30 cc/min) were tested on two different days. To test the measurement sensitivity of the probe, flow rates between 0 and 10 cc/min, and 15 and 20 cc/min were tested at intervals of 1 cc/min. From the phantom study it was concluded that the probe displayed good measurement repeatability, as the trend in perfusion as a function of inlet flow rates for both days was found to be the same. It was also found that the data collected using the probe yielded significantly different perfusion estimates for different flow rates, as statistical analyses show that the average perfusion differences between flow rates are truly independent within a 90% confidence interval, for flow differences above 4 cc/min. It was found that for flow rates below 4 cc/min the probe sensitivity was significantly reduced. For the in vivo study it was concluded that the probe can be used to obtain estimates of perfusion from rats. This preliminary study also served to establish proper experimental protocols for future tests. / Master of Science

A non-invasive blood perfusion system has been developed and tested in a phantom tissue and an animal model. The system uses a small sensor with a laminated flat thermocouple to measure the heat transfer response to an arbitrary thermal event (convective or conductive) imposed on the tissue surface. Blood perfusion and contact resistance are estimated by comparing heat flux data with a mathematical model of the tissue. The perfusion system was evaluated for repeatability and sensitivity using both a phantom tissue test stand and exposed rat liver tests. Perfusion in the phantom tissue tests was varied by controlling the flow of water into the phantom tissue test section, and the perfusion in the exposed liver tests was varied by temporarily occluding blood flow through the portal vein. The phantom tissue tests indicated that the probe can be used to detect small changes in perfusion (0.009 ml/ml/s). The probe qualitatively tracked the changes in the perfusion of the liver model due to occlusion of the portal vein. / Master of Science

La angiogénesis y la neovascularización son procesos biológicos que tienen lugar en los tejidos y que están asociados al aumento de las demandas de oxígeno y nutrientes. En adultos normales estos procesos no suelen ocurrir. Sin embargo, en condiciones de enfermedad, como en inflamaciones o en el desarrollo de tumores, el VEGF (factor de crecimiento vascular endotelial, del inglés vascular endothelial growth factor), ls proteína señalizadora causante de la angiogénesis,está fuertemente expresada. En estas circunstancias se formas rápidamente nuevos vasos y capilares. Esta nueva red vascular es caótica y no presenta una estructura normal, especialmente en el caso de tumores. La cuantificación de la angiogénesis es esencial para evaluar el grado de agresividad de un tumor y la eficacia de los tratamientos. Es necesario desarrollar herramientas fiables y reproducibles que sean sensibles a cambios tempranos, lo cual puede permitir utilizar tratamientos más individualizados. En este sentido, el modelado farmacocinético de imágenes de perfusión por resonancia magnética (RM) es una valiosa herramienta para la evaluación de parámetros como la permeabilidad capilar, el coeficiente de extracción, el volumen intersiticial y el volumen vascular. / Sanz Requena, R. (2010). Methodological developments and clinical applications of pharmacokinetic models in contrast-enhanced magnetic resonance perfusion studies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8422

The Thermoelectric Perfusion Probe is a completely electronic system that cyclically heats and cools tissue to measure blood perfusion. The probe produces the thermal event with a thermoelectric cooler and then measures the resulting heat flux and temperatures: the arterial temperature and the sensor temperature (the temperature between the heat flux gage and the skin). The Thermoelectric Perfusion Probe was validated and calibrated on a phantom tissue test stand, a system that simulates perfusion with known, controlled flow. With the new pressed sensor technology, a thermocouple sealed to a heat flux gage, the sensor temperature and the heat flux are simultaneously recorded. The pressed sensor tests validated the program used to predict perfusion for the Thermoelectric Perfusion Probe. This perfusion estimation program can determine the tissues perfusion regardless of how the thermal event is created (i.e. convective cooling, convective heating, conductive heating). Based on experimentation, the Thermoelectric Perfusion Probe displays good repeatability and sensitivity for continuously measuring perfusion. The sensitivity and repeatability of the Thermoelectric Perfusion Probe was proven when the perfusion estimates were compared to the perfusion estimates predicted by the Convective Perfusion Probe, a previously validated perfusion probe, and the CFD Flow Model, a computational model of the phantom tissue test stand. / Master of Science

Human organoids have potential to revolutionize in vitro disease modeling by providing multicellular architecture and functional that are similar to those in vivo. Nonetheless, organoid-based, high-throughput screening (HTS) of compounds is challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Therefore, we first fabricated a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic culture via injection molding. We established various cell loading methods in hydrogels on the pillar plate. In addition, we investigated the effect of flow on the necrotic core of spheroids in the pillar/perfusion plate. Finally, we developed microarray three-dimensional (3D) bioprinting technology using the pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

Introduction : Le TNF-a, impliqué dans l’inflammation et la défense de l’hôte, aaussi des propriétés anti-tumorales et est utilisé dans le traitement local des sarcomesdes membres. P53 est un anti-oncogène dont la mutation est associée audéveloppement de tumeurs. Des données expérimentales ont démontré une relationentre activité anti-tumorale de TNF-a et le statut de p53. Matériel et méthodes : Notre objectif a été d’étudier en immunohistochimie lestatut de p53 chez 110 patients atteints de sarcomes et traités par TNF-a. Ensuite,dans 8 sarcomes cultivés ex-vivo, nous avons étudié la localisation de l’apoptoseinduite par le TNF-a par microscopie confocale. Puis, dans 9 lignées de sarcomeshumains, nous avons testé la relation p53/TNF-a en abrogeant p53 grâce à un sh-RNA, ou en utilisant des petites molécules telles que CP-31398 ou Nutlin-3a aptes àrestaurer p53. Enfin, pour mieux comprendre les mécanismes de résistance au TNF-a,nous avons mesuré par méthode EMSA la liaison de NF-kB à l’ADN et recherché parRT-PCR quels gènes de l’apoptose étaient différentiellement régulés.Résultats : Le statut muté de p53 corrèle avec la réponse histologique autraitement par TNF-a chez l’homme. L’apoptose induite par le TNF-a est trouvée aussibien au niveau de la cellule endothéliale que de la cellule tumorale. De plus, laréponse au TNF-a est modulée par le statut de p53 puisque dans les lignées étudiées,l’abrogation de p53 supprime celle-ci alors que la réparation d’une activité p53 permetde l’augmenter. Enfin, une potentialisation de l’apoptose induite par TNF-a estobservée lorsqu’il est associé avec CP-31398 ou Nutlin-3a et elle corrèle avec ladiminution de la liaison du NF-kB à l’ADN. Une augmentation de l’expression desgènes RIPK2, TP53BP2 et GADD45 et une diminution de l’expression de TGF-b1 etFAIM est observée lorsque l’association de Nutlin-3a et TNF-a est synergique sur lamort cellulaire.Conclusions : Ces résultats suggèrent que l’utilisation de molécules capablesde restaurer l’activité de p53 peut inverser la résistance des sarcomes des tissusmous au traitement par TNF-a. Dans ce contexte, des études cliniques pourraientexploiter cette approche pour l’utiliser dans le cadre des sarcomes particulièrementrésistants aux traitements conventionnels ainsi que dans d’autres tumeurs. / Introduction: Although named for its antitumor properties, TNF-a is implicatedin a wide spectrum of diseases including chronic inflammation, autoimmunity andcancer. It is used for the loco regional treatment of limb’s sarcoma. P53 is an antioncogenewhose mutation is associated with tumour development. Experimental datademonstrated that TNF-a cytotoxic activity and p53 status are related.Material and methods: Our objective was to study by immunohistochemistrythe p53 status in 110 sarcoma patients treated by isolated limb perfusion with TNF-a. Then, we studied by confocal microscopy in 8 freshly obtained sarcoma tumours,the localization of apoptosis. Finally, in 9 sarcoma cell lines with different p53 status,we tested the p53/TNF-a relationship by abrogating p53 with a sh-RNA and by usingsmall molecules known to restore p53 functions. To better understand the mechanismsof resistance to TNF-a, we measured by EMSA the NF-kB-binding to DNA and withRT-PCR, we explored the regulation of some apoptosis related genes.Results: We demonstrated a relationship between p53 status and thehistological response to TNF-a use in humans. TNF-a induced apoptosis was presentin endothelial cells as well as in the tumour cells. TNF-a cytotoxicity was dependent onthe p53 status since in the cell lines studied, p53 abrogation reduced it and p53restoration allowed it to increase. Moreover, a potentiation of TNF-a cytotoxic effectwas observed when it was combined to CP-31398 or Nutlin-3a. The killing magnitudewas therefore related to the decrease in the NF-kB-binding to DNA when Nutlin-3awas added. A gene expression increase for RIPK2, TP53BP2 and GADD45 and adecrease for TGF-b1 and FAIM was observed if the combined treatment wassynergistic on tumour death cells.Conclusions: These results suggest that the use of compounds able to restorep53 activity could reverse the soft tissue sarcoma’s resistance to TNF-a treatment.Clinical studies should be performed in order to utilize this approach and to use it inthe context of sarcomas that are particularly resistant to conventional treatments.

Le flux sanguin cérébral (CBF) caractérise la micro-circulation et l'irrigation des tissus. Cette information de perfusion cérébrale est utilisé en clinique pour le diagnostic et le suivi thérapeutique de nombreuses maladies. La technique de mesure de CBF la moins invasive est celle par marquage de spins artériels (ASL) où l'eau du sang fait office de traceur. L'objectif de cette thèse, menée dans le cadre d'une convention CIFRE, consistait à faciliter l'utilisation de séquences ASL continues et pseudo-continues (CASL, pCASL) ainsi qu'à améliorer leur performance en pré-clinique. En effet, la mesure quantitative de CBF par ASL est un protocole complexe qui nécessite plusieurs étapes d'ajustements, d'acquisitions et de traitement de données. Dans le but d'alléger ce protocole, un package CASL a été développé en collaboration avec Bruker. Plusieurs étapes d'ajustements et de post-processing ont été automatisées, rendant la génération de cartes CBF relatives et absolues plus aisée. Le champ magnétique élevé des scanners IRM pré-cliniques présente de nombreux avantages mais est également une source de problèmes en ASL. Nous nous sommes intéressés plus particulièrement à deux d'entre eux : l'instabilité du marquage de spins et l'échauffement induit par les séquences ASL. Pour stabiliser le marquage ASL, une stratégie d'optimisation de la séquence pCASL a été développée et testée chez le rat à 9.4 T. Ceci a permis l'obtention d'un marquage robuste, même en situations de shim dégradé. Le package pCASL a été partagé avec dix autres instituts dans le monde. L'échauffement induit lors de séquences CASL et pCASL par le dépôt d'énergie radiofréquence a été caractérisé globalement et localement, dans le cerveau et au niveau des carotides, pour deux configurations d'antenne d'émission. Pour finir, une séquence pCASL encodée en temps a été développée et appliquée à la souris, dans le cadre d'une collaboration avec des équipes néerlandaises du Leiden University Medical Center. Cet outil permet la mesure simultanée de CBF et du temps de transit artériel, un paramètre pouvant refléter des pathologies vasculaires sous-jacentes. / Cerebral blood flow (CBF) characterizes the blood supply to brain tissue. This perfusion-related parameter contributes in diagnosis and therapeutic follow-up in many diseases. The least invasive technique to measure CBF is arterial spin labeling (ASL), where arterial water is used as tracer. The aim of this PhD project, conducted within a CIFRE agreement (Convention Industrielle de Formation par la REcherche), was to increase the performance and to facilitate the use of continuous and pseudo-continuous arterial spin labeling (CASL, pCASL) tools in preclinical studies. CBF quantification by means of ASL is one of the most challenging MRI modalities in terms of the workflow, since additional adjustments, acquisitions and post-processing steps are required. First, to render the workflow smoother for the user, a CASL package has been developed in collaboration with Bruker. This workflow allows easier relative and absolute CBF measurements, thanks to the integration of automated adjustments and reconstruction steps. In a second step, problems arising at high magnetic field were addressed. A strategy to optimize the pCASL labeling sequence in order to obtain robust results was developed and its robustness towards suboptimal shim conditions was demonstrated at 9.4 T in rats. The developed pCASL-package, consisting of three sequences, was shared with ten other institutes worldwide. Another issue encountered at high magnetic fields is heating due to RF power deposition, which was assessed locally in the brain and in the carotids, as well as globally, for the CASL and pCASL sequences and for two different transmit coil configurations. In a third step, time-encoded pCASL was developed in mice in collaboration with teams of the Leiden University Medical Center. This tool enables the simultaneous mapping of CBF and arterial transit time, a parameter that can reflect underlying pathologies such as increased vessel tortuosity or occlusion.

Tissue engineering strategies that combine porous biomaterial scaffolds with cells capable of osteogenesis or bioactive proteins have shown promise as effective bone graft substitutes. Attempts to culture bone tissue-engineering constructs thicker than 1mm in vitro often result in a shell of viable cells and mineralized matrix surrounding a necrotic core. To address this limitation, we developed a perfusion bioreactor system that improves mass transport throughout large cell-seeded constructs. Additionally, we established and validated 3-D computational methods to model flow and shear stresses within the microporosity of perfused constructs. Micro-CT scanning and analysis techniques were used to non-destructively monitor mineral development over time in culture. CFD modeling of axial perfusion through cylindrical scaffolds with a regular microarchitecture revealed a uniform flow field distributed throughout the scaffold. Perfusion resulted in a 140-fold increase in mineral deposition at the interior of 3 mm thick polymer scaffolds seeded with rat bone marrow stromal cells. The total detected mineral volume tripled as the construct length was increased from 3 to 9 mm. Increasing scaffold length to 9 mm did not affect the mineral volume fraction (MVF) within the full volume of each construct. Mineral volume, spatial distribution, density, particle size and particle number were then quantified on cell-seeded constructs in 5 different culture environments. The effect of time varying flow conditions was compared with continuous perfusion as well as two different control cell culture methods in an attempt to enhance mineralized matrix within the constructs. Intermittent elevated perfusion and dynamic culture in an orbital rocker plate produced the greatest amount of mineral within 9 mm long constructs compared to low continuous flow and high continuous flow cases. Together, these studies indicate that dynamic culture conditions enhance construct development with regards to cell viability, mineralized matrix deposition, growth rate, and distribution. Furthermore, these techniques provide a rational approach to selecting perfusion culture conditions that optimize the amount and distribution of mineralized matrix production. Finally, the established perfusion bioreactor, in combination with micro-CT analysis, provides a foundation for evaluating new scaffolds and cell types that may be useful for the development of effective bone graft substitutes.

Thesis--Copenhagen University. / Summary in Danish. Includes bibliographical references (p. 96-120).

Thesis--Copenhagen University. / Summary in Danish. Includes bibliographical references (p. 96-120).