Objectifs : Développer un traitement HIFU (High-Intensity Focused Ultrasound) des anomalies placentaires au moyen d’un transducteur torique. Les essais ont été menés à partir d’un modèle ex-vivo, puis la faisabilité, l’efficacité et l’innocuité du traitement a été évaluée sur un modèle de guenons gestantes. Les premières applications thérapeutiques envisagées à l’échelle humaine, concernent le traitement du syndrome transfuseur-transfusé (STT) et les accrétions placentaires pour lesquelles un protocole d’essai clinique a été établi. Matériels et méthodes : Un transducteur torique fonctionnant à 3 MHz et muni d’une cellule d’imagerie échographique intégrée fonctionnant à 7,5 MHz ont été utilisés. Des simulations numériques de séquences de traitement HIFU ont été menées à partir d’une étude préliminaire sur la caractérisation acoustique du tissu placentaire humain. Ces séquences ont été testées au cours d’une étude ex-vivo sur des placentas humains. Deux modèles ex-vivo ont été conçus. Dans un premier temps, un modèle de traitement extracorporis. Dans un second temps, des traitements HIFU ont été réalisés à des distances variables du transducteur, par modification de la taille du ballonnet, afin de simuler un traitement per-césarienne. Le transducteur était placé au contact de la face foetale du placenta afin de simuler la séreuse utérine. A partir des résultats issus de ces essais ex-vivo, un protocole in-vivo sur des guenons gestantes a été mené afin de valider la faisabilité, l’efficacité et l’innocuité de la réalisation de lésions HIFU dans le placenta de guenons gestantes de manière totalement non-invasive. La qualité du monitoring échographique était évaluée au cours des trois études, et corrélée à l’analyse macroscopique. Une étude histologique a également été menée. Résultats : L’atténuation placentaire a été mesurée à partir de 12 échantillons placentaires humains pour un âge gestationnel compris entre 17 et 40 semaines d’aménorrhées (SA). L’atténuation augmentait en fonction de l’âge gestationnel et était compris entre 0,072 et 0,098 Np.cm-1.MHz-1. Lors d’un premier essai ex-vivo, 33 échantillons placentaires humains ont été inclus et soumis à une séquence HIFU, le temps d’insonification était de 55 secondes, la puissance acoustique utilisée était de 90 Watts. Au total, vingt-cinq lésions élémentaires étaient produites pour un diamètre et une profondeur moyens respectifs de 7,1 ± 3,2 et de 8,0 ± 3,1 millimètres. Huit lésions HIFU ont également été produites à partir de la juxtaposition de 6 tirs, pour un diamètre et une profondeur moyenne respectifs de 23,0 ± 5,0 et 11,0 ± 4,7 millimètres. Aucune lésion située en amont de la lésion produite n’a été observée pour une épaisseur de paroi abdominale similaire à celle d’une guenon gestante (10,8 ± 1,7 millimètres). Dans un second temps, 8 placentas humains pour un âge gestationnel compris entre 39 et 40 SA, ont été soumis à une séquence de traitement HIFU sans interposition de paroi abdominale. Le temps d’exposition était de 75 secondes pour une puissance acoustique de 90 Watts. Les lésions placentaires ont été produites à 2 (n=4), 6 (n=4), 7 (n=4) et 8 (n=7) millimètres de la surface du placenta. Au total, 19 lésions placentaires ont été produites, pour un diamètre et une profondeur moyenne respectifs de 14,6 ± 2,1 et de 14,1 ± 2,3 millimètres. Au cours de l’étude in-vivo, 8 guenons ont été incluses pour un âge gestationnel moyen de 72 ± 4 jours. Les puissances acoustiques utilisées étaient de 65, 80, 110 et 120 Watts pour un temps de traitement de 30, 15, 20 et 20 secondes respectivement. Au total 6 lésions placentaires ont été produites à l’issu de 13 insonifications pour des diamètres moyens de 6,4 ± 0,5 mm, 7,8 ± 0,7 mm et une profondeur moyenne de 3,8 ± 1,5 mm [etc…] / Objectives: To develop a High-intensity Focused Ultrasound (HIFU) treatment for placental abnormalities. Trials were first conducted using an ex-vivo model. Then the safety, feasibility and efficacy were demonstrated using a pregnant monkey model. The first therapeutic applications for human concern the treatment of the twin-to-twin transfusion syndrome (TTTS) and placenta accreta, for which, a clinical trial has already been established. Materials and Methods: A toroidal HIFU transducer, with an integrated ultrasound imaging probe was used. Numerical simulations have allowed identifying HIFU treatment parameters based on a preliminary experiment measuring the acoustic attenuation of human placentae. These HIFU parameters were tested during an ex-vivo study on human placentae. Two models were used. First, an extracorporis model of treatment was developed. Second, a percesarean model was developed. HIFU lesions were performed at different distances from the transducer, by adjusting the quantity of water between the transducer and tissues. The transducer was placed in contact with the fetal side of the placenta in order to simulate the uterine serosa. Using the results of these studies, an in-vivo study was conducted in a pregnant monkey model. The aim was to evaluate the feasibility, the efficacy and the harmlessness of the HIFU treatment applied to the placenta non invasively. The ultrasound monitoring was assessed during these three studies, and was correlated to the macroscopic examination. A histological study was also performed. Results: The placental attenuation was measured using 12 placental samples for a gestational age from 17 to 40 weeks of gestation (WG). The attenuation coefficient increased according to the gestational age, and was ranged from 0,072 to 0,098 Np.cm-1.MHz-1. During the first experimental ex-vivo study, 33 human placental samples were included and treated with HIFU. The treatment parameters were an exposure time of 55 seconds and an acoustic power of 90 Watts. Twenty-five HIFU singles lesions were created with an average diameter and depth of 7.1 ± 3.2 and 8.0 ± 3.1 millimeters, respectively. Eight HIFU lesions were also created by juxtaposing 6 single HIFU lesions. The average diameter and depth of these juxtaposed lesions were 23.0 ± 5.0 and 11.0 ± 4.7 millimeters, respectively. No secondary lesion was observed in overlying abdominal tissues. The thickness of these intervening tissues was similar to a pregnant monkey (10.8 ± 1.7 millimeters). In a second set of experiments, 8 human placentae for a gestational age ranging between 39 and 40 weeks were treated without intervening tissues. The time of exposure was 75 seconds and the acoustic power was 90 Watts. The placental lesions were created at 2 (n=4), 6 (n=4), 7 (n=4) and 8 (n=7) millimeters from the surface of the placenta. In total, 19 placental lesions were created with an average diameter and depth of 14.6 ± 2.1 and 14.1 ± 2.3 millimeters, respectively. Eight pregnant monkeys were included in the in-vivo experiments. The average gestational age was 72 ± 4 days. The placenta was treated non-invasively with acoustic powers of 65, 80, 110 and 120 Watts for a time of exposure of 30, 15, 20 and 20 seconds, respectively. In total, 6 placental lesions were created from 13 insonifications. The average diameters and depths of these lesions were 7.8 ± 0.7 and 3.8 ± 1.5 mm, respectively. No significant variation in maternal or fetal parameters was observed. All placental lesions appear hyperechoic in sonograms and well correlated with the macroscopic measurements. The ultrasound monitoring was better invivo when compared with ex-vivo results. The histological examination demonstrated a well delimited lesion of coagulation in all cases

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Bc. Michaela Jahodová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: In vitro and ex vivo study of drug-drug interactions of antiretrovirals on intestinal ATP-binding drug transporters The absorption of orally administered drugs takes place especially in the intestine, where it can affect by the activity of drug's ABC transporters located on the apical membrane of the intestinal epithelium. Study of drug interactions in intestinal ABC transporters is essential to ensure effective and safe pharmacotherapy. Testing of bi- directional transport on Caco-2 cells is generally the preferred method for in vitro evaluation of substrates and inhibitors of ABC transporters. Drawbacks of the Caco-2 model increase the need and necessity to introduce new models. A great potential is the involvement of ex vivo methodologies in the human or rat intestine. The aim of the work was to introduce an in vitro methodology using the Caco-2 cell monolayer and the ex vivo methodology of precision-cut rat intestinal slices. By the bi-directional transport method, we analyzed drug interactions of the model substrate P-gp and BCRP Rhodamine 123 (RHD123) and clinically-used tenofovir...

Das Monozyten chemotaktische Protein-1 (MCP-1) ist ein CC-Chemokin, das in seiner Rolle als Chemoattraktor auf Monozyten in der Genese von Malignomen eine wesentliche Rolle spielt. Dabei kann es sowohl zur lokalen Tumorabwehr als auch zur Tumorgenese, Tumor-angiogenese und Metastasierung beitragen. Die vorliegende Arbeit untersucht die MCP-1-Inhibition und die Stimulation durch MCP-1 auf die Koloniebildung und die Zytokinexpression von Plattenepithelkarzinomen der Kopf-Hals-Region (HNSCC) im FLAVINO-Assay. Dieser ist ein klonogener, qualitätskontrollierter Ex-vivo-Koloniebildungsassay, der an der Klinik für Hals-Nasen-Ohrenheilkunde der Universität Leipzig etabliert und patentiert wurde und unter flavinschützenden Bedingungen durchgeführt wird. Weiterhin wird die Eignung von MCP-1, Interleukin-6 (IL-6), Interleukin-8 (IL-8) und des Vascular endothelial growth factor (VEGF) als Biomarker in HNSCC, die mithilfe von ELISA in Seren und Kulturüberständen quantifiziert wurden, untersucht. Durch die Stimulation durch MCP-1 und dessen Blockade sowie durch in vivo tolerierbare Konzentrationen von Cisplatin, Docetaxel, Cilengitide und Temsirolimus wurde die Expression der untersuchten Zytokine in den Kulturüberständen der HNSCC unterschiedlich moduliert. Cisplatin und MCP-1 supprimierten die Koloniebildung signifikant, während unter Docetaxel und Temsirolimus eine insignifikante Reduktion und durch Cilengitide eine insignifikante Stimulation der Koloniebildung beobachtet wurde. Die MCP-1-Blockade durch einen Anti-MCP-1-Antikörper führte zu keiner signifikanten Modulation der Koloniebildung. MCP-1 und der Anti-MCP-1-Antikörper senkten die Zytokinexpression, während bis auf Cisplatin alle Zytostatika die Zytokinexpressionen steigerten. Bezüglich der kombinierten Testung der Zytostatika und der MCP-1-Blockade bzw. Stimulation unterschieden sich die Proben, sodass additive, synergistische und antagonistische Effekte resultierten. Da durch MCP-1 gesteuerte tumorassoziierte Makrophagen das Mikromilieu eines Tumors wesentlich beeinflussen, gebührt diesen ebenfalls eine besondere Aufmerksamkeit. In dieser Arbeit wurden unter MCP-1 antitumoröse Effekte beobachtet, sodass weitere klinische Testungen der antitumorösen Wirkung des MCP-1 auf HNSCC lohnenswert erscheinen. Die individuelle Chemoresponse-Testung kann dabei helfen, das biologisch heterogene Verhalten der HNSCC besser zu verstehen. In diesem Sinne wäre die klinische Validierung solcher Testsysteme wertvoll.

Un défi majeur en oncologie clinique est de caractériser avec précision la réponse des patients aux agents thérapeutiques. Actuellement, il n'existe pas de modèles et de tests fiables capable de reproduire précisément une tumeur primaire dans toute sa complexité. Or, ce paramètre est essentiel pour mettre en œuvre une stratégie de médecine personnalisée capable d'identifier le régime de traitement le plus approprié pour un patient particulier dans un délai cliniquement pertinent. Pour répondre à ce besoin, notre groupe a développé un nouveau modèle 3D ex vivo qui repose sur la micro-dissection d'un échantillon de tumeur (MDT) d'un patient et l'utilisation de technologies microfluidiques pour maintenir la viabilité du tissu et le microenvironnement tumoral naturel afin d’évaluer la sensibilité aux traitements dans un délai adapté à la prise de décision clinique. Cette approche permettrait de sélectionner les thérapies les plus efficaces tout en réduisant l'administration de traitements inefficaces associés à des effets secondaires indésirables, ainsi que les coûts de prise en charge des patients. Des travaux précédemment publiés par notre équipe ont montré que la viabilité des cellules cancéreuses situées dans notre modèle de tumeur ex vivo pouvait être caractérisée par microscopie confocale sur l’intégralité du MDT ou par cytométrie de flux sur les MDTs après dissociation enzymatique des cellules. Cependant, ces techniques présentent des limitations en termes de résolution visuelle pour la microscopie confocale et de sensibilité et information spatiale pour la cytométrie de flux. Nous proposons ici d’associer notre modèle 3D de MDTs en microfluidiques à des techniques d’immuno-histopathologie, dans le but d’offrir une évaluation moléculaire, spatiale et quantitative de la réponse de la tumeur au traitement. Pour cela, nous avons optimisé une procédure de lithographie en paraffine de nos systèmes microfluidiques, permettant la production de blocs de micro-étalages micro-réseaux de tissus micro-disséqués (MDTMA). afin de permettre une coloration morphologique du tissu et un marquage de protéines spécifiques pour analyser l'architecture tissulaire, la prolifération et l’apoptose cellulaire au sein des échantillons traités. En outre, nous avons montré que le modèle ex vivo est comparable et corrélé au système de modèle de souris in vivo de référence pour l'essai de chimio-sensibilité. Suite à l’optimisation de ce modèle, nous avons collecté 25 échantillons de tumeurs de patientes atteintes de cancer de l’ovaire, pour réaliser des MDTs et des cultures de cellules primaires afin de comparer les profils transcriptomiques de ces deux modèles avec celui de la tumeur d’origine, et d'analyser les réponses aux traitements et le microenvironnement tumoral. Les données transcriptomiques obtenues par micropuces ARN nous ont permis d'effectuer une analyse bio-informatique des voies de signalisation incluant un groupement hiérarchique non supervisé. Nos résultats montrent que les MDT à chaque point de temps (jour 0, 8 et 15) sont génétiquement similaires à la tumeur primaire par opposition aux cultures cellulaires primaires, et que les principales voies dérégulées sont impliquées dans la réponse cellulaire au stress. Nous avons observé une viabilité élevée des cellules au sein des MDT sur une période de culture de 15 jours. En outre, nous avons déterminé qu'un régime de chimiothérapie (carboplatine et paclitaxel) consistant en une induction thérapeutique de 10 heures suivie d'une période de récupération de 14 heures était idéal pour caractériser la réponse au traitement. Notre analyse de prédiction de la réponse des patients montre que nous avons une corrélation positive élevée d'une efficacité de 95 % entre la réponse ex vivo et la réponse clinique pour les patients appariés. En général, nos résultats suggèrent que notre technique fournit un modèle plus sophistiqué et précis pour récapituler la réponse de la tumeur primaire dans un laps de temps cliniquement adapté, et pourrait servir de plateforme pour tester de nouvelles thérapeutiques, et d'outil d'orientation clinique pour la réponse des patients. / A major challenge in clinical oncology is the inability to accurately predict the patients’ response to therapeutic agents. Currently, there are no reliable models and assays available that reiterate the immense complexity of a primary tumor. These factors are important to implement a personalized medicine strategy capable of identifying the most suitable treatment regimen for a particular patient in a clinically relevant timeframe. To answer this need, our group has developed a novel ex vivo 3D model that relies on the micro-dissection of a patient’s tumor specimen and the utilization of microfluidic technologies to monitor drug sensitivity within a time-frame suitable for clinical decision-making. This approach would allow for better selection of effective therapies and limit the administration of ineffective treatments, further improving treatment outcome of patients while reducing cost and drug-induced toxicities. Previously published work studied that the viability of cancer cells located within the tumor was characterized using two imaging modalities: confocal microscopy and flow cytometry. However, each technique has its own disadvantage, limiting their ability to molecularly characterize the effect of therapeutic agents on cancer cells. Thus, we hypothesize that our 3D ex vivo tumor-derived model coupled to a pathology-like tool would allow for a more comprehensive approach to evaluate tumor response to treatment, providing a readout system to closely mirror the patient’s response, and evaluating molecular mechanisms involved in response to drugs. To address this hypothesis, we optimized a paraffin-embedding lithography procedure allowing the production of micro-dissected tissue micro-array (MDTMA) block to allow morphological and protein-specific staining to analyze the cellular integrity and tissue architecture of treated samples. In addition, we showed that ex vivo model is comparable and correlated to the gold standard in vivo mouse model system for chemosensitivity assay. Moreover, we collected, following informed consent, 25 post-surgical OC patient tumor samples, to form micro-dissected tissues (MDTs), and primary cell cultures for micro-array analysis and characterization of the TME and response prediction. The micro-array data allowed us to perform unsupervised hierarchical clustering and pathway analysis showing that the MDTs at each time-point (day 0, 8 and 15) are genetically similar to the primary tumor as opposed to the primary cell cultures and that main deregulated pathways are involved in cellular response to stress. We observed a high viability of cells within MDTs over a culture period of 15 days. In addition, we determined that a treatment regimen consisting of a 10-hour therapy induction followed by a 14-hour recovery period was ideal for characterizing carboplatin treatment response. Our response prediction analysis of patients shows that we have a high positive correlation of 95% efficiency between ex vivo and clinical response for matched patients. In general, our results suggest that our ex vivo drug response model provides a more sophisticated model to recapitulate primary tumor response in a clinically suitable timeframe that can be exploited further serving, in part, as a platform to test new therapeutics and as a clinical guidance tool for patient response.

Alveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. Here we show that mouse long-term ex vivo expanded AMs (exAMs) maintained a core AM gene expression program, but showed culture adaptations related to adhesion, metabolism and proliferation. Upon transplantation into the lung, exAMs reacquired full transcriptional and epigenetic AM identity, even after several months in culture and could self-maintain long-term in the alveolar niche. Changes in open chromatin regions observed in culture were fully reversible in transplanted exAMs and resulted in a gene expression profile indistinguishable from resident AMs. Our results indicate that long-term proliferation of AMs in culture did not compromise cellular identity in vivo. The robustness of exAM identity provides new opportunities for mechanistic analysis and highlights the therapeutic potential of exAMs.

Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are leveraged to rapidly optimize several parameters of the ex vivo DNA assembly methodology testing lysates from different engineered strains of E. coli, with various buffer components and using titrations of purified cloning enzymes. Finally, in order to complete the cloning suite, a vector expressing the Pyrococcus furiosis (Pfu) DNA polymerase was designed, constructed and expressed in E. coli to create a ‘functionalized lysate’ capable of ex vivo PCR. Not only do we demonstrate ex vivo cloning methodology as a complete cloning package capable of replacing the expensive cloning reagents currently required by synthetic biologists, but also establish ex vivo as an overarching approach for conducting molecular biology.

A comparison of the antimicrobial efficacy of silver diamine fluoride and silver nitrate on various cariogenic bacteria: an ex vivo study By: Reham AlNajjar, D.D.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Dentistry at Virginia Commonwealth University. Virginia Commonwealth University, 2019 Thesis Advisor: William Dahlke, D.M.D., Associate Professor and Chair of Pediatric Dentistry, School of Dentistry Purpose: The use of silver-based antimicrobials is an emerging method for the treatment of dental caries. In this study, the authors compare the efficacy of the two most prominent silver- based therapeutics, silver diamine fluoride (SDF) and silver nitrate (AgNO3), on cariogenic and non-cariogenic multispecies biofilms. Currently there is a lack of studies comparing the efficacy of SDF to AgNO3. Methods: Plaque samples from anterior and posterior tooth sites from children presenting both with early childhood caries and caries-free children were collected, pooled, and utilized to create four ex vivo biofilm systems in artificial saliva. SDF and AgNO3 were administered to these biofilms and bacterial survival was quantified and compared to untreated controls. Results: Each of the four pooled sample types was applied to plates coated in artificial saliva + 1% sucrose. Both SDF and AgNO3 were very effective against plaque derived biofilms when compared to untreated biofilms (P0.05) in the potency of each compound. Conclusions: SDF and AgNO3 significantly inhibit ex vivo cariogenic and non-cariogenic biofilms at similar levels.

Wireless capsule endoscopy (WCE) is a state-of-the-art technology to receive images of human intestine for medical diagnostics. In WCE, the patient ingests a specially designed electronic capsule which has imaging and wireless transmission capabilities inside it. While the capsule travels through the gastrointestinal (GI) tract, it captures images and sends them wirelessly to an outside data logger unit. The data logger stores the image data and then they are transferred to a personal computer (PC) where the images are reconstructed and displayed for diagnosis. The key design challenge in WCE is to reduce the area and power consumption of the capsule while maintaining acceptable image reconstruction. In this research, the unique properties of WCE images are identified by analyzing hundreds of endoscopic images and video frames, and then these properties are used to develop novel and low complexity compression algorithms tailored for capsule endoscopy. The proposed image compressor consists of a new YEF color space converter, lossless prediction coder, customizable chrominance sub-sampler and an efficient Golomb-Rice encoder. The scheme has both lossy and lossless modes and is further customized to work with two lighting modes – conventional white light imaging (WLI) and emerging narrow band imaging (NBI). The average compression ratio achieved using the proposed lossy compression algorithm is 80.4% for WBI and 79.2% for NBI with high reconstruction quality index for both bands. Two surveys have been conducted which show that the reconstructed images have high acceptability among medical imaging doctors and gastroenterologists. The imaging algorithms have been realized in hardware description language (HDL) and their functionalities have been verified in field programmable gate array (FPGA) board. Later it was implemented in a 0.18 μm complementary metal oxide semiconductor (CMOS) technology and the chip was fabricated. Due to the low complexity of the core compressor, it consumes only 43 µW of power and 0.032 mm2 of area. The compressor is designed to work with commercial low-power image sensor that outputs image pixels in raster scan fashion, eliminating the need of significant input buffer memory. To demonstrate the advantage, a prototype of the complete WCE system including an FPGA based electronic capsule, a microcontroller based data logger unit and a Windows based image reconstruction software have been developed. The capsule contains the proposed low complexity image compressor and can generate both lossy and lossless compressed bit-stream. The capsule prototype also supports both white light imaging (WLI) and narrow band imaging (NBI) imaging modes and communicates with the data logger in full duplex fashion, which enables configuring the image size and imaging mode in real time during the examination. The developed data logger is portable and has a high data rate wireless connectivity including Bluetooth, graphical display for real time image viewing with state-of-the-art touch screen technology. The data are logged in micro SD cards and can be transferred to PC or Smartphone using card reader, USB interface, or Bluetooth wireless link. The workstation software can decompress and show the reconstructed images. The images can be navigated, marked, zoomed and can be played as video. Finally, ex-vivo testing of the WCE system has been done in pig's intestine to validate its performance.

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, 2006. / Submitted by Fernanda Weschenfelder (nandaweschenfelder@gmail.com) on 2009-10-05T19:59:50Z No. of bitstreams: 1 Cesar Augusto M e Silva - tese de doutorado.pdf: 12677167 bytes, checksum: fbc5893a3b0a2783f94b0033256bf3d1 (MD5) / Approved for entry into archive by Guimaraes Jacqueline(jacqueline.guimaraes@bce.unb.br) on 2009-11-24T13:24:50Z (GMT) No. of bitstreams: 1 Cesar Augusto M e Silva - tese de doutorado.pdf: 12677167 bytes, checksum: fbc5893a3b0a2783f94b0033256bf3d1 (MD5) / Made available in DSpace on 2009-11-24T13:24:50Z (GMT). No. of bitstreams: 1 Cesar Augusto M e Silva - tese de doutorado.pdf: 12677167 bytes, checksum: fbc5893a3b0a2783f94b0033256bf3d1 (MD5) Previous issue date: 2006 / Introdução: Os fatores mecânicos pulmonares relacionados às vias aéreas e à microvasculatura desempenham papel fundamental no contexto da lesão de isquemia e reperfusão (I/R). O objetivo deste trabalho foi estudar o impacto do flush vascular pulmonar (FVP), da isquemia fria e o efeito de estratégias de recrutamento alveolar sobre as propriedades mecânicas e morfométricas e correlacionálas à função pulmonar durante a reperfusão ex-vivo. Material e Métodos: 128 ratos da raça Wistar foram divididos em 4 grupos (i) controle: pulmões isolados submetidos a 15 minutos de isquemia à temperatura ambiente; (ii) Flush: pulmões isolados submetidos ao flush vascular pulmonar (FVP) com solução de Euro-Collins (EC) e preservados a 4 0C por 10 horas (isquemia fria); (iii) CPT: pulmões isolados submetidos ao FVP com EC e à isquemia fria e que foram recrutados à CPT por 2 minutos antes do início da reperfusão; (iv) VC: pulmões isolados submetidos ao FVP com EC e ventilados com volume corrente (VC) por 10 minutos antes do início da reperfusão. Todos os pulmões foram reperfundidos durante 60 minutos, tempo no qual foram estudadas as trocas gasosas, as propriedades mecânica e hemodinâmica. Ao final da reperfusão, o peso líquido/peso seco foi calculado e a análise morfométrica realizada. Em outra etapa, 40 ratos da raça Wistar foram divididos em 5 grupos: (i) controle: pulmões com sangue; (ii) Flush: pulmões submetidos ao FVP com EC; (iii) Isquemia: pulmões submetidos ao FVP com EC e submetidos à isquemia fria; (iv) CPT: pulmões submetidos ao FVP com EC, submetidos à isquemia fria e recrutados à CPT por 2 minutos; (v) VC: pulmões submetidos ao FVP com EC, submetidos à isquemia fria e ventilados com volume corrente por 10 minutos. A análise das propriedades mecânicas e o estudo morfométrico foram realizados em todos os pulmões dos 5 grupos. Resultados: o FVP e a isquemia fria aumentaram (i) a percentagem de área alveolar colapsada, (ii) a dissipação de energia nos componentes viscoelásticos pulmonares e (iii) a eslastância estática pulmonar. Estes pulmões desenvolveram edema fulminante durante os primeiros 15 minutos de reperfusão. O recrutamento à CPT e a ventilação com VC atenuaram as alterações mecânicas e morfométricas provocadas pela preservação e evitaram a lesão I/R. Durante a reperfusão, as trocas gasosas, as propriedades mecânica e hemodinâmica dos pulmões recrutados foi similar a dos controle. Conclusões: as alterações mecânicas e morfométricas causadas pela preservação pulmonar aumentam a impedância dos pulmões tornando-os mais susceptíveis à lesão I/R, e as manobras de recrutamento alveolar empregadas imediatamente antes do início da reperfusão atenuam as inomogeneidades pulmonares causadas pela preservação e previnem a lesão I/R. ___________________________________________________________________________________ ABSTRACT / Rationale: mechanical factors related to the airways and to the pulmonary microvasculature play a fundamental role in the context of ischemic-reperfusion injury (I/R). The goal of this study is to investigate the impact of pulmonary vascular flush (PVF), cold ischemia and alveolar recruitment on the mechanical and morphometrical properties and to correlate these findings and pulmonary hemodynamics and gas exchange during the reperfusion. Methods: 128Wistar rats were randomically divided into 4 groups: (i) Control: fresh lungs; (ii) Flush: lungs flushed with Euro-Collins solution (EC), preserved at 4 0C for 10 hours (cold ischemia); (iii) TLC: flushed lungs, preserved at 4 0C for 10 hours, inflated to TLC for 2 minutes prior to rapid reperfusion; (iv) VT: flushed lungs preserved at preserved at 4 0C for 10 hours, ventilated with tidal volume during 10 minutes prior to rapid reperfusion. All lungs were subjected to an ex-vivo reperfusion during 60 minutes. In this period, the gas exchange, the mechanical and hemodynamical properties were evaluated and, at the end, the wet-to-dry ratio was calculated and the morphometrical analysis performed. In other phase, 40 Wistar rats were randomically divided in 5 groups: (i) Control: fresh lungs; (ii) Flush: lungs flushed with EC; (iii) Ischemia: flushed lungs, preserved at 4 0C for 10 hours; (iv) TLC: flushed lungs, preserved at 4 0C for 10 hours, inflated to TLC for 2 minutes; (v) VT: flushed lungs preserved at 4 0C for 10 hours, ventilated with tidal volume during 10 minutes. The end-inflation occlusion method was used to evaluate mechanical properties of the lungs. The morphometrcial analysis was also performed. Results: PVF and cold ischemia increased (i) the percentage of alveolar colapsed area; (ii) the energy dissipation on the lung viscoelastic components and (iii) the pulmonary lung elastance. These lungs (ischemic not subjected to alveolar recruitment prior to reperfusion) presented severe pulmonary edema, whereas the function of the recruited lungs were similar to that of fresh lungs. Conclusions: cold preservation increases pulmonary impedance and the huffiness to the I/R injury, and the alveolar recruitment manoeuvres performed before the reperfusion reverts, or at least, decreases the pulmonary inhomogeneities caused by the preservation and warns the I/R injury.

La recherche et développement de dispositifs médicaux avec diverses applications en diagnostiques et en thérapie ont été réalisés. Actuellement, tous les systèmes micro-ondes disponibles d'hyperthermie proposent uniquement des traitements avec une puissance élevée de micro-ondes. Dans cette thèse, un nouveau système d'hyperthermie micro-ondes est étudié pour le bénéfice des fonctions de diagnostic et de thérapie. L'utilisation d'un applicateur avec un niveau très faible et inoffensif de puissance micro-ondes permet de faire le premier diagnostic. Le traitement thérapeutique thermique sera effectué en utilisant le même applicateur avec une puissance micro-ondes élevée et adaptée sur la partie pathologique. Des caractérisations micro-ondes large bande de cinq tissus biologiques différents ont été effectuées à différentes températures avec une méthode de sonde coaxiale ouverte et le modèle de ligne virtuelle. Les expérimentations ex vivo d'hyperthermie micro-ondes avec des puissances de quelques watts à 2,45GHz ont été réalisées sur ces tissus d'épaisseurs variées. L'évolution de la température des tissus a été mesurée en utilisant un capteur infrarouge. Les simulations électromagnétiques et thermiques pour les expérimentations ex vivo d'hyperthermie micro-ondes ont été effectuées en utilisant COMSOL Multiphysics avec la méthode des éléments finis et la symétrie axiale 2D en considérant les tissus variés de différentes épaisseurs et puissances micro-onde incidente. Les simulations du modèle correspondent bien aux mesures. Cette recherche illustre la possibilité d'avoir un câble coaxial souple et adapté à la fois au diagnostic et au traitement pour une thérapie mini invasive. / Research and development of medical devices with various diagnostic and therapeutic applications have been carried out in different countries because of the great advances in electronic and electromagnetic devices during recent decades. However, at present, all of available existing microwave hyperthermia system can just offer treatment, by using high microwave power. In this thesis, a new microwave hyperemia system is researched which could have both diagnostic and therapeutic functions. One single applicator is used to measure dielectric properties of tissue with a very low harmless microwave power for diagnosis first. Then thermal therapeutic treatment will be carried out by using the same applicator with higher and adapted microwave power. Microwave broad band characterization of five different biological tissues at different temperatures with an open–ended coaxial probe method and the virtual line model has been carried out. Ex vivo microwave hyperthermia experiments using microwave power of a few Watts at 2.45GHz have been carried out on five tissues of various thicknesses. Temperature evolution of the biological tissues has been measured by using an infra-red senor. Electromagnetic and thermal simulations for ex vivo microwave hyperthermia experiment have also been achieved by using COMSOL Multiphysics software with 2D axisymmetrical finite–element method and considering different tissues of various thicknesses and incident microwave powers. Simulation results correlate well with the experimental ones. This research, illustrates the possibility to have a flexible and feasible coaxial cable for both diagnosis and treatment for a minimally invasive therapy.