Dans l'imagerie moléculaire par ultrasons, des microbulles sont fonctionnalisées avec des ligands. Après injection intraveineuse, ces microbulles ciblées s'accrochent aux marqueurs présents sur l'endothélium tumoral et permettent une détection non-invasive. Dans cette thèse, l'imagerie moléculaire par ultrasons a été développée pour la détection de la nétrine-1, qui est surexprimée dans 70% des cancers du sein et promeut la survie cellulaire. Une nouvelle thérapie moléculaire interférant avec la nétrine-1 a été développée et nécessite l'identification des patientes qui pourront bénéficier de ce traitement. Avec l'imagerie moléculaire de la nétrine-1, il a été possible de discriminer les tumeurs positives pour la nétrine-1 des tumeurs négatives. Par sa capacité à détecter de manière spécifique la nétrine-1 présentée sur l'endothélium des tumeurs, cette technique d'imagerie pourrait donc devenir un test d'accompagnement pour la thérapie d'interférence de la nétrine-1 chez les patientes atteintes de cancer du sein.La destruction ciblée des microbulles par ultrasons induit la cavitation et la sonoporation qui perméabilisent le tissu et facilite la délivrance locale de médicaments. De plus, cette destruction ciblée peut induire l'infiltration de cellules immunitaires et la libération d'antigènes tumoraux, déclenchant une réponse immunitaire anti-tumorale. Dans cette thèse, nous avons quantifié l'activation de la réponse immunitaire dans le carcinome hépatocellulaire, suivant la délivrance de nanoparticules chargés en microARN-122 et anti-microARN-21. Dans les nœuds lymphocytaires tumoraux, une baisse d'expression des cytokines pro-tumorales et une augmentation d'expression des cytokines anti-tumorales ont été observées, suggérant une réponse thérapeutique positive. L'approche thérapeutique de destruction ciblée des microbulles par ultrasons pour la délivrance de micro-ARN s'avère donc être un outil immuno-modulatoire puissant / Ultrasound molecular imaging uses microbubbles as ultrasound contrast agents which are functionalized with targeting ligands. Upon intravenous injection, targeted microbubbles bind to molecular markers presented on the tumor endothelium and enable the non-invasive assessment cancer-related biomarkers. In the present thesis, ultrasound molecular imaging was developed for detection of netrin-1, which is upregulated in 70% of metastatic breast cancer and promotes cell survival. A newly developed netrin-1 interference therapy requires the identification of patients who overexpress the target protein and, could benefit from anti-netrin-1 therapy. In vivo imaging of netrin-1 showed a significantly increased imaging signal in netrin-1-positive breast tumors compared to netrin-1-negative breast tumors and normal mammary glands. The results suggest that ultrasound molecular imaging allows accurate detection of netrin-1 on the endothelium of netrin-1-positive tumors and has the potential to become a companion diagnostic for netrin-1 interference therapy in breast cancer patients.Ultrasound-targeted microbubble destruction triggers cavitation and sonoporation thereby permeabilizing the tissue and facilitating local drug delivery. Further, immune cell infiltration and tumor antigen release are induced and trigger anti-tumor immune responses. In the present thesis, ultrasound-targeted microbubble destruction-mediated delivery of anti-cancer microRNA-122 and anti-microRNA-21 is studied for immune response activation in hepatocellular carcinoma, in which the immune microenvironment is deregulated. Tumor lymph nodes showed pro-tumor cytokine downregulation and anti-tumor cytokine upregulation, suggesting an overall positive therapy response with regard to the tumor immunology. The results identified ultrasound-targeted microbubble destruction-mediated miRNA delivery as a potent immuno-modulatory therapeutic approach

Macroalgal beds provide the ecological foundations for most shallow reef ecosystems in temperate environments. With distinctive canopies primarily of brown laminarian algae (northern hemisphere), or laminarian or fucalean algae (southern hemisphere), in many areas these habitats are at risk from human activity. Overexploitation, pollution, and other effects of coastal activities have resulted in significant habitat loss in coastal ecosystems, and human-induced climate change is now seen as a major threat to ecosystem health in marine systems. Understanding the impact of climate change is particularly important for habitat-forming ecosystem engineers like kelps, as these species form the basis of hierarchically organised communities and play a fundamental role in determining community structure and ecological processes. South eastern Australia has experienced increases in marine temperatures at nearly four times the global average, and there is now evidence that, in some locations, macroalgae communities are retreating in a manner consistent with ocean warming. Successful management of marine systems requires understanding ecosystem processes, particularly the patterns and magnitude of production. Macroalgal communities often show relatively low resistance to disturbance, yet rapid recovery once disturbances are removed, hence they are generally highly dynamic in response to environmental perturbations. As a result, macroalgae are likely to play an increasingly important role in buffering the short term/dynamic effects of climate change on temperate reef communities.Knowledge of the productivity of seaweed-dominated temperate reef systems is largely a synthesis from studies conducted over small spatial scales utilising a variety of methods that generally measure different characteristics of both individual seaweeds and collectively. As a result of the diversity of measurement methods, estimates of gross primary productivity (GPP), production potential, and macroalgal biomass for temperate reefs are numerous and variable. This can lead to challenges for ecologists attempting to amalgamate research findings to facilitate long-term, broad-scale perspectives or compare short-term research between spatially separated communities. However, to date there has been relatively little research to compare measurement approaches and quantify differences in productivity estimates across the different techniques.The present research provides a unique investigation into some of the techniques and methodology involved in measuring primary productivity in marine systems, particularly kelp forests, using the macroalgae Ecklonia radiata, Phyllospora comosa and Macrocystis pyrifera as study species. The work is based on both field and laboratory exploration of productivity measurements and associated parameters. In situ measurements of primary productivity (diel oxygen modelling, benthic oxygen exchange chambers) or PSII electron transport (PAM fluorometry) are compared, and the possibility of using acoustics as a means of quantifying oxygen production at large scales is explored, as has already been applied in seagrass beds. This thesis also provides an in depth investigation of the effect of variability in sampling methodology with regards to interpretation of PAM fluorometry-derived parameters. Chapter 2 investigates the acoustic properties of Ecklonia radiata. The density, sound speed and resulting adabiatic compressibility of E. radiata tissue were investigated in the laboratory. Four methods were developed and trialled to determine the intrinsic sound speed of Ecklonia radiata tissue based on measurement of the time of flight of an ultrasonic pulse, while compressibility was calculated from density measurements. The results show that Ecklonia radiata sound speed and density are higher, and compressibility lower, than that of seawater. Properties varied according to size and tissue type and the variation likely reflected differences in cell type, packing and structure as well as the concentrations of alginates and other carbohydrates. These are important considerations for acoustic propagation and the results provide valuable inputs for future acoustic work. Chapter 3 focuses on the acoustic modelling of different scenarios of primary production in a shallow water rocky reef environment of Fortescue Bay (Canoe Bay), Tasmania, where E. radiata dominates the canopy. In February 2012, the environment was continuously probed by acoustic signal transmission and monitored by a comprehensive set of oceanographic sensors with the aim to assess the potential for acoustics to quantify excess oxygen production in bubble form. Ray-theory acoustic modelling results indicate that ecologically-significant void fractions of oxygen in the canopy layer from production would be clearly seen in diel variation of propagation features such as the energy decay rate of the medium impulse response. The model can then be used to invert empirical data for retrieving void fraction. However, comparative analysis of part of FORTES 12 data and model suggests that no large excess of bubbles was produced by photosynthesis under the present environmental conditions, in contrast to earlier observations made in seagrasses. As a result, the use of acoustics as a means of measuring primary productivity in kelp could not be further explored during the course of this research.Chapter 4 provides a unique comparison of the estimates of photosynthetic O2 production rates in an Ecklonia radiata dominated community using three different measurement methods: diel oxygen GPP models, benthic oxygen exchange chambers, and electron transport rate from PAM fluorometry which is usually interpreted as a measure of production potential. All three methods were run concurrently in situ in Fortescue Bay, Tasmania. The first diel oxygen model was fitted to in situ measures of dissolved oxygen (DO) in the environment and demonstrated a good fit, however, a consequence of this approach is that large variation in oxygen production was predicted at low PAR levels. A second model was created which utilised an explicit relationship between DO production and PAR, but it didn’t represent DO at the surface as well as the first model. Importantly, the two models indicate similar daily production rates of the seaweed bed (all species combined) that are ~ 2 times that predicted for the kelp alone based on incubations in the benthic chambers and scaling for the average size of adult kelp sporophytes and their population density. Oxygen evolution from incubation of sporophytes in benthic chambers and PAM fluorometry derived electron transport rates showed similar patterns, but the results indicate that the latter method may overestimate potential photosynthesis. The results suggest that diel oxygen modelling, benthic oxygen exchange chambers and PAM fluorescence can all provide good indications of productivity in shallow water marine environments. However, care must be taken in interpretation of results as each method differs in the type of productivity estimates it provides. As a direct measure of total seaweed production per unit area of reef, estimates from models based on empirical measures of environmental DO have much to recommend them.Chapter 5 details a final analysis investigating the effects of diurnal, seasonal and latitudinal variability in ambient light on PAM-derived parameters, as well as possible effects associated with depth, within- and between-alga variation in PSII performance, and latitudinal effects unrelated to the light climate. This research was based on field measurements undertaken in Tasmania, Western Australia and New South Wales, Australia in both summer and winter during 2012 and 2013, focussing on Ecklonia radiata, Macrocystis pyrifera and Phyllospora comosa. Photosynthetic characteristics of all species were highly dependent on the time of day, depth, latitude/region, season, and part of the thallus from which measurements were taken. Patterns dependent on time-of-day, depth and thalli placement varied with season and/or geographic region, and the nature of these patterns varied between species. It is clear from this work that efforts to standardise approaches to taking measurements of seaweeds using PAM fluorometry will be essential if measurements are to be compared meaningfully across studies.The key findings of this thesis are: (1) a first determination of the acoustic properties of E. radiata tissue which enable the development of scattering models to interpret scientific echosounder data collected in kelp beds; (2) a Gaussian beam/finite element beam code (Bellhop) with detailed environmental input and a huge number of beams can predict the acoustic character of a shallow water rocky reef and bubble layers with low-frequency effective sound speed; (3) the model allows prediction of the acoustic energy decay rates due to various scenarios of ecologically-relevant photosynthetic O2 production rates; (4) day vs night acoustic measurement and model data comparisons challenge void fraction predictions made from well established measurements and methods; (5) diel oxygen modelling, benthic oxygen exchange chambers and PAM fluorescence can all provide good indications of productivity, however, understanding the limitations of each method is essential when interpreting the results as the measurements they provide are not directly comparable; and (6) applying a consistent sampling methodology is a key consideration when planning research utilising PAM fluorometry as diurnal, seasonal, and latitudinal variability, as well as effects associated with depth and within- and between-alga variation in PSII performance will have significant impact on PAM-derived parameters. The results of this work give valuable insight into the advantages and disadvantages involved with several main techniques currently utilised to measure production of macroalgal/seagrass beds, and the challenges faced by ecologists attempting to interpret results and compare research between methods and across studies. Last but not least, this study provides important and relevant information on the potential use of acoustics as a future means of determining productivity of benthic habitat on large scales in marine environments. The work presented herein will assist in both development and interpretation of future study of productivity in marine systems. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished

La barrière hémato-encéphalique (BHE) est une protection naturelle du système nerveux central. Son étanchéité constitue néanmoins un frein à de nombreuses thérapies médicamenteuses. Elle peut être temporairement perméabilisée grâce à une exposition à des ultrasons, couplée à une injection de microbulles dans la circulation sanguine. Dans ce manuscrit, l'ouverture de la BHE avec un dispositif ultrasonore non focalisé et implantable est étudiée. Une méthode automatique de quantification du volume d'ouverture grâce aux images IRM issues d'une étude clinique de phase 1/2a chez des patients atteint d'un glioblastome multiforme récurrent a été développée et validée. Une corrélation entre la probabilité d'ouverture et la pression acoustique locale a été trouvée. L'activité de cavitation des microbulles a été étudiée in vitro pour affiner la compréhension de son lien avec l'ouverture de la BHE. L'incertitude de quantification de cette activité à l'aide d'un capteur mono-élément utilisé passivement (PCD) à travers le crâne a été évaluée. Une correction se basant sur la position du PCD par rapport à la source de cavitation a été proposée et validée. L'influence du volume couvert par un nuage de cavitation dans le champ ultrasonore non focalisé sur les amplitudes des signaux enregistrés par le PCD pendant le traitement clinique a été discutée. Deux méthodes de localisation et de différenciation de sources de cavitation multiples dans un contexte transcrânien ont été évaluées par simulations et in vitro / The blood-brain barrier (BBB) is a natural protection of the central nervous system. However, it limits the delivery of many drugs to the brain tissues. It can be temporarily disrupted by ultrasound exposure combined with intravenous injection of microbubbles. In this manuscript, BBB disruption with an implantable unfocused ultrasound device is studied. An automatic method for quantifying the volume of BBB disruption using MR images from a phase 1/2a clinical study in patients with reccurent glioblastoma was assessed and validated. A correlation between the probability of disruption and the local acoustic pressure was found. Microbubbles cavitation activity was studied in vitro to better understand its effect on BBB disruption. The uncertainty on the amplitudes of cavitation signals recorded with a passive single-element detector (PCD) through the skull was quantified. A position-based correction of the PCD signal was assessed and validated. The effect of the volume of a cavitation cloud in the unfocused ultrasound field on the signal amplitude recorded by the PCD during the clinical treatment was discussed. Two methods for localizing and discriminating cavitation sources in a transcranial context were evaluated by simulations and in vitro

[pt] Nos últimos anos, as vantagens do uso de agentes de contraste de ultrassom (UCA) com distribuição de tamanho monodisperso foram destacadas. Caracterizadas por um coeficiente de variação (CV) inferior a 5 por cento, as microbolhas monodispersas têm o potencial de melhorar a qualidade das imagens de ultrassom (melhorando a relação sinal/ruído e reduzindo os efeitos de sombra). Também facilita o monitoramento da freqüência de ressonância das microbolhas, abrindo possibilidades nas áreas de imagem molecular e medições de pressão não-invasiva. Além disso, as bolhas monodispersas podem otimizar a entrega de drogas, genes e gases terapêuticos (por exemplo, sonotrombólise, sonoporação, abertura da barreira hemato-encefálica). No entanto, até agora, ao contrário das bolhas polidispersas, a liofilização de populações monodispersas de microbolhas, sem deteriorar sua monodispersividade, continuam sendo um desafio. Assim, hoje, as bolhas monodispersas não podem ser armazenadas nem transportadas. Isto representa um gargalo para seu uso em aplicações clínicas. Tentativas feitas para resolver o problema têm usado solventes tóxicos, levantando questões regulatórias O objetivo do presente trabalho foi de desenvolver uma nova técnica de liofilização para microbolhas monodispersas que não degradassem sua distribuição de tamanho, ou suas propriedades acústicas, sem o uso de solventes tóxicos. Como primeira etapa do projeto, foram fabricados dispositivos microfluídicos com focalização de escoamento (flow-focusing) para produzir microbolhas com distribuição de tamanho altamente monodispersa (CV menor que 5 por cento). Durante esta etapa, foi realizada a otimização da formulação das microbolhas e dos materiais crioprotetores. Foi realizada a caracterização geométrica de duas populações de microbolhas com diâmetros médios de 40 micrômetros e 5 micrômetros . Com o uso de uma câmera de alta velocidade acoplada a um microscópio ótico, imagens de todas as etapas do processo de liofilização das microbolhas foram capturadas e analisadas, visando controlar a distribuição de tamanho e a taxa de produção das microbolhas. As etapas do processo de liofilização consistiram na produção, coleta, congelamento, liofilização e ressuspensão. O desenvolvimento de uma nova técnica de recuperação, onde as microbolhas eram armazenadas em monocamadas, resultou em uma redução drástica da interação entre as bolhas durante a liofilização. Desta forma, foi possível preservar a monodispersão durante o processo de liofilização, resultando em um CV menor que 6 por cento para a população de microbolhas ressuspensas. Os ensaios de microscópio eletrônico de varredura ambiental (ESEM) demonstraram uniformidade nas cascas das microbolhas liofilizadas com uma espessura de parede estimada em 70nm. Na segunda etapa do projeto, foi realizada uma caracterização da resposta acústica de retrodifusão das microbolhas liofilizadas em forma de PVA, em comparação com as microbolhas recém produzidas e as microbolhas polidispersa SonovueTM disponíveis comercialmente. Primeiramente, a resposta acústica de retrodifusão das microbolhas foi avaliada em duas configurações diferentes: a célula centimétrica (recipiente grande - 45mmx10mmx30mm), e o milli-canal (sistema confinado no qual o líquido está em repouso - 10 mmx35 mmx1 mm). Usando um transdutor acústico focalizado com frequência de 2,25MHz, as respostas acústicas das microbolhas, na forma de frequência e amplitude de ressonância fundamental, antes e depois do processo de liofilização, foram comparadas para a população de bolhas de 5 micrômetros de diâmetro. Verificou-se que a variação de amplitude e frequência de ressonância fundamental das bolhas estava dentro da faixa de incerteza experimental, sugerindo que suas propriedades acústicas foram preservadas. Observamos também, de acordo com a literatura, que existe uma dependência linear entre a concentração da população de microbolhas (sem liofilização) e a amplitude do coeficiente de retrodifusão. Posteriormente, foi feita uma comparação da resposta de retrodispersão acústica para bolhas monodispersas e polidispersas. Também, de acordo com a literatura, observamos uma amplitude no sinal de resposta das bolhas monodispersas de 8 a 10 vezes maior que a das bolhas polidispersas, para a mesma concentração in vitro. Também foi possível observar a menor incerteza no monitoramento do pico de ressonância fundamental das bolhas e uma menor largura de banda para a população de bolhas monodispersas. Finalmente, utilizando a abordagem de imagens de matriz universal de ultrassom, desenvolvida no Institut Langevin, a resposta acústica de retrodifusão da população monodispersa liofilizada e polidispersa foi avaliada em um tecido artificial com impedância acústica similar ao tecido humano. Os resultados preliminares reforçam os resultados das medições acústicas de retrodifusão na célula centimétrica e no milicanal, nos quais a população monodispersa apresentou uma largura de banda significativamente reduzida em comparação com a ampla largura de banda da população polidispersa. O presente trabalho apresentou com sucesso uma nova técnica desenvolvida para liofilizar as microbolhas monodispersas sem degradar suas propriedades geométricas e acústicas. Assim, propusemos uma nova geração de agentes de contraste ultrassom na forma de um pó liofilizado estável que pode ser transportado e armazenado por meses e ressuspenso para uso em aplicações clínicas. / [en] In recent years, the advantages of using ultrasound contrast agents (UCA) with monodisperse size distribution have been highlighted. Characterized by a coefficient of variation (CV) lower than 5 percent, monodisperse microbubbles have the potential to improve the quality of ultrasound images (improving signal-to-noise ratio and reducing shadowing effects. It also facilitates microbubble resonance frequency monitoring, opening possibilities in the areas of molecular imaging and non-invasive pressure measurements. In addition, monodisperse bubbles can optimize the drugs, genes, and therapeutic gas delivery (e.g. sonotrombolysis, sonoporation, blood-brain barrier opening). However, thus far, contrarily to polydisperse bubbles, freeze-drying monodisperse populations of fresh microbubbles, without deteriorating their monodispersity, remains a challenge. Thereby, today, monodisperse bubbles can neither be stored nor transported. This represents a bottleneck for their use in clinical applications. Attempts made to solve the problem have used toxic solvents, raising regulatory issues The objective of the present work was to develop a new freeze-drying technique for monodisperse microbubbles that did not degrade their size distribution, or their acoustic properties, without the use of toxic solvents. As the first step of the project, flow-focusing microfluidic devices were fabricated to produce microbubbles with highly monodisperse size distribution (CV less than 5 percent). During this step, the optimization of the microbubble formulation and cryoprotectant materials was performed. Geometric characterization of two of microbubbles with mean diameters of 40 Micrometers and 5 Micrometers was performed. With the use of a high-speed camera coupled to an optical microscope, images of all stages of the freeze-drying process of the microbubbles were captured and analyzed, aiming to control the size distribution and production rate of the microbubbles. The steps of the freeze-drying process consisted of production, collection, freezing, lyophilization, and resuspension. The development of a new retrieval technique, where the microbubbles were stored in monolayers, resulted in a drastic reduction of the interaction between the bubbles during lyophilization. In this way, it was possible to preserve the monodispersity during the freeze-drying process, resulting in a CV less than 6 percent for the resuspended microbubble population. Environmental scanning electron microscope (ESEM) assays demonstrated uniformity in the shells of the freeze-dried microbubbles with an estimated wall thickness of 70nm. In the second stage of the project, a characterization of the backscatter acoustic response of the freeze-dried monodisperse PVA-shelled microbubbles, in comparison with freshly produced microbubbles, and commercially available polydisperse microbubbles SonovueTM was conducted. Firstly, the backscatter acoustic response of the microbubbles was evaluated in two different setups: the centimetric cell (large container - 45mmx10mmx30mm), and the milli-channel (confined system in which the liquid is at rest - 10 mmx35 mmx1 mm). Using a focused acoustic transducer with a frequency of 2.25MHz, the acoustic responses of the microbubbles, in the form of the fundamental resonance frequency and amplitude, before and after the freeze-drying process was compared for the bubble population of 5 Micrometers diameter. It was found that the variation of amplitude and fundamental resonance frequency of the bubbles were within the experimental uncertainty range, suggesting that their acoustic properties were preserved. We also observed, in agreement with the literature, that there is a linear dependence between the concentration of the microbubble population (without freeze-drying) and the amplitude of the backscatter coefficient. Subsequently, a comparison of the acoustic backscatter response was performed for monodisperse and polydisperse bubbles. Also, in agreement with the literature, we observed an amplitude in the response signal of the monodispersed bubbles of 8 to 10 times higher than that for the polydispersed ones, for the same in vitro concentration. It was also possible to observe the lower uncertainty in monitoring the fundamental resonance peak of the bubbles and a smaller bandwidth for the monodispersed bubble population. Finally, using the universal ultrasound matrix imaging approach, developed at Institut Langevin, the backscatter acoustic response of the freeze-dried monodisperse and polydisperse population was evaluated in a phantom mimicking tissue. The preliminary results reinforce the findings from the backscatter acoustic measurements in the centimetric cell and the milli-channel, in which the monodisperse population presented a significantly reduced bandwidth in comparison with the wide bandwidth of the polydisperse population. The present work successfully presented a new technique developed to freezedry monodisperse microbubbles without degrading their geometrical and acoustic properties. Thus, we proposed a new generation of ultrasound contrast agents in the form of a stable freeze-dried powder that can be transported and stored for months and resuspended for use in clinical applications. / [fr] Ces dernieres années, les avantages de l utilisation d agents de contraste ultrasonores (ACU) à distribution de taille monodisperse ont été mis en évidence. Caractérisées par un coefficient de variation (CV) de leur diamètre inférieur à 5 Pour cent, les microbulles monodisperses ont le potentiel d améliorer la qualité des images ultrasonores (amélioration du rapport signal/bruit et réduction des effets d ombre). Elles facilitent également le contrôle de la fréquence de résonance des microbulles, ouvrant des possibilités dans les domaines de l imagerie moléculaire et des mesures de pression non invasives. En outre, les bulles monodisperses peuvent optimiser l administration de médicaments, de gènes et de gaz thérapeutiques (par exemple, sonotrombolyse, sonoporation, ouverture de la barrière hémato-encéphalique). Cependant, jusqu à présent, contrairement aux bulles polydisperses, la lyophilisation de populations monodisperses de microbulles fraiches, sans détérioration de leur monodispersité, reste un défi. Ainsi, aujourd hui, les bulles monodisperses ne peuvent être ni stockées ni transportées. Cela représente un goulot d étranglement pour leur utilisation dans des applications cliniques. Les tentatives faites pour résoudre ce problème ont utilisé des solvants toxiques, ce qui soulève des problèmes de réglementation. L objectif du présent travail était de développer une nouvelle technique de lyophilisation des microbulles monodisperses qui ne dégrade pas leur distribution de taille, ni leurs propriétés acoustiques, et ce sans utiliser de solvants toxiques. La première étape du projet a consisté à fabriquer des dispositifs microfluidiques de focalisation du flux pour produire des microbulles avec une distribution de taille hautement monodisperse (CV moins que 5 Pour cent). Au cours de cette étape, l optimisation de la formulation des microbulles et des matériaux cryoprotecteurs a été réalisée. La caractérisation géométrique de deux microbulles avec des diamètres moyens de 40 micrometres et 5 micrometres a été menée. A l aide d une caméra à haute vitesse couplée à un microscope optique, des images de toutes les étapes du processus de lyophilisation des microbulles ont été capturées et analysées, dans le but de contrôler la distribution de taille et le taux de production des microbulles. Les étapes du processus de lyophilisation comprenaient la production, la collecte, la congélation, la lyophilisation et la remise en suspension. Le développement d une nouvelle technique de récupération, ou les microbulles étaient stockées en monocouches, a permis de réduire considérablement l interaction entre les bulles pendant la lyophilisation. De cette maniere, il a été possible de préserver la monodispersité pendant le processus de lyophilisation, ce qui a permis d obtenir un CV moins que 6 Pour cent pour la population de microbulles remise en suspension. Les analyses au microscope électronique à balayage environnemental (ESEM) ont démontré l uniformité des enveloppes des microbulles lyophilisées avec une épaisseur de paroi estimée à 70nm. Dans la deuxième étape du projet, une caractérisation de la réponse acoustique (en rétrodiffusion) des microbulles monodisperses lyophilisées et enveloppées de PVA a été réalisée et comparée avec celles de microbulles fraichement produites et de microbulles polydisperses disponibles dans le commerce (SonovueTM). Tout d abord, la réponse acoustique en rétrodiffusion des microbulles a été évaluée dans deux configurations différentes : la cellule centimétrique (grand récipient - 45mmx10mmx30mm), et le milli-channel (systeme confiné dans lequel le liquide est au repos - 10 mmx35 mmx1 mm). En utilisant un transducteur acoustique focalisé avec une fréquence de 2.25MHz, les réponses acoustiques des microbulles (fréquence de résonance fondamentale et amplitude) avant et après le processus de lyophilisation ont été comparées pour la population de bulles de 5 micrometres de diamètre. Il a été constaté que la variation de l amplitude et de la fréquence de résonance fondamentale des bulles se situait dans la plage d incertitude expérimentale, ce qui suggère que leurs propriétés acoustiques ont été préservées. Nous avons également observé, en accord avec la littérature, qu il existe une dépendance linéaire entre la concentration de la population de microbulles (sans lyophilisation) et l amplitude du coefficient de rétrodiffusion. Par la suite, une comparaison de la réponse acoustique a été effectuée pour des bulles monodisperses et polydisperses. Aussi, en accord avec la littérature, nous avons observé une amplitude du signal de réponse des bulles monodisperses de 8 à 10 fois supérieure à celle des bulles polydisperses, pour une même concentration in vitro. Il a également été possible d observer une plus faible incertitude dans le suivi du pic de résonance des bulles et une plus petite largeur de bande pour la population de bulles monodisperses. Enfin, en utilisant une approche originale dite d imagerie matricielle par ultrasons, développée à l Institut Langevin, la réponse acoustique de la population lyophilisée monodispersée et polydispersée a été évaluée dans un fantôme simulant les tissus. Les résultats préliminaires renforcent les conclusions des mesures acoustiques menées dans la cellule centimétrique et le canal millimétrique, dans lesquelles la population monodisperse présente une largeur de bande significativement réduite par rapport à la grande largeur de bande de la population polydisperse. Le présent travail a permis le développement d une nouvelle technique pour lyophiliser des microbulles monodispersées sans dégrader leurs propriétés géométriques et acoustiques. Ainsi, nous avons proposé une nouvelle génération d agents de contraste ultrasonores se présentant sous la forme d une poudre lyophilisée stable qui peut être transportée et stockée pendant des mois et remise en suspension pour être utilisée dans des applications cliniques.

A administração intravenosa de solução salina hipertônica (HSS) induz alterações sistêmicas circulatórias como o aumento da pressão arterial e do volume circulante efetivo, além de ter efeitos locais sobre a microcirculação. No presente estudo foram analisados os efeitos produzidos pela administração de solução salina hipertônica 7,5% sobre a hemodinâmica do tumor através de estudos de imagem funcional e posteriormente, foi avaliado o seu potencial de otimizar a entrega de moléculas no tumor. A velocidade do sangue nos vasos tumorais estimada por Ultrassom Color Doppler foi aumentada após a injeção da HSS em comparação ao controle PBS em tumores de melanoma (B16F10 (p=0,019), SK-MEL-147 (p =0,028)) e de mama (4T1 (p=0,015)). Este mesmo efeito não foi observado nas artérias segmentarias do rim (p=0,476). Ultrassonografia com contraste por microbolhas (CEUS) foi realizada em xenoenxertos de tumor de melanoma (B16F10), carcinoma de cólon (HCT-116) e mama (MDA-MB-231), e como controle foi realizada imagem no rim e no músculo nos animais portadores destes tipos tumorais (n=3 por grupo). Após a injeção da HSS, o volume relativo de sangue foi aumentado nos tumores B16F10 (p=0,022) e HCT-116 (P = 0,039), mas o mesmo não foi observado com o tumor MDA-MB-231 (p=0,186). Além disso, não houve alterações nos tecidos normais (rim p = 0,957; músculo p = 0,104). Todos os testes estatísticos foram bicaudais. Quando a HSS foi utilizada como veículo para entrega de moléculas de baixo peso molecular como cisplatina e doxorrubicina no tratamento de tumores B16F10 e 4T1 respectivamente, não houve aumento da eficácia terapêutica, avaliada através do crescimento tumoral e peso dos tumores. O efeito da HSS sobre a retenção de macromoléculas nos tumores SK-Mel- 147 e 4T1, avaliado através de imagem por epifluorescência do contraste ótico IR-783, não foi suficientemente notório para rejeitar a hipótese nula. Assim, a HSS induz um aumento transitório na velocidade do sangue e do volume sanguíneo, de maneira relativamente seletiva para os tumores avaliados, com exceção do MDA-MB-231. Portanto, esta pode ser uma estratégia útil para aumentar a entrega de moléculas e otimizar tanto o efeito terapêutico, quanto o diagnóstico por imagem / Intravenous administration of Hypertonic saline solution (HSS) induces systemic circulatory changes including blood pressure rising, effective circulating volume increase as well as local effects on microvasculature. We analyzed the effects produced by Hypertonic Saline 7,5% administration on tumor hemodynamics through functional imaging studies as well as whether it enhances molecular delivery in tumor tissue when used as a vehicle. Blood velocity assessed by Color Doppler Ultrasound was increased after HSS injection compared to PBS in the following tumors: B16F10 (p=0,019), SKMEL- 147 (p=0,028) and 4T1 (p=0,015). No statistical difference was observed on the segmental kidney arteries (p=0,476). Dynamic Contrast enhanced ultrasound (CEUS) was done in B16F10, HCT-116 and MDA-MB-231 tumor xenografts, kidney and muscle tissues (n=3 per group). After HSS injection, relative blood volume was increased in B16F10 (p=0,022) and HCT-116 (p=0,039) but not on MDA-MB-231 (p=0,186). Changes on normal tissues were not statistically different (kidney p=0,957; muscle p=0,104). All statistical tests were two-sided. Administration of HSS as a vehicle for low molecular weight molecules cisplatin and doxorubicin in the treatment of B16F10 and 4T1 tumors respectively had no significant improvement of therapeutic efficacy, estimated by tumor growth and tumor weight measurements. Effect of HSS over retention of macromolecules in tumors SK-Mel-147 and 4T1, evaluated by epifluorescence imaging of the optical contrast IR- 783 was not large enough to reject the null hypothesis. HSS induces a transient increase in velocity of the blood as well as the blood volume that is relatively selective for the evaluated tumors with exception of MDA-MB-231. Data suggest that HSS administration might be a useful strategy to increase the delivery of molecules and optimize both therapy and diagnostic imaging

A administração intravenosa de solução salina hipertônica (HSS) induz alterações sistêmicas circulatórias como o aumento da pressão arterial e do volume circulante efetivo, além de ter efeitos locais sobre a microcirculação. No presente estudo foram analisados os efeitos produzidos pela administração de solução salina hipertônica 7,5% sobre a hemodinâmica do tumor através de estudos de imagem funcional e posteriormente, foi avaliado o seu potencial de otimizar a entrega de moléculas no tumor. A velocidade do sangue nos vasos tumorais estimada por Ultrassom Color Doppler foi aumentada após a injeção da HSS em comparação ao controle PBS em tumores de melanoma (B16F10 (p=0,019), SK-MEL-147 (p =0,028)) e de mama (4T1 (p=0,015)). Este mesmo efeito não foi observado nas artérias segmentarias do rim (p=0,476). Ultrassonografia com contraste por microbolhas (CEUS) foi realizada em xenoenxertos de tumor de melanoma (B16F10), carcinoma de cólon (HCT-116) e mama (MDA-MB-231), e como controle foi realizada imagem no rim e no músculo nos animais portadores destes tipos tumorais (n=3 por grupo). Após a injeção da HSS, o volume relativo de sangue foi aumentado nos tumores B16F10 (p=0,022) e HCT-116 (P = 0,039), mas o mesmo não foi observado com o tumor MDA-MB-231 (p=0,186). Além disso, não houve alterações nos tecidos normais (rim p = 0,957; músculo p = 0,104). Todos os testes estatísticos foram bicaudais. Quando a HSS foi utilizada como veículo para entrega de moléculas de baixo peso molecular como cisplatina e doxorrubicina no tratamento de tumores B16F10 e 4T1 respectivamente, não houve aumento da eficácia terapêutica, avaliada através do crescimento tumoral e peso dos tumores. O efeito da HSS sobre a retenção de macromoléculas nos tumores SK-Mel- 147 e 4T1, avaliado através de imagem por epifluorescência do contraste ótico IR-783, não foi suficientemente notório para rejeitar a hipótese nula. Assim, a HSS induz um aumento transitório na velocidade do sangue e do volume sanguíneo, de maneira relativamente seletiva para os tumores avaliados, com exceção do MDA-MB-231. Portanto, esta pode ser uma estratégia útil para aumentar a entrega de moléculas e otimizar tanto o efeito terapêutico, quanto o diagnóstico por imagem / Intravenous administration of Hypertonic saline solution (HSS) induces systemic circulatory changes including blood pressure rising, effective circulating volume increase as well as local effects on microvasculature. We analyzed the effects produced by Hypertonic Saline 7,5% administration on tumor hemodynamics through functional imaging studies as well as whether it enhances molecular delivery in tumor tissue when used as a vehicle. Blood velocity assessed by Color Doppler Ultrasound was increased after HSS injection compared to PBS in the following tumors: B16F10 (p=0,019), SKMEL- 147 (p=0,028) and 4T1 (p=0,015). No statistical difference was observed on the segmental kidney arteries (p=0,476). Dynamic Contrast enhanced ultrasound (CEUS) was done in B16F10, HCT-116 and MDA-MB-231 tumor xenografts, kidney and muscle tissues (n=3 per group). After HSS injection, relative blood volume was increased in B16F10 (p=0,022) and HCT-116 (p=0,039) but not on MDA-MB-231 (p=0,186). Changes on normal tissues were not statistically different (kidney p=0,957; muscle p=0,104). All statistical tests were two-sided. Administration of HSS as a vehicle for low molecular weight molecules cisplatin and doxorubicin in the treatment of B16F10 and 4T1 tumors respectively had no significant improvement of therapeutic efficacy, estimated by tumor growth and tumor weight measurements. Effect of HSS over retention of macromolecules in tumors SK-Mel-147 and 4T1, evaluated by epifluorescence imaging of the optical contrast IR- 783 was not large enough to reject the null hypothesis. HSS induces a transient increase in velocity of the blood as well as the blood volume that is relatively selective for the evaluated tumors with exception of MDA-MB-231. Data suggest that HSS administration might be a useful strategy to increase the delivery of molecules and optimize both therapy and diagnostic imaging

Background: Addressing oil spills is crucial to protect the marine environment (Etkin, 2021). While physical and mechanical recovery methods have proven effective in controlling surface oil slicks (Doshi, 2018), subsurface challenges remain unaddressed. There is a need for low-cost, effective, and environmentally friendly solutions for subsurface oil removal. Bioinspired designs, based on nature’s evolutionary optimization, could offer promising solutions to oil spills. Objectives: The primary objective was to explore innovative and bioinspired approaches for effectively capturing and eliminating oil droplets from subsurface environments. The study aims to pioneer breakthroughs in biomimetic technologies for subsurface oil recovery. The objectives include developing a system inspired by humpback whales using bubbles, creating a fiber-based system inspired by copepods, and exploring sound as a separation technique for emulsions. Additionally, to decode the fluid mechanics within each capture system, unraveling the optimal processes responsible for successful oil droplet capture and separation in the emulsion simulation. And finally to assess system performance and potential for real-world scaling beyond the lab setting. Methods: A controlled environment simulating post-spill scenarios was established using different types of oil (crude oil, canola oil, fish oil). The emulsions were then exposed to three technology systems: micro-bubble redirection system, brush capture system, and sound wave modulations. The efficiency of oil removal and capture from the subsurface was measured using absorbent weight for the micro-bubble systems and lipid extraction for capture brush efficiency. High-speed camera images were taken to track oil droplet redirection in a flow tank, and ImageJ software to analyze droplet characteristics for effective control. Results: The analysis demonstrated that the Micro-Bubble air flotation method emerges as a highly efficient solution for post-spill oil recovery, consistently demonstrating exceptional performance. Cylinder-Ring Bubble Air Flotation method achieves a remarkable 72.4% recovery rate for canola oil, while fish oil exhibits a 14.0% recovery rate after 3 hours, highlighting the influence of oil viscosity. Be- 4 yond mere buoyancy, air bubbles showcase versatility, redirecting and containing oil droplets. The Micro-Bubble Redirection System, quantitatively assessed in a controlled environment, proves to be a significant breakthrough in controlling oil dispersion in aquatic settings. The biomimetic brush exhibited substantial oil capture capability. The fiber capture system at 360 RPM for 1-minute cycles, repeated 50 times, achieved over 46% oil removal. The modified brush at the same speed and duration captured over 19% of total oil. Post-treatment, the average size of oil droplets increased from 4.5 m to 5.5 m, showcasing changes in droplet size distribution with the fiber capture system. The sonic treatment effectively separated the majority of oil from water, revealing small oil droplets (x = 0.026 mm) in the central emulsion. This phenomenon warrants further investigation as a potential alternative to chemical surfactants. Conclusion: The development of biomimetic tools for oil spill clean-up represents a significant advancement in environmental protection. By addressing subsurface oil droplets, these methods contribute to safeguarding marine food webs from oil contamination. This study underscores the importance of innovative, natureinspired solutions in tackling complex environmental challenges. / Contexte : La lutte contre les déversements de pétrole est cruciale pour atténuer leur impact sur l’environnement marin (Etkin, 2021). Alors que les méthodes de récupération physique et m´ecanique se sont révélées efficaces pour contrôler les nappes de pétrole en surface (Doshi, 2018), les d´efis liés aux couches sous-marines restent non résolus. Il existe un besoin de solutions économiques, efficaces et respectueuses de l’environnement pour l’élimination du pétrole en sous-surface. Les conceptions bioinspirées, basées sur l’optimisation évolutive de la nature, pourraient offrir des solutions prometteuses aux déversements de pétrole. Objectifs : L’objectif principal était d’explorer des approches innovantes et bioinspirées pour capturer et éliminer efficacement les gouttelettes de pétrole des environnements sousmarins. L’étude vise à initier des percées dans les technologies biomimétiques pour la récupération du pétrole en sous-surface. Les objectifs incluent le développement d’un système inspiré des baleines à bosse utilisant des bulles, la création d’un système basé sur les fibres inspiré des copépodes, et l’exploration du son comme technique de séparation des émulsions. De plus, décoder la mécanique des fluides dans chaque système de capture, en démêlant les processus optimaux responsables de la capture et de la séparation réussies des gouttelettes de pétrole dans la simulation d’émulsion. Enfin, évaluer les performances du système et son potentiel de mise à l’échelle dans le monde réel au-delà du cadre du laboratoire. Méthodes : Un environnement contrôlé simulant des scénarios post-déversement a été établi en utilisant différents types de pétrole (pétrole brut, huile de colza, huile de poisson). Les émulsions ont ensuite été exposées à trois systèmes technologiques : système de redirection à micro-bulles, système de capture par brosse, et modulations des ondes sonores. L’efficacité de l’élimination et de la capture du pétrole en sous-surface a été mesurée en utilisant le poids absorbant pour les systèmes à micro-bulles et l’extraction des lipides pour l’efficacité de la brosse de capture. Des images de caméra haute vitesse ont été prises pour suivre la redirection des gouttelettes de pétrole dans 1 un réservoir à écoulement, et le logiciel ImageJ pour analyser les caractéristiques des gouttelettes pour un contrôle efficace. Résultats : L’analyse a démontré que la méthode de flottation d’air à microbulles émerge comme une solution très efficace pour la récupération d’huile après une fuite, montrant de manière cohérente des performances exceptionnelles. La méthode de flottation d’air à cylindre-anneau atteint un remarquable taux de récupération de 72,4% pour l’huile de canola, tandis que l’huile de poisson présente un taux de récupération de 14,0 % après 3 heures, mettant en évidence l’influence de la viscosité de l’huile. Au-delà de la simple flottabilité, les bulles d’air montrent leur polyvalence en redirigeant et en contenant les gouttelettes d’huile. Le système de redirection à microbulles, évalué de manière quantitative dans un environnement contôlé, s’avère être une avancée significative dans le contrôle de la dispersion de l’huile dans les milieux aquatiques. La brosse biomimétique a montré une capacité de capture substantielle de l’huile. Le système de capture de fibres à 360 tours par minute pendant des cycles d’1 minute, répété 50 fois, a atteint plus de 46% d’élimination de l’huile. La brosse modifiée à la même vitesse et durée a capturé plus de 19 % de l’huile totale. Après traitement, la taille moyenne des gouttelettes d’huile est passée de 4,5 m à 5,5 m, mettant en évidence des changements dans la distribution de la taille des gouttelettes avec le système de capture de fibres. Le traitement sonique a séparé efficacement la majorité de l’huile de l’eau, révélant de petites gouttelettes d’huile (x = 0,026 mm) dans l’émulsion centrale. Ce phénomène mérite une investigation plus approfondie en tant qu’alternative potentielle aux agents tensioactifs chimiques. Conclusion : Le développement d’outils biomimétiques pour le nettoyage des déversements de pétrole représente une avancée significative dans la protection de l’environnement. En s’attaquant aux gouttelettes de pétrole en sous-surface, ces méthodes contribuent à protéger les réseaux alimentaires marins de la contamination par le pétrole. Cette étude souligne l’importance de solutions innovantes et inspirées par la nature pour relever les défis environnementaux complexes.