Ultrasound is one of the most widely used modalities in medical imaging because of its high cost-effectiveness, wide availability in hospitals, generation of real-time images, and use of nonionizing radiation. However, the image quality can be insufficient in some patients. Introducing a contrast agent (CA), which comprises a suspension of 2–6 mm-sized microbubbles, improves the image quality and thus the image analysis. At present, contrast-enhanced ultrasound is frequently used during standard clinical procedures such as kidney, liver, and cardiac (echocardiography) imaging. Multimodality and targeted imaging are future areas for ultrasound CAs. Multimodality imaging may improve diagnostics by simultaneously providing anatomical and functional information. Targeted imaging may allow for identification of particular diseases. The work within this thesis focused mainly on a novel multimodal polymer-shelled CA with the potential to be target specific. In Study I, the acoustic response was determined in a flow phantom by evaluating the contrast-to-tissue-ratio when using contrast sequences available in clinical ultrasound systems. This study showed that a high acoustic pressure is needed for optimal visualization of the polymer-shelled CA. In Study II, the in vivo performance of this CA was evaluated in a rat model, and the blood elimination time and subcellular distribution were determined. In Study III, the efficiency in endocardial border delineation was assessed in a pig model. The polymer-shelled CA had a significantly longer blood circulation time than the commercially available CA SonoVue, which is favorable for target-specific CA, in which a long circulation time increases the probability of target-specific binding. Transmission electron microscopic analysis of tissue sections from liver, kidney, spleen and lungs, obtained at different time points after CA injection showed that macrophages were responsible for the elimination of the polymer-shelled CA. A higher dose of the polymer-shelled CA was needed to obtain similar endocardial border delineation efficiency as that obtained using SonoVue. The results of Studies I–III demonstrate that the polymer-shelled CA has potential applicability in medical imaging. Current guidelines for contrast-enhanced echocardiography are limited to cases of suboptimal image quality or when there is a suspicion of structural abnormalities within the left ventricle. It may be hypothesized that the wider use of contrast-enhanced echocardiography may help to detect some diseases earlier. Study IV assessed the diagnostic outcomes after contrast administration in patients without indications for CA use. The myocardial wall motion score index and ejection fraction were evaluated by experienced and inexperienced readers, and a screening for left ventricular structural abnormalities was performed. More cases of wall motion and structural abnormalities were detected in the contrast-enhanced analysis. Intra- and interobserver variability was lower with the use of CAs. This study suggests that the more widespread use of CAs instead of the current selective approach may contribute to earlier detection of cardiovascular disease. / <p>QC 20150401</p>

Radiation therapy is administered to more than 50% of patients diagnosed with cancer. Mechanisms of interaction between radiation and tumour cells are relatively well understood on a molecular level, but much remains uncertain regarding how radiation interacts with the tumour as a whole. Recent studies have suggested that tumour response to radiation may in fact be regulated by endothelial cell response, consequently stressing the role of tumour blood vessels in radiation treatment response. As a result, various treatment regimens have been proposed to strategically combine radiation with vascular targeting agents. A great deal of effort has been aimed towards developing efficient vascular targeting agents. Nonetheless, no optimal method has yet been devised to strategically deliver such agents. Recent evidence suggesting that these drugs may “normalize” tumour blood vessels and enhance radiosensitivity, is supporting experiments where anti-angiogenic drugs are combined with cytotoxic therapies such as radiotherapy. In contrast, ultrasound-stimulated microbubbles have recently been demonstrated to enhance radiation therapy by biophysically interacting with endothelial cells. When combined with single radiation doses, these microbubbles are believed to cause localized vascular destruction followed by tumour cell death. Finally, a new form of ‘pro-angiogenics’ has also been demonstrated to induce a therapeutic tumour response. The overall aim of this thesis is to study the role of tumour blood vessels in treatment responses to single-dose radiation therapy and to investigate radiation-based vascular targeting strategies. Using pharmacological and biophysical agents, blood vessels were altered to determine how they influence tumour cell death, clonogenicity, and tumour growth, and to study how these may be optimally combined with radiation. Three-dimensional high-frequency power Doppler ultrasound was used throughout these studies to investigate vascular response to therapy.

Objetivo: Avaliar a eficácia da ultrassonografia com contraste (US com contraste) de segunda geração no diagnóstico diferencial entre oclusão e pseudo-oclusão de artéria carótida interna cervical (ACI) comparativamente à ultrassonografia com Doppler (US Doppler) utilizando a angiotomografia computadorizada (angio-TC) como padrão-ouro. Materiais e Métodos: Estudo prospectivo realizado entre junho de 2006 e junho de 2008 com 72 pacientes sintomáticos (57; 15) e 78 com ACIs aparentemente ocluídas pela US Doppler. Esses pacientes foram submetidos inicialmente à US Doppler e, em seguida, à US com contraste. Todos os pacientes realizaram posteriormente a angio-TC, utilizada como padrão-ouro. A correlação entre os métodos foi feita através de estudo duplo cego. Resultados: A sensibilidade, a especificidade e a acurácia da US com contraste foram respectivamente de 100%; 90,5% e 97,4%. Quando comparada à angio-TC, a quantidade de falsas oclusões pela US Doppler foi de 26,9% (21/78), (p < 0,001), enquanto que pela US com contraste foi de 2,6% (2/78) (p = 0,500). A US com contraste obteve melhor resultado que a US Doppler, com diferença estatisticamente significante (p < 0,001). Conclusão: A US com contraste é tão eficaz quanto a angio-TC e superior à US Doppler no diagnóstico diferencial entre oclusão e pseudo-oclusão da ACI / Purpose: Evaluate the efficacy of second-generation contrast ultrasound (CEUS) for distinguishing the diagnosis of cervical internal carotid artery (ICA) occlusion from the one of pseudo-occlusion when compared with Doppler Ultrasound (DUS) utilizing computerized angiotomography (CTA) as the gold standard. Material and Methods: A prospective study was performed between June 2006 and June 2008 with 72 symptomatic patients (57 males; 15 females) and 78 ICAs apparently occluded by DUS. These patients were initially subjected to DUS and then to CEUS. All patients went through CTA later on, used as the gold standard. Correlation between the methods was made by means of a double-blind study. Results: The sensitivity, specificity and accuracy of CEUS were taken as 100%, 90.5% and 97.4% respectively. When compared with CTA the amount of false occlusions by DUS was 26.9% (21/78), (p < 0.001) whereas by CEUS 2.6% (2/78) (p = 0.500) was seen. Far better results were obtained with CEUS than with DUS with a significant discrepancy (p < 0.001). Conclusion: CEUS shows to be as effective as CTA but better than DUS for distinguishing the diagnosis of ICA occlusion from the one of pseudo-occlusion

Nous avons étudié la stabilisation de microbulles d’air par les tensioactifs semi-fluorés CF3(CF2)n-1(CH2)mOP(O)(OH)2 (FnHmPhos, où n = 8, 10, m = 2) et des nanoparticules magnétiques d’oxyde de fer et de ferrite de cobalt. Ces nouveaux objets combinant propriétés acoustiques et magnétiques pourraient servir d’agent de contraste à la fois en échographie et en imagerie IRM. Nous avons étudié des propriétés physico-chimiques des tensioactifs en solution et à l’interface aireau. Nous avons montré par tensiométrie dynamique et par des études de films de Langmuir que les tensioactifs F8H2Phos et F10H2Phos forment des monocouches dont le module élastique est très élevé, ce qui favorise la stabilisation de bulles de petite taille ainsi que de bulles non-sphériques. Nous avons proposé une approche de dégonflement/gonflement contrôlé en faisant varier la pression dans la bulle par la température et nous avons étudié le froissement de la paroi des bulles pendant le dégonflement. Des microbulles portant des nanoparticules ont été obtenues dans une suspension des nanoparticules d’oxyde de fer ou de ferrite de cobalt en présence de F8H2Phos ou F10H2Phos, en déstabilisant la suspension par NaCl, ce qui permet de diminuer la barrière électrostatique entre les nanoparticules et la surface des bulles et de favoriser l’adsorption. Les microbulles obtenues sont stables pendant plus d’une semaine grâce à la paroi rigide des nanoparticules. / The stabilization of air microbubbles by semi-fluorinated surfactants CF3(CF2)n-1(CH2)mOP(O)(OH)2 (FnHmPhos, where n = 8, 10, m = 2) and iron oxide nanoparticles was studied in order to design novel microbubbles that possess both acoustical and magnetic properties. Such microbubbles have potential applications as dual constrast agents by enhancing the signals from both ultrasonography and MRI imaging. We have studied physic-chemical properties of the surfactants in solution and at water-air interface. We have demonstrated by dynamic tensiometry and studies of Langmuir monolayers that F8H2Phos and F10H2Phos form highly elastic interfacial films that are favorable for long-term stabilization of small bubbles and bubbles of non-spherical shape. We designed an approach of temperature-controlled pumping/shrinking of the microbubbles and demonstrated the correlation of the collapse structures of the bubble shell with microbubble stability. The stabilization of microbubbles by iron oxide or cobalt ferrite nanoparticles was achieved by destabilizing the nanoparticle suspension in the presence of F8H2Phos or F10H2Phos. Under these conditions the nanoparticles were adsorbed onto the surface of the microbubbles forming a rigid shell which enhanced their stability and lengthened their lifetime to greater than one week.

Introdução: O diabetes melito (DM) está associado com alterações na reserva de fluxo coronariano a nível microcirculatório e a ecocardiografia sob estresse com perfusão em tempo real (EPMTR) é uma técnica útil para avaliação não invasiva dessas alterações. O objetivo deste estudo foi avaliar se o controle do DM teria influência sobre os valores da Reserva de Fluxo Microvascular (RFM) em pacientes livres de coronariopatia obstrutiva. Métodos: Estudamos 30 pacientes com DM tipo 2 (GD) e 11 pacientes saudáveis do grupo controle (GC). A RFM foi avaliada pela EPMTR utilizando contraste a base de microbolhas. Os diabéticos foram estudados na fase descompensada (Fase 1) e após a otimização do tratamento quatro meses depois (Fase 2). Analisamos três parâmetros na quantificação miocárdica: Volume relativo de sangue do miocárdio (AN), velocidade do fluxo () e fluxo miocárdico absoluto (ANx). Todos os pacientes realizaram angiotomografia de coronárias (64 detectores) para confirmar a ausência de coronariopatia obstrutiva. Os grupos foram pareados por idade, sexo, peso, índice de massa corpórea e separados os pacientes com melhora(GCM) dos níveis de hemoglobina glicosilada maiores que 1% (valor absoluto) e os sem melhora(GSM). Resultado; durante a EPMTR na Fase 1 foram: Valores (s-1): 1,16±0,59 (GCM) vs.1,72±1,08 (GSM) vs. 2,33±1,75 (GC), com p < 0,001 e valores ANx(dBs-1): 1,53±0,83 (GCM) vs. 2,08 ± 1,33 (GSM) vs. 2,61±1,66 (GC) com p < 0.001. Na Fase 2 obtivemos valores de (s-1): 1,84±1,11 (GCM) vs. 1,29±0,76 (GSM) vs. 2,20±1,53(GC) com p < 0.001 e valores ANx(dBs-1): 1,70 ± 1,01 (GCM) vs.1,43 ± 0,87 GSM) vs. 2,69 ± 1,57 (GC) com p < 0.001. Conclusão: Pacientes diabéticos tipo 2 com controle clínico inadequado apresentam redução na reserva de fluxo microvascular. Uma melhora dos níveis de hemoglobina glicosilada maior que 1% está associada a uma melhora na perfusão miocárdica / Background: Diabetes mellitus (DM) is associated with alterations in coronary flow reserve on microvascular circulation. Real-time myocardial contrast echocardiography has proven to be a useful method for non-invasive evaluation of microvascular alterations. The objective of this study was to assess whether the control of diabetes would influence the values of Microvascular Flow Reserve (MFR) in patients free of obstructive coronary artery disease (CAD). Methods: Thirty patients were studied with DM (DG) and eleven healthy subjects (CG). MFR was determined by quantitative contrast echocardiography during dipyridamole stress using intravenous microbubbles based contrast. Diabetic individuals were studied in a decompensated state (Phase 1) and after optimization of medical treatment four months later (Phase 2). We evaluated three parameters of myocardial blood flow quantification. Relative myocardial blood volume (AN), blood flow velocity () and myocardial absolute flow (ANx ). All patients underwent computed coronary angio-tomography (64 Slices) to determine the absence of obstructive CAD. The groups were paired by age, sex, weight, body mass index and separated patients that improvement (IG) in levels of glycosylated hemoglobin greater than 1% (absolute value) and those that showed no improvement (NIG). Results: and ANx reserve values in phase 1 were respectively: 1.163±0.587 (IG) vs.1.724±1,077 (NIG) vs. 2.328±1.752 (CG), with p < 0.001 and ANx(dBs-1) values: 1.527±0.828 (IG) vs. 2.080±1.328 (NIG) vs. 2.609±1.659 (CG) with p < 0.001. Values in Phase 2: (s-1): 1.839±1.112 (IG) vs. 1.284±0.761 (NIG) vs. 2.199±1.528 (CG) with p < 0.001 and ANx(dBs-1) values: 1.696±1.012 (IG) vs. 1.426±0.866 (NIG) vs. 2.687±1.574 (CG) with (p < 0.001). Patients that reached the goal HbA1C levels had a significant enhancement in coronary reserve (from 10.42±2.03% to 8.73±1.77%; p<0.001). Conclusion: These results suggest that diabetic individuals with poor blood glucose control and no obstructive coronary artery disease have impaired MFR. Improvement in glycosylated hemoglobin greater than 1% is associated with increase in microvascular function

La sonoporation, associant les ultrasons (US) et les microbulles (MB) permet une délivrance localisée des molécules thérapeutiques (e.g., acides nucléiques, molécules chimio-thérapeutiques), avec une efficacité thérapeutique élevée et un faible taux de toxicité. Cependant, les mécanismes impliqués dans le transfert de ces molécules restent jusqu’alors mal connus. Les travaux présentés dans ce manuscrit de thèse ont pour objectif de mettre en évidence l’importance du contact microbulle/cellule et les forces de radiation au cours de la sonoporation et s’intéressent par ailleurs, à l’étude des mécanismes sous-jacents durant le processus de sonoporation ainsi que les conséquences cellulaires (membranaire et intracellulaire) engendrées. Ces études sont réalisées par microscopie électronique, et comparent deux lignées cellulaires U-87 MG et MDA-MB-231. Nous nous intéresserons également aux effets du sérum sur l’efficacité de la sonoporation dans la délivrance de molécules (gènes), mais aussi aux changements du cytosquelette et son implication au cours du processus de sonoporation. / Sonoporation combines ultrasound and microbubbles, and promises a local delivery of therapeutic molecules (i.e., nucleic acids, chemotherapeutic molecules) with a high therapeutic efficacy and a low toxicity level. However, the mechanisms involved in the molecules uptake remain hitherto unclear. The work presented in this thesis manuscript aims to highlighting the processes of action of sonoporation by exploring the importance of microbubble/cell contact and the radiation forces and the study of underlying mechanisms during sonoporation and the generated cellular consequences (membrane and intracellular), by electron microscopy, and by comparing two cell lines U-87 MG and MDA-MB-231. We also explore the serum effects on the efficiency of sonoporation in the delivery of molecules (genes) but also the changes in the cytoskeleton and its involvement during sonoporation.

This work deals with preparation of microbubble suspension from a mixture of phospholipids, palmitic acid and polyethylene glycol. Properties of prepared systems were studied using bubble tensiometry and dynamic light scattering method and were compared with commercial contrast agent SonoVue®. Suspensions were prepared in various conditions including different atmosphere and increased temperature in some steps of preparation and different solution. Effect of polyethylene glycol addition on surface activity of the system was studied. Surface activity of phospholipids was insignificant. Surface tension decreased with increasing concentration and molecular weight of polyethylene glycol in the system. Effect of different atmosphere and increased temperature showed no substantial trend. It emerged that dynamic light scattering is not suitable for this type of samples because of high polydispersity and phase separation of the system.

Dans les tumeurs solides, l'hypoxie est un mécanisme de résistance à la radiothérapie bien connu. Il a déjà été démontré que, lorsque les microbulles (MB) sont exposées à une impulsion ultrasonore (US), celles-ci peuvent induire une vasodilatation dans les tissus musculaires. De plus, une impulsion thérapeutique peut être délivrée localement dans la tumeur en dirigeant le faisceau US. Cette approche est donc proposée comme thérapie provasculaire ciblée, guidée par l’imagerie ultrasonore dans les tumeurs afin de réduire l'hypoxie avant la radiothérapie. Le contrôle de la vasodilatation est induit par la production d'oxyde nitrique (NO) par la voie de signalisation cellulaire du eNOS dans les cellules endothéliales. Il a été démontré que l'augmentation de l'ATP extracellulaire active la voie de signalisation du eNOS. Il a aussi été démontré que l’oscillation des MB sous l’effet des US libèrent de l'ATP lorsque le tissu musculaire est traité. Cependant, les effets des différentes conditions ultrasonores et de MB sur la libération d'ATP n'ont pas encore été étudiés. Nous émettons donc l'hypothèse qu'il existe des conditions permettant de maximiser l’activation des voies de signalisation purinergiques (ATP) et d'optimiser leur durée d’activation pour une réponse provasculaire optimale. Les motivations de ce projet sont de tester divers paramètres et d'étudier les interactions MB/cellules dans des conditions d'écoulement, qui sont généralement difficile à mettre en place lorsqu'on utilise des boîtes de Pétri. Pour quantifier plus facilement les voies de signalisation, nous avons créé des puces microfluidiques avec quatre canaux parallèles dans lesquels des cellules ont pu être cultivées. Avec quatre canaux traités lors d’une même impulsion ultrasonore, nous avons aussi augmenté le nombre de données à traiter et nous pouvons observer les effets de plusieurs impulsions lorsque les MB étaient dans un écoulement. En outre, la puce que nous avons développé est capable de donner une concentration en MB différente dans chaque canal afin de pouvoir tester quatre concentrations de MB différente dans des conditions d’écoulement. Les objectifs de ce projet de maîtrise sont donc les suivants : (1) concevoir la puce microfluidique ; (2) être capable de cultiver des cellules dans les canaux microfluidiques ; (3) créer des protocoles pour mesurer la libération d'ATP et la viabilité cellulaire après une impulsion ultrasonore ; (4) observer la capacité de la puce à donner différentes concentrations de MB dans chaque canaux en conditions d’écoulement. Lors de la conception de la puce microfluidique, nous avons créé un environnement dans lequel les quatre canaux de la puce ont des concentrations différentes de microbulles fluides. Ainsi, nous avons atteint les objectifs du projet. Nous avons réussi à introduire dans le canal microfluidique des cellules endothéliales de cordon ombilical humain (HUVEC) et une lignée cellulaire de cancer du sein (4T1). Les monocouches cellulaires créées par chacune des deux lignées cellulaires ont été traitées avec succès par une impulsion thérapeutique ultrasonore lors de l’injection de MB. Nos résultats montrent qu'une augmentation du nombre de cycles et de la pression, libère plus d'ATP et induisent une mortalité cellulaire plus importante. En outre, nous avons établi un lien entre la libération d'ATP et la mortalité cellulaire en comparant différentes impulsions thérapeutiques ultrasonore. Cette analyse a permis de dégager deux tendances. Avec des impulsions à faible énergie, la libération d'ATP est augmenté et on constate une très faible augmentation de la mort cellulaire ; inversement, avec des impulsions à plus forte énergie, la libération d'ATP et la mortalité cellulaire ont augmentés et on atteint un plateau. Ainsi, nos résultats confirment que différents mécanismes de libération d'ATP peuvent être déclenchés par les thérapies MB et US. / In solid tumors, hypoxia is a well-known resistance mechanism to radiation therapy. It was previously shown that microbubbles (MBs), when exposed to an ultrasound pulse (US) can cause vasodilation in muscle tissue. Conceptually, the therapeutic pulse can be localized on the tumor by steering the US beam. This approach is therefore proposed as a targeted image-guided provascular therapy in tumors to reduce hypoxia before radiotherapy. However, the effects of US and MB conditions on the relative increase in tumor perfusion remain largely unknown. Vascular control is managed by the production of nitric oxide (NO) through the eNOS pathway inside the endothelial cells. Increases in extracellular ATP have been shown to be a signaling event for the activation of this pathway. Fittingly, MB and US have been shown to release ATP when muscle tissue was treated. However, the effects of therapeutic US and MB parameters on the treatment have not yet been described. We, therefore, hypothesize that there are conditions that will maximize the purinergic signaling pathways (ATP) and optimize their time course for an optimal provascular response. The motivation for this project came from the desire to test various parameters and study MB/cell interactions in flowing conditions, which are typically limited when using petri dish setups. To quantify more easily the signaling pathways, we created microfluidic chips with four parallel cell coated channels. This chip allowed us to increase the throughput when using a single US exposure in static conditions and with the ability to support multiple US exposures with MB replenishment in flowing conditions. Also, the custom-made chip multiplexes the bubble concentration to obtain four channels with different flowing microbubble concentrations. The goals of this master’s project were thus: (1) to design the microfluidic chip; (2) to demonstrate the capacity for cell culture; (3) create protocols for measuring ATP and cell viability after therapeutic pulses; (4) to demonstrate repeatable flowing conditions with the multiplexed MB concentration. On the design of the microfluidic chip, we were successful at creating an environment where four of the four channels in the chip have different concentrations of flowing microbubbles. Thus, fulfilling the project's goals. We have succeeded in seeding both Human Umbilical Vein Endothelial Cells (HUVECs) and a breast cancer cell line (4T1) into the microfluidic channel. The cell monolayers created by both cell lines were successfully treated with an US and MB therapeutic pulse. Our results support that an increase in both, cycles and pressure, release more ATP and cause more cell death. Further, we linked ATP release to cell death by comparing different therapeutic pulses. From this analysis, two trends appeared. With lower energy pulses, ATP release increased sharply with a very small increase in cell death; conversely, with higher energy pulses, ATP release continued to increase with cell death but reached a plateau. Thus, our results support that different mechanisms of ATP release can likely be triggered by MB and US therapy.

<p>  </p> <p>Fouling is a severe problem for food processing equipment, including heat exchanger, filtration system, etc., which can decrease heat transfer efficiency, increase pressure drop, and affect food quality and safety. To ensure process efficiency and product quality, regular cleaning is necessary. On manufacturing lines in the food industry, cleaning is usually performed by cleaning-in-place (CIP) operations, which mainly comprise water rinse, alkaline wash and acid wash steps. Although CIP can ensure uniform cleaning of equipment, lower costs associated with labor and plant downtime, and improve personnel safety, it consumes large amounts of energy, chemicals and water and thus affects the environmental sustainability.</p> <p>Microbubbles (MBs) are fine gas bubbles with very different physicochemical properties from millimeter-sized bubbles, including longer residence time in liquid, higher mass transfer rate, larger surface-area-to-volume ratio, and generation of free radical when bubbles collapse. In addition, MBs feature hydrophobic liquid-gas interface, allowing hydrophobic and amphipathic substances to attach to and spread on bubble surfaces. MBs have been used in cleaning processes, such as oil flotation and fresh produce washing; however, their applications in CIP operations in food processing have not been explored.</p> <p>The objective of this dissertation is to develop a novel CIP operation with the incorporation of MBs for cleaning of food processing equipment. MBs were incorporated into rinsing water to clean milk deposit fouled on heat transfer surface. A computational fluid dynamics model was built to predict the contact frequency of MBs with deposit and further identify the flow conditions that provided maximum MB-deposit contact. Moreover, MBs were confirmed to be able to attach to milk deposit by microscopic imaging. Rinsing with MB-incorporated water noticeably enhanced the deposit removal at Re of 4392 and 5403, by 27−31%. For cleaning of microfiltration membrane reversibly fouled by palm oil-in-water emulsions as model wastewater, although adding MBs into alkaline wash increased the membrane flux recovery by 235%, increasing the crossflow velocity of MB-incorporated liquid did not guarantee the enhancement in cleaning performance. Lastly, a MB-assisted full CIP process was tested on an ultrafiltration system used for milk concentration. MB-assisted CIP showed an increased cleaning efficiency with up to 72% higher flux recovery than conventional CIP, and alkaline wash with MBs added was the major step accounting for enhanced protein removal.</p> <p>Overall, this dissertation proves the effectiveness of MBs in cleaning of different types of food deposits, and provides groundwork knowledge of MB incorporation into CIP operations for different food processing equipment. The results are expected to guide the scale-up of MB-assisted CIP processes that can reduce the water and chemical usage in food manufacturing sectors, ultimately improving both economic and environmental sustainability of the food industry.</p>

Thesis deals with ultrasonographic contrast examinations, that are performed to assess tissue perfusion and non-invasive ultrasound method speckle tracking, overcoming the weaknesses of Doppler techniques used to scanning the movement of the tissue.