Tesis por compendio / [ES] La necesidad de confinar y manipular micro-objetos tiene aplicaciones en múltiples áreas de la ciencia y tecnología. Actualmente, existen diversas técnicas para lograr este objetivo, y una de las más destacadas es el uso de las llamadas pinzas ópticas, que se han convertido en una herramienta ampliamente utilizada en laboratorios de todo el mundo. Este trabajo de investigación se centra en el fascinante campo del atrapamiento y manipulación de micro-objetos, con un enfoque destacado en la combinación de elementos ópticos difractivos y la técnica de pinzas ópticas. Esta combinación permite un aumento de la versatilidad de los sistemas experimentales de pinzas ópticas. Los avances presentados en esta tesis tienen aplicaciones en una amplia gama de campos, desde la nanotecnología hasta la biología celular. Como lentes difractivas implementadas en los sistemas de pinzas ópticas, se introducen las lentes difractivas Kinoform basadas en la secuencia aperiódica m-Bonacci. Estas lentes permiten atrapar múltiples partículas simultáneamente y manipularlas tridimensionalmente en dos planos focales diferentes, lo que amplía significativamente las posibilidades de investigación y desarrollo en diversas disciplinas. Además, se aborda la generación de múltiples trampas ópticas mediante lentes Kinoform cuadrifocales basadas en otra secuencia aperiódica conocida como Silver Mean, permitiendo atrapar partículas en cuatro planos focales de manera simultánea. Este avance mejora significativamente la versatilidad de los sistemas de pinzas ópticas. Adicionalmente el uso de vórtices multiplexados en un sistema de pinzas ópticas, permite atrapar de manera independiente múltiples partículas y transferir momento angular. Estos avances abren nuevas posibilidades en la construcción de micromotores y aplicaciones de micro-ensamblaje. Un efecto asociado a las trampas ópticas es la generación de microburbujas, en la actualidad estas se han convertido en objeto de estudio debido a la facilidad de generación y a sus posibles aplicaciones como agentes de transporte de partículas o micro-objetos. Aprovechando este efecto en esta tesis se implementa una técnica de atrapamiento que emplea fuerzas termoforéticas en la captura y manipulación de microburbujas en líquidos. Esto constituye otro avance importante en el campo del atrapamiento tridimensional. Por último, se desarrolla un laboratorio virtual utilizando COMSOL Multiphysics para simular el atrapamiento acústico, lo que permite a los estudiantes interactuar con el sistema y comprender mejor este fenómeno. Este enfoque educativo proporciona herramientas valiosas para la comprensión y análisis de la manipulación de partículas, lo que beneficia a los estudiantes de pre-grado y grado interesados en este campo. En conjunto, todos estos avances representan contribuciones significativas en el campo del atrapamiento y manipulación de partículas, en particular a través de las pinzas ópticas, promoviendo el progreso tecnológico y científico en diversas disciplinas y brindando oportunidades educativas para futuras generaciones de investigadores y científicos. A lo largo del desarrollo de esta tesis, se han creado nuevos elementos difractivos que superan ciertas limitaciones y aumentan las capacidades de las pinzas ópticas, abriendo nuevas perspectivas de aplicación para tecnologías preexistentes. / [CA] La necessitat de confinar i manipular microobjectes té aplicacions a múltiples àrees de la ciència i la tecnologia. En l'actualitat, hi ha diverses tècniques per assolir aquest objectiu, i una de les més destacades és l'ús de les anomenades pinces òptiques, que han esdevingut una eina molt utilitzada en laboratoris de tot el món. Aquest treball de recerca se centra en el fascinant camp de la captura i la manipulació de microobjectes, destacant la combinació d'elements òptics difractius i la tècnica de les pinces òptiques. Aquesta combinació permet augmentar la versatilitat dels sistemes experimentals de pinces òptiques. Els avenços presentats en aquesta Tesi tenen aplicacions en una àmplia gamma de camps, des de la nanotecnologia a la biologia cel·lular. Com a lents difractives implementades en sistemes de pinces òptiques, es presenten les lents difractives Kinoform basades en la seqüència aperiòdica m-Bonacci. Aquestes lents permeten atrapar simultàniament múltiples partícules i manipular-les tridimensionalment en dos plans focals diferents, fet que amplia significativament les possibilitats de recerca i desenvolupament en diverses disciplines. A més, s'aborda la generació de múltiples trampes òptiques utilitzant lents Kinoform quadrifocals basades en una altra seqüència aperiòdica coneguda com a Silver Mean, que permet atrapar partícules en quatre plans focals simultàniament. Aquest avenç millora significativament la versatilitat dels sistemes de pinces òptiques. A més, l'ús de vòrtexs multiplexats en un sistema de pinces òptiques permet atrapar múltiples partícules de manera independent i transferir el moment angular. Aquests avenços obren noves possibilitats en la construcció de micromotors i aplicacions de microassemblatge. Un efecte associat a les trampes òptiques és la generació de microbombolles, actualment aquestes s'han convertit en objecte d'estudi a causa de la facilitat de generació i de les seves potencials aplicacions com a agents de transport de partícules o microobjectes. Aprofitant aquest efecte, aquesta Tesi implementa una tècnica d'atrapament que utilitza forces termoforètiques en la captura i manipulació de microbombolles en líquids. Això constitueix un altre avenç important en el camp de l'atrapament tridimensional. Finalment, es desenvolupa un laboratori virtual utilitzant COMSOL Multiphysics per simular l'atrapament acústic, cosa que permet als estudiants interactuar amb el sistema i comprendre millor aquest fenomen. Aquest enfocament educatiu proporciona eines valuoses per a la comprensió i l'anàlisi de la manipulació de partícules, cosa que beneficia els estudiants de grau i postgrau interessats en aquest camp. En conjunt, tots aquests avenços representen contribucions significatives al camp de l'atrapament i la manipulació de partícules, particularment a través de pinces òptiques, promovent el progrés tecnològic i científic en diverses disciplines i proporcionant oportunitats educatives per a futures generacions d'investigadors i científics. Al llarg del desenvolupament d'aquesta Tesi, s'han creat elements difractius nous que superen certes limitacions i augmenten les capacitats de les pinces òptiques, obrint noves perspectives d'aplicació per a tecnologies preexistents. / [EN] The need to confine and manipulate micro-objects has applications in multiple areas of science and technology. Currently, there are several techniques to achieve this goal, and one of the most prominent is the use of the so-called optical tweezers, which have become a widely used tool in laboratories around the world. This research work focuses on the fascinating field of micro-object capture and manipulation, highlighting the combination of diffractive optical elements and the optical tweezers technique. This combination allows to increase the versatility of the experimental optical tweezers systems. The advances presented in this thesis have applications in a wide range of fields, from nanotechnology to cell biology. As diffractive lenses implemented in optical tweezers systems, Kinoform diffractive lenses based on the aperiodic m-Bonacci sequence are presented. These lenses allow multiple particles to be trapped simultaneously and manipulated three-dimensionally in two different focal planes, which significantly expands the possibilities for research and development in various disciplines. In addition, the generation of multiple optical traps is addressed using quadrifocal Kinoform lenses based on another aperiodic sequence known as Silver Mean, which allows particles to be trapped in four focal planes simultaneously. This advance significantly improves the versatility of optical tweezer systems. In addition, the use of multiplexed vortices in an optical tweezer system allows multiple particles to be trapped independently and angular momentum to be transferred. These advances open up new possibilities in micromotor construction and microassembly applications. One effect associated with optical traps is the generation of microbubbles, currently these have become an object of study due to the ease of generation and their potential applications as transport agents for particles or micro-objects. Taking advantage of this effect, this thesis implements an trapping technique that employs thermophoretic forces in the capture and manipulation of microbubbles in liquids. This constitutes another important advance in the field of three-dimensional trapping. Finally, a virtual laboratory is developed using COMSOL Multiphysics to simulate acoustic trapping, allowing students to interact with the system and better understand this phenomenon. This educational approach provides valuable tools for the understanding and analysis of particle manipulation, benefiting undergraduate and graduate students interested in this field. Taken together, all of these advances represent significant contributions to the field of particle trapping and manipulation, particularly through optical tweezers, promoting technological and scientific progress in various disciplines and providing educational opportunities for future generations of researchers and scientists. Throughout the development of this thesis, new diffractive elements have been created that overcome certain limitations and increase the capabilities of optical tweezers, opening new application perspectives for pre-existing technologies. / We acknowledge the financial support from Ministerio de Ciencia e Innovación (grants PID2019-107391RB-I00 and PID2022-1142407NB-I00), Generalitat Valenciana (grant PROMETEO/2019/048 and CI-PROM/2022/30), and Universitat Politècnica de València (PAID-01-20-25), Spain. We acknowledge the financial support from CONACyT (grant A1-S-28440). / Muñoz Pérez, FM. (2024). Diseño y caracterización experimental de sistemas de atrapamiento y manipulación de micro-objetos mediante técnicas ópticas, térmicas y acústicas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203436 / Compendio

The feasibility of applying a recently developed liquid-liquid extraction method termed Predispersed Solvent Extraction (PDSE) in an <i>ex situ</i> pump-and-treat system to remove trace amounts of dissolved chlorinated solvents like perchloroethylene (PCE) and trichloroethylene (TCE) from contaminated ground water has been investigated. In PDSE, the solvent is comminuted into globules with diameters ranging from submicron to 100 microns, and stabilized by a surfactant film prior to contact with the aqueous feed. These stabilized globules, termed oil-core aphrons (OCAs), disperse readily in water since water is the continuous phase in systems where the oil-water phase ratios can be as high as 9. Due to their fine particle size and large surface area, high extraction mass-transfer rates are achieved with minimal mixing. OCA phase separation from water can be expedited with microbubble foam flotation. Experiments in this investigation focused on PDSE process development for this groundwater remediation application. Distribution coefficients for PCE and TCE in possible OCA solvents were determined experimentally and agree with published calculated values. Various surfactant/solvent OCA formulations using the aforementioned solvents were examined with emphasis on creating a weakly stable dispersion which would maximize extraction efficiency yet destabilize sufficiently to permit rapid flotation with minimum losses in the raffinate. Accelerating phase separation, hence solvent recovery, through dispersion chemical destabilization with salts, coagulants, and flocculants at varied pH was examined with and without microbubble flotation. The presence of OCAs in the aqueous phase reduced vapor phase concentrations of PCE as much as 96% and was assessed through apparent Henry's Law constants. TCE concentrated in dodecane OCA extract was degraded with a CO-dehydrogenase enzyme complex to cis 1,2-dichloroethylene, trans 1,2-dichloroethylene, and 1,1-dichloroethylene, and vinyl chloride as a possible means of destroying TCE dissolved in the extract. Based on the implications of these experiments, the development of a PDSE <i>ex situ</i> pump-and-treat system appears technically feasible and a conceptual process layout has been provided. / Master of Science

Thesis (MScEng)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Patients in critical care with lung injuries need to be assisted with regards to breathing function, but current methods are not applicable for all situations. The most common method, Extracorporeal Membrane Oxygenation (ECMO) is an expensive procedure and requires trained staff to operate the equipment at all times. Lung injury may lead to the inability of the lungs to be perfused and the blood oxygenated by tracheal intubation, whereas mechanical ventilators can injure the lungs further. Especially at risk are preterm neonates, where congenital disorders or complications during birth render ECMO the only viable option. Respiratory Assist Catheters (RACs) could be used as an alternative because they do not place extra stress on the lungs, are easy to implement, cost-effective and are available for immediate use in clinical settings or in first aid situations. The development of such a device requires knowledge of possible oxygenation methods as well as the risks involved in implementing such a device. The possibility of oxygenating the blood via microbubbles by means of a RAC is promising due to the high gas transfer rates common in bubble oxygenators. It is the aim of this study to develop a prototype that could function as a RAC and to evaluate the feasibility of oxygenation by using microbubbles. The method used to design a prototype included selection of various materials and finalization of a design to be tested. The tests selected were in vivo tests and ex vivo tests using animal models to investigate the dissolution times of the microbubbles, as well as the physiological effects of an intravenously placed device. Measurements of oxygen saturation of the blood in arterial blood (SaO2), venous blood (SvO2) and pulmonary pressure allowed the oxygen transfer rates and risks involved to be evaluated, and also gave an indication regarding the formation dynamics of microbubbles in the blood. An in vitro test was also performed with the aim of determining the rate of dissolving of oxygen, and hence to give an indication regarding microbubble dissolution times. Mathematical simulations based on the dissolution rate of oxygen in venous blood confirmed the abovementioned results. The tests and simulations were analysed in order to evaluate the feasibility of intravenously oxygenating the blood using microbubbles. Approximate bubble dissolution times were an indicator of the feasibility of the concept and showed that very large bubble dissolution times renders intravenous bubble oxygenation unfeasible. These large dissolution times also lessen the possibility of implementing bubble oxygenation in an intravenous device. / AFRIKAANSE OPSOMMING: Pasiënte wat a.g.v. longbeserings in hoë-sorg behandel word het hulp nodig om asem te haal, maar bestaande metodes werk nie in alle omstandighede nie. Die mees algemene metode is ekstrakorporeale membraan suurstofverbinding (Extracorporeal Membrane Oxygenation (ECMO)), maar hierdie metode is duur en het voltyds opgeleide personeel nodig om dit te beheer. Longbeserings kan lei tot die onvermoë van die longe om bloed te ontvang en ook dat die bloed suurstof kry d.m.v. trageale intubasie. Meganiese ventilators kan die longe verder beskadig. Vroeggebore babas word blootgestel aan risiko’s veral waar oorerflike afwykings/steurnisse aanwesig is of komplikasies tydens geboorte en dus die EMCO die enigste lewensvatbare opsie maak. Kateters wat asemhaling aanhelp (Respiratory Assist Catheters (RACs)) kan as alternatief gebruik word aangesien dit nie ekstra spanning op die longe plaas nie, maklik is om te implementeer, koste-effektief is en beskikbaar is vir onmiddellike gebruik in kliniese omstandighede of in noodhulpsituasies. Die ontwikkeling van hierdie tipe toestel vereis kennis van moontlike suurstofverbindingsmetodes en ook die risiko’s verbonde aan die implementering van die toestel. Die moontlikheid om die bloed van suurstof te voorsien d.m.v. mikroborrels deur die RAC lyk belowend a.g.v. die hoë gasoordrag-koers wat algemeen is by borrel suurstofverbinders. Hierdie studie het ten doel om ʼn prototipe te ontwikkel wat kan dien as ʼn RAC en ook om die lewensvatbaarheid van suurstofverbinding met mikroborrels te bepaal. Die metode wat gebruik is om die prototipe te ontwerp sluit in die kies van verskeie materiale en die finalisering van die ontwerp wat getoets moet word. Die geselekteerde in vivo en ex vivo toetse is afgeneem deur gebruik te maak van dier-modelle om sodoende ondersoek in te stel na die oplossing van die mikroborrels en ook die fisiologiese gevolge van die toestel wat binne die aar geplaas is. Metings van die suurstofversadiging van bloed in slagaarbloed (SaO2), aarbloed (SvO2) en pulmonêre druk het toegelaat dat die koers en risiko’s verbonde aan suurstofoordrag geëvalueer word. Hierdie metings gee ook ’n aanduiding van die vormingsdinamika van die mikroborrels in die bloed. ’n In vitro toets is gedoen met die doel om die koers te bepaal van die oplossing van suurstof, en dus ’n aanduiding te gee van die tyd verbonde aan die oplossing van die mikroborrels. Wiskundige simulasies gebaseer op die oplossingskoers van suurstof in are het die bogenoemde toetse bevestig. Die toetse en simulasies is geanaliseer om die lewensvatbaarheid te bepaal om suurstof binne-aars te verskaf deur mikroborrels. Geskatte tye waarteen die borrels oplos is as aanduiding gebruik vir die lewensvatbaarheid van die konsep en ook die moontlike inwerkingstelling van die binne-aarse toestel.

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

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

INTRODUÇÃO: O transplante de pâncreas é a opção definitiva para a manutenção do estado normoglicêmico permanente nos portadores de diabetes mellitus tipo 1. O meio de contraste de ultra-som é uma metodologia capaz de avaliar a perfusão tecidual, mas não há um estudo para a avaliação do padrão de perfusão do pâncreas transplantado normal e patológico. A importância da avaliação da perfusão do enxerto e a aplicação do meio de contraste por microbolhas foram os motivos para a realização desta pesquisa. Este estudo, desenvolvido no Instituto de Radiologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo no período de novembro de 2004 a setembro de 2006 objetivou: A) estabelecer os padrões de vascularização do enxerto nos pacientes normais e naqueles com suspeita de complicações; B) comparar os achados de exames laboratoriais, uso ou não de insulina exógena e situação clínica dos pacientes com os achados da ultra-sonografia modo-B e com meio de contraste por microbolhas; C) Estabelecer o valor da ultra-sonografia com meio de contraste de microbolhas (USMCM) na avaliação do pâncreas transplantado. CASUÍSTICA e MÉTODOS: Vinte e seis pacientes foram submetidos ao exame de ultra-sonografia com contraste em um total de trinta estudos, 20 (66,7%) em homens e 10 (33,3%) em mulheres. A idade dos pacientes variou de 25 a 51 anos, com média de idade de 40 anos (+ 7,3 anos). O tempo médio da realização do transplante até a realização do estudo variou de um dia a 63 meses (mediana de 24 meses). Ao modo-B, o pâncreas foi analisado quanto a ecogenicidade, dimensões (avaliação qualitativa e quantitativa) e contornos. Após a administração do meio de contraste, observou-se o tempo de chegada do meio de contraste no pâncreas, o padrão e a intensidade de realce. Após esta avaliação foi realizada uma classificação baseada nos achados do modo-B e contraste nas seguintes possibilidades: padrão de perfusão normal, alterações agudas (rejeição, pancreatite ou trombose) ou alterações crônicas (rejeição crônica). RESULTADOS: Observou-se associação estatisticamente significante entre a ecogenicidade e a situação clínica (p=0,010); ecogenicidade e uso de insulina (p= 0,021); dimensões (avaliação qualitativa) e situação clinica (p=0,011); dimensões (avaliação qualitativa) e uso de insulina (p=0,028); padrão de realce (p=0,024) e intensidade do realce com a situação clínica (p=0,039). Houve associação estatisticamente significante também entre o uso ou não de insulina exógena com a perfusão do enxerto (p=0,014) e a hipótese diagnóstica (ultra-som) (p=0,001). CONCLUSÃO: Os padrões de vascularização do pâncreas transplantado normais e naqueles com suspeita de complicações foram estabelecidos. Os critérios de ecogenicidade e a avaliação qualitativa das dimensões do pâncreas ao ultra-som modo-B se mostraram adequados na diferenciação entre estudos normais e alterados. A ultra-sonografia com meio de contraste de microbolhas foi útil na diferenciação entre estudos normais e alterados do pâncreas transplantado, utilizando os critérios de padrão do realce, intensidade do realce e perfusão do enxerto na fase arterial. / INTRODUCTION: Pancreatic transplantation is the long-term therapeutic option for maintaining normoglycemic status in patients with type 1 diabetes mellitus. Sonographic contrast medium allows to evaluate tissue perfusion but there are no studies comparing this parameter in normal and pathologic pancreatic grafts in the literature at the time of this writing. The importance of assessment of graft perfusion and the potential role of contrast-enhanced sonography with microbubbles in this regard were the reasons for this research. The study conducted at the Instituto de Radiologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo between November, 2004 and September, 2006 had the following objectives: A) to establish patterns of graft vascularization in normal patients and in those with suspected abnormalities; B) to compare grayscale mode and contrastenhanced sonographic findings with clinical status, laboratory results, and need for exogenous insulin; C) to define the role of contrast-enhanced sonography with microbubbles in the evaluation of pancreatic transplantation.MATERIALS AND METHODS: Twenty-six patients underwent a total of 30 sonographic examinations with microbubbles, 20 (66.7%) men and 10 (33.3%) women. Age range was 25 to 51 years, with a mean of 40 years (+ 7.3 years). Time elapsed between transplantation and imaging ranged from 1 day to 63 months (median, 24 months). Using grayscale mode, pancreas was assessed for echogenicity, dimensions (both quantitatively and qualitatively) and contours. Following intravenous contrast medium administration, time for enhancement of the pancreatic graft, pattern and intensity of enhancement were documented. A classification system based on grayscale and contrast-enhanced sonographic findings was designed, with the following categories: normal perfusion pattern, acute changes (rejection, pancreatitis or thrombosis) and chronic changes (chronic rejection). RESULTS: There was a statistically significant association between echogenicity and clinical status (p=0.010); echogenicity and need for exogenous insulin (p=0.021); dimensions (qualitative criteria) and clinical status (p=0.011); dimensions (qualitative criteria) and need for exogenous insulin (p=0.028); pattern (p=0.024) and intensity of enhancement versus clinical status (p=0.039). There was also statistically significant association between need for exogenous insulin and graft perfusion (p=0.014), and sonography-based diagnosis (p=0.001). CONCLUSION: The study provided patterns of vascularization in normal pancreatic grafts and in patients with suspected abnormalities. Distinction of normal and abnormal pancreatic grafts was possible using echogenicity and qualitative analysis of graft size on grayscale mode. Contrast-enhanced sonography with microbubbles also contributed to differentiation between normal and abnormal pancreatic grafts, using pattern and intensity of enhancement and perfusion of the graft during the arterial phase.

INTRODUÇÃO: O transplante de pâncreas é a opção definitiva para a manutenção do estado normoglicêmico permanente nos portadores de diabetes mellitus tipo 1. O meio de contraste de ultra-som é uma metodologia capaz de avaliar a perfusão tecidual, mas não há um estudo para a avaliação do padrão de perfusão do pâncreas transplantado normal e patológico. A importância da avaliação da perfusão do enxerto e a aplicação do meio de contraste por microbolhas foram os motivos para a realização desta pesquisa. Este estudo, desenvolvido no Instituto de Radiologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo no período de novembro de 2004 a setembro de 2006 objetivou: A) estabelecer os padrões de vascularização do enxerto nos pacientes normais e naqueles com suspeita de complicações; B) comparar os achados de exames laboratoriais, uso ou não de insulina exógena e situação clínica dos pacientes com os achados da ultra-sonografia modo-B e com meio de contraste por microbolhas; C) Estabelecer o valor da ultra-sonografia com meio de contraste de microbolhas (USMCM) na avaliação do pâncreas transplantado. CASUÍSTICA e MÉTODOS: Vinte e seis pacientes foram submetidos ao exame de ultra-sonografia com contraste em um total de trinta estudos, 20 (66,7%) em homens e 10 (33,3%) em mulheres. A idade dos pacientes variou de 25 a 51 anos, com média de idade de 40 anos (+ 7,3 anos). O tempo médio da realização do transplante até a realização do estudo variou de um dia a 63 meses (mediana de 24 meses). Ao modo-B, o pâncreas foi analisado quanto a ecogenicidade, dimensões (avaliação qualitativa e quantitativa) e contornos. Após a administração do meio de contraste, observou-se o tempo de chegada do meio de contraste no pâncreas, o padrão e a intensidade de realce. Após esta avaliação foi realizada uma classificação baseada nos achados do modo-B e contraste nas seguintes possibilidades: padrão de perfusão normal, alterações agudas (rejeição, pancreatite ou trombose) ou alterações crônicas (rejeição crônica). RESULTADOS: Observou-se associação estatisticamente significante entre a ecogenicidade e a situação clínica (p=0,010); ecogenicidade e uso de insulina (p= 0,021); dimensões (avaliação qualitativa) e situação clinica (p=0,011); dimensões (avaliação qualitativa) e uso de insulina (p=0,028); padrão de realce (p=0,024) e intensidade do realce com a situação clínica (p=0,039). Houve associação estatisticamente significante também entre o uso ou não de insulina exógena com a perfusão do enxerto (p=0,014) e a hipótese diagnóstica (ultra-som) (p=0,001). CONCLUSÃO: Os padrões de vascularização do pâncreas transplantado normais e naqueles com suspeita de complicações foram estabelecidos. Os critérios de ecogenicidade e a avaliação qualitativa das dimensões do pâncreas ao ultra-som modo-B se mostraram adequados na diferenciação entre estudos normais e alterados. A ultra-sonografia com meio de contraste de microbolhas foi útil na diferenciação entre estudos normais e alterados do pâncreas transplantado, utilizando os critérios de padrão do realce, intensidade do realce e perfusão do enxerto na fase arterial. / INTRODUCTION: Pancreatic transplantation is the long-term therapeutic option for maintaining normoglycemic status in patients with type 1 diabetes mellitus. Sonographic contrast medium allows to evaluate tissue perfusion but there are no studies comparing this parameter in normal and pathologic pancreatic grafts in the literature at the time of this writing. The importance of assessment of graft perfusion and the potential role of contrast-enhanced sonography with microbubbles in this regard were the reasons for this research. The study conducted at the Instituto de Radiologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo between November, 2004 and September, 2006 had the following objectives: A) to establish patterns of graft vascularization in normal patients and in those with suspected abnormalities; B) to compare grayscale mode and contrastenhanced sonographic findings with clinical status, laboratory results, and need for exogenous insulin; C) to define the role of contrast-enhanced sonography with microbubbles in the evaluation of pancreatic transplantation.MATERIALS AND METHODS: Twenty-six patients underwent a total of 30 sonographic examinations with microbubbles, 20 (66.7%) men and 10 (33.3%) women. Age range was 25 to 51 years, with a mean of 40 years (+ 7.3 years). Time elapsed between transplantation and imaging ranged from 1 day to 63 months (median, 24 months). Using grayscale mode, pancreas was assessed for echogenicity, dimensions (both quantitatively and qualitatively) and contours. Following intravenous contrast medium administration, time for enhancement of the pancreatic graft, pattern and intensity of enhancement were documented. A classification system based on grayscale and contrast-enhanced sonographic findings was designed, with the following categories: normal perfusion pattern, acute changes (rejection, pancreatitis or thrombosis) and chronic changes (chronic rejection). RESULTS: There was a statistically significant association between echogenicity and clinical status (p=0.010); echogenicity and need for exogenous insulin (p=0.021); dimensions (qualitative criteria) and clinical status (p=0.011); dimensions (qualitative criteria) and need for exogenous insulin (p=0.028); pattern (p=0.024) and intensity of enhancement versus clinical status (p=0.039). There was also statistically significant association between need for exogenous insulin and graft perfusion (p=0.014), and sonography-based diagnosis (p=0.001). CONCLUSION: The study provided patterns of vascularization in normal pancreatic grafts and in patients with suspected abnormalities. Distinction of normal and abnormal pancreatic grafts was possible using echogenicity and qualitative analysis of graft size on grayscale mode. Contrast-enhanced sonography with microbubbles also contributed to differentiation between normal and abnormal pancreatic grafts, using pattern and intensity of enhancement and perfusion of the graft during the arterial phase.

Recombinant Pichia pastoris is one of the important methylotropic yeast due to its robustness and ability to produce hormones like human chorionic gonadotropin (hCG), luteinizing hormone (LH) extracellularly. High growth on glycerol and strong protein expression on methanol by insertion of alcohol oxidase (AOX) promoter demand the fermentation to be a multistage operation. Methylotropic pathway demands more oxygen as methanol has to be converted to formaldehyde with half mole of oxygen. Moreover as fermentation progresses cell density in the reactor also increases. In case of Pichia pastoris fermentation cell density usually reaches very high (above 100 gm/lit) at the end of fermentation. Both these contribute in the increased oxygen demand in the fermentation and oxygen transfer turns out to be a limiting step. The present study focuses on the oxygen transfer process and its improvement in the fermentation. Oxygen transfer in bioreactor is a multistep process and involves different kinetic as well as mass transfer steps. In case of fermentation especially at high cell densities, oxygen transfer from bubbles to the broth becomes limiting step. The interface transport is governed by many physical as well as kinetic parameters. It is essential to screen these parameters from the whole set to identify the key parameters. Sensitivity analysis is carried out by using Metabolic Control Analysis (MCA) to quantify the effects of different parameters. It is found that bubble size and oxygen partial pressure are two such key parameters which can be manipulated. Use of pure oxygen to increase partial pressure and thereby solubility of oxygen in broth is a common approach. This work focuses on bubble size manipulation to increase the oxygen transfer rates.The idea behind this work is on to generate micron sized bubbles and utilize them effectively in the fermentation. There are many techniques reported to generate microbubble dispersions. In this work ’Spinning Disc microbubble Generator’ is fabricated to generate microbubbles. A flat disc surrounded by baffles with 5 mm gap in between, when subjected to 5000 rpm generates microbubbles. Some modifications are done to the set up to achieve desired properties of the bubbles. The bubbles generated fall in the range of 30-300 micron with mean size of about 60 micron. Use of Tween-20 surfactant stabilize the bubbles and hence offer a good resistance to coalescence and breakage. The liquid fraction in the bubbles can be as high as 40%. Contineous addition of this dispersion unnecessarily can dilute the fermentation broth. To overcome this volume constrain, a recirculation system is designed. Microbubble dispersion is added contineously to the reactor and equivalent fermentation broth is pumped back to the microbubble generator to achieve steady state to the liquid volume in both the vessels. Mass transfer studies with microbubbles show the potential of microbubble dispersion (MBD) to enhance mass transfer significantly. Decrease in volumetric mass transfer coefficient (KLa) due to surfactant is overcompensated by the increase in the interfacial area and net effect is, potential enhancement in KLa. The enhance- ment factor, that is, ratio of mass transfer coefficient with MBD to mass transfer coefficient with conventional sparging, is obtained to be about 4 to 5. Prior to utilization of bubbles in the recirculation system, cells are checked for the shear sensitiveness. Negligible lysis losses and almost no effect on growth patterns in shake flask culture confirm that the cells used are mechanically stable at operating conditions. Better growth patterns in shake flask are observed when microbubbles are pumped for predetermined duration in the broth. It shows possible use of MBD as oxygen carriers. Glycerol batch phase with MBD and conventional sparging is studied at different initial cell densities. Conventional sparging fails to grow the cells and Dissolved Oxygen (DO) levels close to zero suggest high oxygen demands which can not be sustained by conventional sparging. The same batch is run using MBD. Reasonably good growth patterns are observed. DO levels are well above 70% for most of the time during operation. High oxygen demand which can not be sustained by conventional sparging alone can be sustained by MBD. In this way in high den- sity cultures utilization of MBD can be a good alternative to fulfill required oxygen demand in fermentation.

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.