Manual handling of biological cells is routine in most laboratories. This is gradually changing with the development of robotic cell handling systems, and micro-scale lab-on-chip devices. Attempts were made to develop devices that automate or assist cell handling in the context of a bovine nuclear transfer (NT) system. The system, a zona-free bovine NT cloning system, formed a baseline reference for tool design and performance evaluation. Bovine NT can, as other cell handling procedures, be improved by rapid and precise cell positioning. Improvements in cell handling can increase the quantity of cells processed, and the uniformity of conditions the cells are subject to during processing. Tools were developed for two areas of cell handling: cell fusion and cell transportation. Designs suitable for implementation in microscale lab-on-chip systems were evaluated. Tool development was predominantly experimental, assisted by numerical modelling. The experimental investigation concerned device fabrication and operational performance. A number of cell handling tool designs were built and tested. Coplanar electrodes are not commonly used in bovine NT and reports on their efficacy were not available. These electrodes, which are simple to fabricate, were tested to determine fusion rates achievable in comparison with those of the baseline procedure. A novel fusion device, the micropit, was designed to assist bovine cell pairing and electrofusion. It was initially uncertain whether this device was capable of achieving cell fusion. Tests were conducted; and cell fusion and micro-positioning were demonstrated, as was an increase in biological cell processing throughput. Many miniaturised lab-on-chip systems rely on cell transportation. One illustration in the baseline procedure is the on-chip transport of cells to the cell fusion device. Potential cell transport mechanisms for a miniature cloning system were evaluated by prototype construction and testing. These mechanisms included travelling wave dielectrophoresis and capillary fluid actuation. To facilitate automation of on-chip cell transportation, a low cost electrically isolated cell detection system was developed based on a DVD pick-up unit. Various obstacles that were encountered during the course of device construction are noted, as are the fabrication methods employed.

Monoploid potato genotypes represent plant material that is free from the "genetic load" of lethal and severely deleterious alleles normally present in the highly heterozygous cultivated potato species. Field evaluations enabled the identification of agronomically superior monoploid potato genotypes from a population of more than 100 anther-derived monoploids. Chemical fusion and electrofusion between pairs selected from 31 superior monoploids resulted in the production of three different groups of intermonoploid somatic hybrids. The hybridity of somatic hybrid plants and calluses was confirmed through PCR-based amplification of simple sequence repeat (SSR) sequences in the potato genome. Polymorphic SSR loci between the monoploid parents of a particular group of somatic hybrids were used to separate true somatic hybrids (heterozygous at the loci) from parental somaclones regenerating from unfused protoplasts (homozygous for one parental band at the loci). One group of somatic hybrids (SH1, SH2 and SH2B) was of particular interest because it resulted from the fusion of a S. phureja monoploid to a high acetylleptinidine-producing monoploid derived from an F1 hybrid between S. chacoense and S. phureja. The leptine acetylleptinidine (ALD) is produced only by some accessions of S. chacoense Bitt. and provides resistance to feeding by the Colorado potato beetle (Leptinotarsa decemlineata Say) when present in sufficient concentrations. The somatic hybrids produced moderate levels of ALD in leaves and stems (roughly 60% that of a high ALD-producing S. chacoense clone). Pollinations of SH1, SH2 and SH2B by several diploid and tetraploid potato clones resulted in three fruit on SH2, one fruit on SH2B and no fruit on SH1. Two resulting progeny populations of SH2 [SH2A = SH2 × S. andigena 8-1 (4x); SH2P = SH2 × S. phureja 66AP11-53 (2x)] expressed higher fertility than the original somatic hybrids and were sexually crossed as both male and female parents to S. tuberosum cv. Atlantic. All of the SH2 progeny populations expressed acetylleptinidines, albeit at lower levels than the SH2 somatic hybrid, providing strong evidence that the genes controlling acetylleptinidine production are dominant. Variation for ALD expression in the SH2 progeny indicated one or a few genes with additive effect controlling the ALD trait. In addition, the choice of male parent in sexual crosses to SH2 affected subsequent ALD expression in progeny populations. The SH2 progeny represent an important first step towards transferring acetylleptinidines to cultivated potato. SH1, SH2 and SH2B appeared to be negatively affected by an unusually high ploidy (hexaploid, 6x). Field-grown plants produced many tubers (mean = 35) of low weight (mean = 10.4 g) and were stunted in appearance. Anther culture of SH2 yielded triploid regenerants (3x). These regenerants may be more phenotypically normal than the original somatic hybrids because of lower ploidy. Segregation of SSR alleles in the triploid anther culture regenerants provided evidence that the hexaploid somatic hybrid SH2 genome is comprised of four homologous genomes of CP2-103 (the high leptine-producing monoploid) and two homologous genomes of 13-14 203 (the S. phureja monoploid). / Master of Science

Nosso objetivo e estudar os principais mecanismos de foto absorção no ANTPOT.232 TH entre 7 e 60 Mev, ressonâncias gigantes e mecanismo do quase-deuteron, associados ao decaimento por fissão. Para isso, fizemos medidas da seção de choque de eletrofissão no ANTPOT.232 TH, entre as energias de 7 a 60 Mev, no laboratório do acelerador linear do IFUSP. Utilizamos, na analise, o formalismo dos fótons virtuais, que relaciona a seção de choque de fotofissão a seção de choque de eletrofissão. Foi utilizada a seção de choque de fotofissão medida em livermore e calculamos a seção de choque de fotoabsorção e2 (t=0 e t=1), além da seção de choque de fotoabsorção obtida via quase-deuteron. Verificamos que a contribuição predominante para a seção de choque de eletrofissão e a da componente de dipolo elétrico, sendo as demais contribuições (quadrupolo elétrico e modelo modificado do quase-deuteron) uma ordem de grandeza inferior a esta. / Our objective is to study the main photoabsorption mechanisms in 232T]n between 7 and 60 Mev, namely, Giant Resonances and Quasi-Deuteron, associated to fission decay. We performed electrofission measurements at the linear accelerator of IFUSP. In the analysis, the Virtual Photon Formalism, that relates the photofission cross section to the electrofission cross section, was used. In the calculations, the photofission cross section measured at Livermore Laboratory was used. The E2 photoabsorption and quasi-deuteron photoabsorption cross sections were calculated. We concluded that the most important contribution to the electrofission cross section comes from the El- giant resonance component, the other ones (E2 giant resonance component and quasi-deuteron mechanisms) being 1 or 2% of total electrofission cross section.

La fusion cellulaire est une méthode de génération de cellules hybrides combinant les propriétés spécifiques des cellules mères. Initialement développée pour la production d’anticorps, elle est maintenant aussi investiguée pour l’immunothérapie du cancer. L’électrofusion consiste à produire ces hybrides en utilisant un champ électrique pulsé. Cette technique présente de meilleurs rendements que les fusions chimiques ou virales, sans introduire de contaminant. L’électrofusion est actuellement investiguée en cuve d’électroporation où le champ électrique n’est pas contrôlable avec précision et le placement cellulaire impossible, produisant de faibles rendements binucléaires. Afin d’augmenter le rendement et la qualité de fusion, la capture et l’appariement des cellules s’avèrent alors nécessaires.Notre objectif a été de développer et de réaliser des biopuces intégrant des microélectrodes et des canaux microfluidiques afin de positionner et d’apparier les cellules avant leur électrofusion. Une première structure de piégeage se basant sur des plots isolants et l’utilisation de la diélectrophorèse a été réalisée. Afin d’effectuer des expérimentations sous flux, une méthode de scellement des canaux, biocompatible et étanche a été développée. Puis, le milieu d’expérimentation a été adapté pour l’électrofusion. En confrontant les résultats des expériences biologiques aux simulations numériques, nous avons pu démontrer que l’application d’impulsions électriques induisait la diminution de la conductivité cytoplasmique. Nous avons ensuite validé la structure par l’électrofusion de cellules. Un rendement de 55% avec une durée de fusion membranaire de 6 s a été obtenu. Dans un second temps, nous avons proposé deux microstructures de piégeage pour l’électrofusion haute densité. La première se base sur un piégeage fluidique, alors que la seconde, utilise ladiélectrophorèse sans adressage électrique à l’aide de plots conducteurs. Jusqu’à 75% des cellules fusionnent dans cette dernière structure. Plus de 97% des hybridomes produits sont binucléaires. Le piégeage étant réversible, les hybridomes peuvent ensuite être collectés pour des études ultérieures. / Cell fusion is a method to generate a hybrid cell combing the specific properties of its progenitor cells. Initially developed for antibody production, it is now also investigated for cancer immunotherapy. Electrofusion consists on the production of hybridoma using electric pulses. Compared to viral or chemical methods, electrofusion shows higher yields and this system is contaminant free. Actually, electrofusion is investigated in electroporation cuvettes, where the electric field is not precisely controllable and cell placement impossible, resulting in low binuclear hibridoma yields. To improve the fusion quality and yield, cell capture and pairing are necessary.Our objective was the development and realization of biochips involving microelectrodes and microfluidic channels to place and pair cells prior to electrofusion. A first trapping structure based on insulators and the use of dielectrophoresis has been achieved. In order to perform fluidic experiments, a biocompatible irreversible packaging was developed. Then, the experimental medium was optimized for electrofusion. Confronting the biological experiments and the numerical simulations, we showed that the application of electric pulses leads to a decrease of the cytoplasmic conductivity. The microstructure was validated by cell electrofusion. A yield of 55%, with a membrane fusion duration of 6 s has been achieved. Secondly, we proposed two trapping microstructures for high density electrofusions. The first one is based on a fluidic trapping while the second one uses dielectrophoresis, free of electric wiring, thanks to conductive pads. Up to 75% of paired cells were successfully electrofused with the conductive pads. More than 97% of the hybridoma were binuclear. The trapping being reversible, the hybridoma can be collected for further analysis.

Notre sujet de recherche vient d'une demande de cliniciens pour obtenir en grande quantité des hybridomes issus de la fusion entre cellules cancéreuses et cellules dendritiques. Ces travaux s'insèrent dans la recherche contre le cancer par un procédé proche de la vaccination, appelé immunothérapie du cancer. Il existe actuellement des dispositifs commercialisés, mais ceux-ci ne parviennent pas à produire de façon importante des hybridomes de qualités. L'idée était donc de se placer à l'échelle des cellules, pour interagir sur elles de façon précise, tout en bénéficiant de la miniaturisation pour contrôler un grand nombre de cellules en parallélisant le dispositif. Restait à choisir quelles forces utiliser pour agir sur les cellules pour les déplacer, les placer et les fusionner, car à l'échelle microscopique le rapport des forces sont différents de ceux à notre échelle. Nous avons opté pour l'emploi de la microfluidique pour le transport des cellules, de la diélectrophorèse pour les positionner et de l'électrofusion pour les fusionner. Ce projet est à la conjonction de savoir-faire dans les domaines de la microtechnologie, de l'interaction champs électriques-cellules et de la biologie cellulaire.

Nosso objetivo e estudar os principais mecanismos de foto absorção no ANTPOT.232 TH entre 7 e 60 Mev, ressonâncias gigantes e mecanismo do quase-deuteron, associados ao decaimento por fissão. Para isso, fizemos medidas da seção de choque de eletrofissão no ANTPOT.232 TH, entre as energias de 7 a 60 Mev, no laboratório do acelerador linear do IFUSP. Utilizamos, na analise, o formalismo dos fótons virtuais, que relaciona a seção de choque de fotofissão a seção de choque de eletrofissão. Foi utilizada a seção de choque de fotofissão medida em livermore e calculamos a seção de choque de fotoabsorção e2 (t=0 e t=1), além da seção de choque de fotoabsorção obtida via quase-deuteron. Verificamos que a contribuição predominante para a seção de choque de eletrofissão e a da componente de dipolo elétrico, sendo as demais contribuições (quadrupolo elétrico e modelo modificado do quase-deuteron) uma ordem de grandeza inferior a esta. / Our objective is to study the main photoabsorption mechanisms in 232T]n between 7 and 60 Mev, namely, Giant Resonances and Quasi-Deuteron, associated to fission decay. We performed electrofission measurements at the linear accelerator of IFUSP. In the analysis, the Virtual Photon Formalism, that relates the photofission cross section to the electrofission cross section, was used. In the calculations, the photofission cross section measured at Livermore Laboratory was used. The E2 photoabsorption and quasi-deuteron photoabsorption cross sections were calculated. We concluded that the most important contribution to the electrofission cross section comes from the El- giant resonance component, the other ones (E2 giant resonance component and quasi-deuteron mechanisms) being 1 or 2% of total electrofission cross section.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12944号 / 論医博第2096号 / 新制||医||1010(附属図書館) / 32203 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 川口 義弥, 教授 横出 正之, 教授 稲垣 暢也 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Det absolut vanligaste materialet vid nybyggnad av dricksvattenledningar var år 2008 polyeten. Flera VA-huvudmän i Sverige och Norge upptäckte dock att vissa av dessa ledningar gick sönder efter bara några få år i drift, trots det faktum att de skulle ha en drifttid på upp mot 150 år. Flera VA-bolag gick då ihop och skapade en branschorganisation under namnet 4S med syftet att stimulera till utveckling av ledningsmaterialet. Organisationen visste dock inte hur utbrett problemet med läckande PE-ledningar var och hade bara indikationer om bakomliggande orsaker. Det vill säga att PE-ledningar av stora dimensioner gick sönder efter få år i drift på grund av fel på skarven. Uppgiften med detta examensarbete var att ta fram ett statistiskt underlag som påvisar hur omfattande problemet var och kunna hitta samband som visar var och varför problemen uppkom. Ett frågeformulär som beskrev för VA-huvudmännen vilken information som eftersöktes för att kunna göra en bra undersökning sattes ihop och skickades ut. De data som sen inkom sammanställdes i ett dokument i programmet Excel för att få en enhetlig och övergripande bild. Resultaten som kom in visade sig dock ha stora brister och det var svårt att dra några statistiskt säkerställda slutsatser. Det fanns dock antydan till att problemet var på skarvar och i mindre dimensioner, oftast på servisledningar. Problembilden som uppkom under arbetets gång var att de data som tillhandahölls oftast var antingen bristfälligt eller obefintligt, och något VA-huvudmännen måste arbeta mer med. Det gick heller inte att se några ålderssamband, det vill säga att PE-ledningarna skulle gå sönder efter kort eller lång tid i drift. / The most common material used when producing new water pipes in Sweden in 2008 was polyethylene. A couple of pipe owners in Sweden discovered that pipes made out of PE who only had been operating a few years broke, despite the fact they are supposed to have a service life of 150 years. The pipe owners therefore created an organization named 4S with the target to develop the PE pipe system. The problem was that the organization did not know how widespread the problem with leaking PE pipes was and they only had indications of why it is caused. The objective with this project was to collect statistics to show how widespread the problem is and to find where and why the problem arises. A couple of question that described to the pipe owners what kind of data that was needed, on order to make a good examination, was put together and sent out. The data that came in was put together in an Excel document to get a better picture of the problem. The results that came in proved to have major flaws and it was difficult to draw statistically based conclusions. The results, however, showed a hint that the problem mainly was in the joints and in smaller dimensions. The biggest problem was that the received data most often was either inadequate or non-existent, and the pipe owners have to work harder improving the system. The statistics also cannot find any age related problem.

An advanced finite element (FE) model of the electrofusion welding of thermoplastic pipes has been developed using the ABAQUS FE package. The heat transfer analysis is coupled with thermal deformation analysis to include the time-dependent closure of the initial gap between the pipe and fitting. The effect of radial melt movement into the interface is modelled using a new `virtual material movement¿ technique. The predicted results (temperature distribution in the weld region, melt affected zones and gap closure time) are compared with experimental data and good agreement is found

En este trabajo de tesis, se presentan diversos estudios experimentales, desarrollados prin cipalmente en pez cebra pero también en ostra del Pacífico, que persiguen la puesta a punto de técnicas relevantes para la utilización de estas dos especies en el campo de la biomedicina, la toxicogenómica y la acuicultura como modelo experimental. En pez cebra, se han puesto a punto y testado: Técnicas de vitrificación de tejido de aleta caudal, de blastómeras (en microvolúmenes) y de tejido gonadal. Técnica de quimerismo de la línea germinal en estadio MBT, con una penalización previa por radiación ultravioleta de los embriones receptores. Técnica de quimerismo larvario (larvas de 48-72 h) utilizando como donantes células testiculares obtenidas de individuos adultos y previamente criopreservadas. Técnica de transplante nuclear utilizando como donantes núcleos de células somáticas adultas y larvarias. Técnica de electroactivación en medio iónico de oocitos de pez cebra. In ostra del Pacífico, se han puesto a punto y testado: Evaluación de los cambios estacionales en la calidad oocitaria y espermática en ostra del Pacífico. Técnica de electrofusión cigótica de cigotos obtenidos por fecundación in vitro en ostra del Pacífico. Técnicas de vitrificación de tejido de aleta caudal, de blastómeras (en microvolúmenes) y de tejido gonadal. En relación con este grupo de técnicas, señalar el diferente nivel de dificultad de cada una de ellas e incluso de los resultados alcanzados. Así, la vitrificación de tejido de la aleta caudal no ha supuesto problema adicional respecto a la aplicación de dicha técnica en los últimos años a tejidos epiteliales de cinco especies de mamíferos, lo que da idea de la versatilidad en el uso de dicha técnica de vitrificación básica. Por contra, la criopreservación de blastómeras ha supuesto un verdadero reto, ya que los patrones de permeabilidad celular en esta especie acuática es muy diferente a la característica en mamíferos. / Cardona Costa, J. (2010). EMBRYOLOGICAL AND MICROMANIPULATION TECHNIQUES IN ZEBRAFISH (Danio rerio) AND PACIFIC OYSTER (Crassostrea gigas) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8326 / Palancia