Diversos tipos celulares nas mais variadas condições têm sido usados como doadores de núcleo para a TN. Ainda não está claro se o estado fisiológico destas células afeta o posterior desenvolvimento dos embriões. Neste trabalho, testou-se a hipóese que fibroblastos bovinos em processo de MCP podem ser reprogramados na transferência nuclear. Fibroblastos foram cultivados até atingirem 60% de confluência, sincronizados por restrição de soro durante 24h e em seguida a MCP foi analisada por citometria de fluxo com a ténica da Anexina V/Iodeto de propídeo. Células Anexina positivas (MCP) e Anexinanegativas (Vivas) foram separadas por citometria de fluxo e utilizadas para a TNS. Céulas não coradas e não separadas no citômetro serviram como controle (Controle). Os embriões reconstruídos foram avaliados quanto à fusão, clivagem (2º dia de cultivo), blastocisto (7º dia) e prenhez (D30, D60 e nascimento). O índice de MCP dos blastocistos obtidos foi determinado. Os resultados foram analisados pela ANOVA ou teste de X2 com nível de significância de 5%. Não houve efeito nas taxas de fusão (p>0,05). Embriões reconstruídos com células MCP tiveram menor taxa de clivagem e formação de blastocistos (72,7% e 18,8%, respectivamente) em comparação ao grupo reconstruído com células Vivas (83,4% e 34,7%, respectivamente; p<0,05), não diferindo dos embriões Controle (77,3% e 27,3%, respectivamente; p>0,05). O índice de MCP do grupo de embriões MCP foi similar aos índices dos embriões clonados a partir de células Vivas e Controle (p>0,05). Após a transferência para receptoras, os grupos MCP, Vivas e Controle não diferiram com relação à taxa de prenhez aos 30d (18,1%, 13,3% e 27,5%, respectivamente; p>0,05). Entretanto aos 60d, a perda gestacional no grupo MCP (25%) foi inferior a do grupo Vivas (100%) e Controle (62,5%). Somente um nascimento, do grupo MCP (4,5% dos embriões transferidos), foi obtido no experimento. Conclui-se que células em processo de MCP, podem ser reprogramadas quando utilizadas como doadoras de núcleo na técnica de transferência nuclear, podendo estabelecer gestações e nascimentos, entretanto houve um efeito prejudicial nas taxas de desenvolvimento embrionário até o estádio de blastocisto assim como houve aumento do índice de MCP nos embriões reconstruídos. / It is not clear if the physiological status of the cells can affect further embryonic development in NT. We hypothesized that adult bovine fibroblasts in PCD can be reprogrammed when used as nuclear donors for cloning. Fibroblasts were cultivated until 60% confluency, synchronized by serum starvation for 24 h and stained with Annexin V and Propidium iodide (PI) by flow citometry. Annexin positive cells (PCD cells) and Annexin negative cells (Live cells) were sorted and used for NT. Unsorted, unstained cells were used as control (Control cells). After reconstruction, fusion, cleavage (day 2 of culture), blastocyst (day 7) and pregnancy rates (day 30, 60 and birth) were recorded. Apoptotic index of the embryos was determined by TUNEL. Data were analyzed with ANOVA and Chi-square test with 5% of significance level. There was no effect on fusion rates (p>0.05). Embryos reconstructed with PCD cells had lower cleavage and blastocyst rates (72.7 and 18.8%, respectively) compared with embryos reconstructed with Live cells (83.4 and 34.7%, respectively; p<0.05). Apoptotic index in embryos produced from cells in PCD was similar compared to embryos produced from Live and Control cells (p>0.05). Pregnancy rates were similar between cloned groups on day 30 after embryo transfer (p>0.05). However it was observed a reduced pregnancy loss in PCD group on day 60 (25%) compared with Control (62.5%) and Live (100%) groups. Only one calf, from PCD cells (4.5% of the transferred embryos), has been obtained in this experiment. In conclusion, it was showed that cells in PCD process can be reprogrammed when used as nuclear donors after NT producing even live animals. However, a negative effect on embryonic development and an increase in the apoptotic index of these embryos was observed.

Philosophiae Doctor - PhD / The estimated 5 × 10³⁰ prokaryotic cells inhabiting our planet sequester some 350–550 Petagrams (1 Pg = 1015 g) of carbon, 85–130 Pg of nitrogen, and 9–14 Pg of phosphorous, making them the largest reservoir of those nutrients on Earth (Whitman et al. 1998). However, reports suggest that only less than 1% of these microscopic organisms are cultivable (Torsvik et al. 1990; Sleator et al. 2008). Until recently with the development of metagenomic techniques, the knowledge of microbial diversity and their metabolic capabilities has been limited to this small fraction of cultivable organisms (Handelsman et al. 1998). While metagenomics has undoubtedly revolutionised the field of microbiology and biotechnology it has been generally acknowledged that the current approaches for metagenomic bio- rospecting / screening have limitations which hinder this approach to fully access the metabolic potentials and genetic variations contained in microbial genomes (Beloqui et al. 2008). In particular, the construction of metagenomic libraries and heterologous expression are amongst the major obstacles. The aim of this study was to develop an ultra-high throughput approach for screening enzyme activities using uncloned metagenomic DNA, thereby eliminating cloning steps, and employing in vitro heterologous expression. To achieve this, three widely used techniques: cell-free transcription-translation, in vitro compartmentalisation (IVC) and Fluorescence Activated Cell Sorting (FACS) were combined to develop this robust technique called metagenomic in vitro compartmentalisation (mIVC-FACS). Moreover, the E. coli commercial cell-free system was used in parallel to a novel, in-house Rhodococcus erythropolis based cell-free system. The versatility of this technique was tested by identifying novel beta-xylosidase encoding genes derived from a thermophilic compost metagenome. In addition, the efficiency of mIVC-FACS was compared to the traditional metagenomic approaches; function-based (clone library screening) and sequence-based (shotgun sequencing and PCR screening). The results obtained here show that the R. erythropolis cell-free system was over thirty-fold more effective than the E. coli based system based on the number of hits obtained per million double emulsions (dE) droplets screened. Six beta-xylosidase encoding genes were isolated and confirmed from twenty-eight positive dE droplets. Most of the droplets that were isolated from the same gate encoded the same enzyme, indicating that this technique is highly selective. A comparison of the hit rate of this screening approach with the traditional E. coli based fosmid library method shows that mIVC-FACS is at least 2.5 times more sensitive. Although only a few hits from the mIVC-FACS screening were selected for confirmation of beta-xylosidase activity, the proposed hit rate suggests that a significant number of positive hits are left un-accessed through the traditional clone library screening system. In addition, these results also suggest that E. coli expression system might be intrinsically sub-optimal for screening for hemicellulases from environmental genomes compared to R. erythropolis system. The workflow required for screening one million clones in a fosmid library was estimated to be about 320 hours compared to 144 hours required via the mIVC-FACS screening platform. Some of the gene products obtained in both screening platforms show multiple substrate activities, suggesting that the microbial consortia of composting material consist of microorganisms that produce enzymes with multiple lignocellulytic activities. While this platform still requires optimisation, we have demonstrated that this technique can be used to isolate genes encoding enzymes from mixed microbial genomes. mIVC-FACS is a promising technology with the potential to take metagenomic studies to the second generation of novel natural products bio-prospecting. The astonishing sensitivity and ultra-high throughput capacity of this technology offer numerous advantages in metagenomic bio-prospecting. / National Research Foundation (NRF)

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

La mer Méditerranée en période stratifiée est une zone ultra-oligotrophe carencée en P. La production procaryotique hétérotrophe étant, comme la production primaire, souvent limitée par la biodisponibilité en phosphate (P), en Méditerranée, les flux d'assimilation de Pi et d'incorporation de leucine dans les protéines ont été particulièrement étudiés dans ce travail de thèse. Cette étude est focalisée sur la mesure des flux spécifiques d'assimilation des populations picoplanctoniques (Synechococcus = Syn, Prochlorococcus=Proc, picophytoeucaryotes=Pic, procaryotes hétérotrophes=Hprok) et sur les contribution à l'échelle cellulaire aux flux globaux, au travers de l'estimation de leurs aptitudes de compétition vis-vis de la limitation en Pi, en combinant le marquage radioactif et le tri cellulaire par caryométrie en flux. Les données ont été acquises dans le cadre de la campagne transméditerranéenne multiidisciplinaire BOUM (jui-juillet 2008). Toutes les populations étudiée participent significativement aux flux d'assimilation de Pi dans le compartiment microbien, lequel est dominé par les Hprok en surface et les cyanobactéries autour du maximum profond de chlorophylle et jusqu'au bas de la zone euphotique : Proc au large et Syn en zone moins ultraoligotrophe. Les caractéristiques cinétiques de ces mêmes populations ont montré qu'avec ses fortes valeurs de Vmax et de Kt+Sn, Syn est la plus capable des populations à répondre rapidement à des apports pulsés en Pi, tandis que Proc et Hprok présentent une forte affinité pour le Pi, i.e. de bonnes aptitudes de compétition aux plus faibles concentrations. La population des Proc aux flux d'incorporation total de leucin n'est pas négligeable (jusqu'à 30%). Compte tenu du caractère ubiquiste et du succès écologique (en abondance) des Proc dans les océans, il semble que la mixotrophie soir une stratégie suivie très adaptée aux environnement oligotrophes. La forte participation (jusqu'à 42 %) des Hprok à faible contenu en acides nucléiques (bactéries de type LNA) par rapport à celle de la communauté totale a été mise en évidence, en particulier dans les eaux de surface, et confirme la forte contribution de ce groupe cytométrique à la production procarytique hétérotrophe dans les systèmes oligotrophes. / The Mediterranean Sea is know as an oligotrophic P-depleted area, where reduced exogenous nutriment suplly to the euphotic zon push microorganisms into intense competition for nutritive resources. For instance, heterotrophic prokaryotic production may, like photosynthesis, be limited by the availability of phosphate (Pi). Thus, this study aimed at investigating Pi assimilation and leuvine incorporation into proteins among different picoplanktonic populations ; heterotrophic prokaryotes (Hprok), Synechococcus (Syn), Prochlorococcus (Proc) and picophytoplankton. More precisely, cell-specific Pi assimilation rates and cell-specific leucine incorporation rates were determined using flow cytometry coupled to cell storing. Data were acquired during the trans-Mediterranean cruise BOUM (June-July 2008). All microbial populations sorted significantly participated to Pi assimilation fluxes, which were dominated, in terms of contribution to the whole-sea water activity, by Hprok wihin surface layers and cyanobacteria in vicinity of the deep chlorophyl maximun (Proc in open sea and Syn at more coastal stations ) and below.Specific kinetic characteristics of Pi uptake showed Syn to be adapted to Pi pulses, owing to their heigher Vmax and k+Sn. At the opposite, Proc and Hprok were more adapted to lower concentrations due to a better affinity for Pi. Proc., detected at the vicinity of the deep chlorophyll maximum, participated actively to leucine incorporation rates of the bulk community (up to 30 %), confirming mixotrophic capacity as a survival strategy in oligotrophic environments. Hprok cells with low nucleic acid content (LNA cells) contributed up to 42% to the bulk leucine incorporation prokaryotic production in oligotrophic waters

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20806号 / 医博第4306号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 髙橋 良輔, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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No description available.

This thesis extends an existing cell population modelling framework to investigate two different hypotheses for what drives the phenomenon of cell sorting, which is the spontaneous self-reorganization of cell populations. This behaviour cause cells to find their way back into their original configuration after they have been scrambled. Original tissue function may also be regained. The modelling framework is called discrete Laplacian cell mechanics (DLCM), and models cell movement on a lattice as a result of pressure differences. The first hypothesis suggests that cells exhibit type-specific adhesion properties which cause cells of the same type to adhere more to each other than to cells of a different kind. The other, more recent, hypothesis explains cell sorting behaviour as a consequence of interfacial tension, where cells of different types exhibit larger tension between them compared to cells of the same type. Adhesion is implemented as a passive force between cells of the same type, which counteract the pressure-driven events, while interfacial tension is implemented as pressure sources arising due to contact with cells of a different type. This thesis investigates whether these additions on the scale of individual cells can be sufficient to induce sorting behaviour on the cell population scale. Subsequently the suitability of implementing these effects in the DLCM framework can be evaluated. Starting from a scrambled cell configuration of two types, the results show that differential adhesion can result in the cell population sorting into smaller clusters, with the addition of Brownian motion improving the sorting ability significantly. Differential interfacial tension as it is implemented here demonstrates the effect of dissociation between cells of different type, but this is not sufficient to achieve sorting. The behaviour can be likened to a form of localized Brownian motion where more unsorted areas are prone to more movement events. Therefore, differential tension is not deemed suitable within the DLCM framework on its own. The cohesive effect of differential adhesion together with the dissociative effect of differential interfacial tension proved to work well together, acheiving a high degree of sorting both overall and compared to the case of only differential adhesion with some Brownian motion. Full separation into one distinct cell mass for each cell type present could not be achieved.

Sorting cells means manipulating them. This induces biological responses of the cells, resulting in functionalities not representing the previous state of the cells, but indicating effects of sorting procedures. Namely in cases that negative selection is not possible, isolated cells are distinct to their previous characteristics. This is true for bead-based sorting or flow cytometric cell separation and heavily skews functional markers of target cells. Of course, this is a limitation for any following investigation of these cells.

No description available.