Conselho Nacional de Desenvolvimento Científico e Tecnológico / In this work we presented the idea to perform excision assays using the piggyBac transposable element as enzyme supplier and the inverted terminal sequences of the element, both necessary for mobilization of a transposable element. Drosophila S2 cells were electroporated to perform insertion of two different plasmids in the cytoplasm of cells, a plasmid carrying the terminal inverted repeats of piggyBac element flanking a GFP gene and other with the transposase coding sequence enzyme which recognizes the terminal inverted repeats, excise of the region where the element is and insert it into another locus. This is a vector-helper system, in which a fragment is excised from a plasmid with the help of the transposase located in the other. Conventional PCR was used to verify excision events showing a 200bp amplification region where the fragment was excised and a region 3kb amplification reagion at times when the fragment was full, ie, it has not mobilized. The qPCR technique was used to quantify the excision of this fragment, carrying out comparisons of the amount of plasmid DNA recovered from the S2 cells after the end of experiment with serial dilutions of the original plasmids carrying the ITRs, which was used as standard. The results showed that the technique involving electroporation and qPCR is feasible and can be used to quantify mobilization of transposable elements. Paralleling with existing tools for this type of quantification, qPCR shows up as a very sensitive technique of detection mobilization, as well as a low cost technique budget. / Neste trabalho apresentamos a ideia de realizar ensaios de excisão utilizando o elemento transponível piggyBac como fornecedor da enzima e das sequências terminais invertidas do elemento, ambos necessários para mobilização. Células S2 de Drosophila melanogaster foram eletroporadas para que houvesse inserção de dois diferentes plasmídeos no citoplasma das células, um plasmídeo portando as repetições terminais invertidas do elemento piggyBac flanqueando um gene GFP e o outro com a sequência codificadora da enzima transposase, a qual reconhece as repetições terminais invertidas e excisa o elemento da região onde está inserido, num sistema vector-helper, em que um fragmento é excisado de um plasmídeo com ajuda da transposase localizada no outro. PCR convencional foi usado para verificar os eventos de excisão, mostrando uma região de amplificação de 200pb nos casos de excisão do fragmento e uma região amplicada de 3kb, nas ocasiões em que o fragmento ficou inteiro, ou seja, não foi mobilizado. A qPCR foi utilizada para quantificar a excisão desse fragmento, realizando comparações da quantidade de DNA plasmidial recuperado das células S2 após o término do experimento com diluições em série do plasmídeo com as ITRs, que foi utilizado como standard. Os resultados mostraram que a técnica envolvendo eletroporação e qPCR é exequível e pode ser utilizada para quantificar mobilização de elementos transponíveis. Fazendo um paralelo com as ferramentas já existentes para esse tipo de quantificação, qPCR mostra-se como uma técnica bastante sensível de detecção de mobilização, bem como uma técnica de baixo custo orçamentário.

La variabilité est une propriété intrinsèque aux systèmes biologiques, essentielle pour l'évolution et l'embryogénèse. Souvent considérée comme du bruit, ce n'est que récemment que l'aléatoire des processus biologiques a commencé à être systématiquement étudié. Cette thèse pose les questions suivantes : qu'est-ce qu'un développement normal ? Quel est l'étendue et le rôle de la variabilité dans la robustesse et la résilience du développement embryonnaire ?Ces questions sont posées pour le lapin (Oryctolagus cuniculus) et l'oursin (Paracentrotus lividus et Sphaerechinus granularis).Nous nous sommes aussi intéressé à la quantification du déterminisme de la variabilité embryonnaire à l'aide d'oursins jumeaux et de lapins clonés.La mesure des comportements cellulaires est effectuée sur des lignages cellulaires obtenus à partir d'imagerie 3D+temps. Nous montrons que les oursins jumeaux peuvent se développer selon trois phénotypes différents, jamais observés chez le normal, avant de converger vers une blastula d'apparence normale. De plus, les comparaisons entre et au sein des pairs de jumeaux montrent que le phénotype et la survie ne dépend que de l'histoire individuelle des embryos.Nos mesures quantitatives des pairs de jumeaux amènent des questions ouvrant de nouveaux horizons de recherche : les jumeaux sont-ils robustes ou résilient ?Le développement pré-implantatoire des lapins a été étudié sur cinq embryons numériques (trois sauvages et deux clones), du stade 32-cellules à l'éclosion.Nous montrons que les divisions asymétriques internes et externes régulent la variation du nombre de cellules internes ainsi que la taille de la masse cellulaire.De plus, la variation du nombre de cellules internes est plus grande que pour les cellules externes, ce qui semble directement lié au taux de morts cellulaires.Notre hypothèse est que le potentiel de bon développement des clones est assuré par une grande plasticité épigénétique des cellules donneuses.Ce travail espère définir des méthodes et des concepts fondateurs pour une exploration quantitative et une modélisation multi-échelle de la morphogénèse animale. / Variability is an intrinsic characteristic of biological systems, essential for evolution and embryogenesis.Considered as noise for centuries, it is only recently that the stochasticity of biological processes has began to be systematically explored.The present thesis addresses the following questions: What is a normal development?What is the extent and role of variability in developmental robustness and resilience?We tackle these issues in rabbit (Oryctolagus cuniculus) and sea urchin (Paracentrotus lividus and Sphaerechinus granularis).We also aimed to quantify determinism and stochasticity of developmental variability by means of sea urchin twins and cloned rabbits.Variations in cell behaviors were investigated through reconstruction of cell lineage from 3D+time imaging.We showed that sea urchin twins can follow three different developmental paths never observed in normal embryo, before converging to normal looking blastula.Moreover, comparisons between and within pairs of twins revealed that phenotype and survival depend on individual history alone.Our quantitative observation of twin pairs raises question opening a future line of research: are twins robust or resilient?Rabbit preimplantation development was explored with five digital specimens (three wild-types and two clones) from the 32-cell stage to hatching.We showed that inner and outer asymmetric divisions regulate the variation of inner cell number and may control inner cell mass size.In addition, the variation of inner cell number in clones is higher than outer cells which seems to be directly correlated to their cell death ratio.Our current hypothesis is that the potential to lead to viable clones requires plasticity of donor's epigenetic state.This work is expected to ground concepts and methods for a quantitative exploration and further multilevel modeling of morphogenetic processes.

Embryonic stem (ES) cells have the ability to maintain pluripotency and self-renewal during in vitro maintenance, which is a key to their clinical applications. ES cells are a model in developmental biology studies due to their potential to differentiate in vitro. Understanding critical pathways of pluripotency, self-renewal, and differentiation during early embryonic development is important for the evaluation of the therapeutic potential of ES cells because of their ability for tumor transformation due to genetic and epigenetic instability acquired during in vitro culture maintenance. Single tandem repeats are sequences of DNA that have been implicated in the deregulation of gene expression in different human conditions. Understanding the origin of repetitive sequence instability and functions in the genome allow characterization of early genomic instability signals in ES cell pluripotency, differentiation, and tumor transformation pathways. The hypothesis of this study was that genetic stability, in repetitive sequences, located near embryonic developmental genes is responsible for pluripotency, self-renewal, differentiation, and chromatin assembly and could be a signal for adaptation, differentiation, or transformation of ES cells in vitro. Our result showed instability in specific repetitive sequences which increased during ES cell passages and embryoid body differentiation in vitro. ES cells displayed significant mean frequencies of genomic instability in repetitive regions that lead to ES cells pluripotency, self-renewal maintenance, or cell lineage specialization. The present study reports potentially biomarkers for identifying accumulation of genomic instability in specific genes that may contributes to adaptation of ES cells and could be the switch that initiates early ES cell lineage commitment in vitro. Determining genetic and epigenetic modifications, including single tandem repeat instability, gene expression changes, and chromatin modifications, is essential for elucidating possible molecular mechanisms of genomic instability and determining novel molecular characterization for diagnostic purposes to ensure ES cell stability and integrity that could potentially lead to use of ES cell derivatives that could then be a safe source needed for regenerative medicine applications

No description available.

Ácidos graxos podem atuar na apoptose ativando e/ou desativando sinais que regulam este processo. Células Jurkat (linhagem de linfócitos-T) foram tratadas com ácido palmítico (PA) (50, 100 e 150µM) e avaliada a ativação de vias de morte e de sobrevivência. O tratamento com PA induziu apoptose pela ativação da via intrínseca de maneira dose-dependente. A exposição destas células ao PA elevou a expressão do receptor de insulina e de GLUT-4, obtendo-se correlação positiva com a apoptose. O mesmo foi observado em linfócitos humanos expostos ao PA e de linfócitos de ratos em jejum. O tratamento das células Jurkat com insulina após exposição ao PA promoveu a ativação da via de sinalização insulínica. O PA aumentou a captação de glicose, mas observou-se diminuição de sua oxidação e o acúmulo de lípides. Assim, a via de sinalização da insulina e o metabolismo de glicose não oxidativo são estimulados como parte de uma coordenada resposta de sobrevivência de linfócitos expostos ao PA em baixas concentrações, mas em altas concentrações ocorre apoptose. / Fatty acids affect apoptosis pathway activating or deactivating signals that regulate this process. Jurkat cells (T-lymphocyte lineage) were treated with sub-lethal concentrations of palmitic acid (PA) (50, 100 e 150µM) and the cell death and survival signaling pathways were investigated. PA induced apoptosis in a dose dependent manner activating the intrinsic pathway. Jurkat cells exposed to PA showed increased insulin receptor and GLUT-4 expression and a positive correlation with apoptosis was obtained. Similar effect was observed in human lymphocytes exposed to PA and lymphocytes from fasted rats. Insulin treatment of Jurkat cells after PA exposure promoted the activation of the insulin signaling pathway. Glucose uptake was increased in the presence of PA, however, its oxidation was diminished and ncreased of lipids accumulation. Therefore, insulin signaling and non oxidative glucose metabolism are stimulated as a part of a coordinated response to survival of lymphocytes exposed to low concentration of PA but in high concentration it induces apoptosis.

Ácidos graxos podem atuar na apoptose ativando e/ou desativando sinais que regulam este processo. Células Jurkat (linhagem de linfócitos-T) foram tratadas com ácido palmítico (PA) (50, 100 e 150µM) e avaliada a ativação de vias de morte e de sobrevivência. O tratamento com PA induziu apoptose pela ativação da via intrínseca de maneira dose-dependente. A exposição destas células ao PA elevou a expressão do receptor de insulina e de GLUT-4, obtendo-se correlação positiva com a apoptose. O mesmo foi observado em linfócitos humanos expostos ao PA e de linfócitos de ratos em jejum. O tratamento das células Jurkat com insulina após exposição ao PA promoveu a ativação da via de sinalização insulínica. O PA aumentou a captação de glicose, mas observou-se diminuição de sua oxidação e o acúmulo de lípides. Assim, a via de sinalização da insulina e o metabolismo de glicose não oxidativo são estimulados como parte de uma coordenada resposta de sobrevivência de linfócitos expostos ao PA em baixas concentrações, mas em altas concentrações ocorre apoptose. / Fatty acids affect apoptosis pathway activating or deactivating signals that regulate this process. Jurkat cells (T-lymphocyte lineage) were treated with sub-lethal concentrations of palmitic acid (PA) (50, 100 e 150µM) and the cell death and survival signaling pathways were investigated. PA induced apoptosis in a dose dependent manner activating the intrinsic pathway. Jurkat cells exposed to PA showed increased insulin receptor and GLUT-4 expression and a positive correlation with apoptosis was obtained. Similar effect was observed in human lymphocytes exposed to PA and lymphocytes from fasted rats. Insulin treatment of Jurkat cells after PA exposure promoted the activation of the insulin signaling pathway. Glucose uptake was increased in the presence of PA, however, its oxidation was diminished and ncreased of lipids accumulation. Therefore, insulin signaling and non oxidative glucose metabolism are stimulated as a part of a coordinated response to survival of lymphocytes exposed to low concentration of PA but in high concentration it induces apoptosis.

There are approximately 600,000 cases of delayed or aberrant fracture healing in people each year, with a small subset of these fractures experiencing disunion. Dietary phosphate deficiency has been shown to impair oxidative phosphorylation and decrease BMP-2 mediated chondrogenic differentiation during fracture healing. Prior studies using pre-committed chondro-progenitor ATDC5 cell line grown in phosphate deficient media showed that energy consumption was linked to protein production and collagen hydroxylation but inversely related to matrix mineralization. The goal of this study was to further define the relationship between energy consumption and BMP-2 mediated stem cell chondrogenic differentiation and further examine how dietary phosphate, and promotion of collagen hydroxylation via ascorbate availability effected these processes. C3H10T1/2 murine cells, a multi-potential cell line, were expanded in pre-differentiation growth medium (DMEM with 10% FBS and 1% Pen/Strep). Once cells reached 60% confluence (day 0), they were grown in differentiating media (α-MEM with 5% FBS and 1X insulin-transferrin-selenium) containing either 100% (1mM) or 25% (0.25mM) inorganic phosphate (Pi), ± 200ng/mL BMP-2(BMP), and ±0.2 mM L-ascorbic acid (AA). In total, there were 8 groups with varying combinations of these three substances. Intracellular lipid, total DNA, protein, and hydroxyproline (HP) content were examined. Chondrocyte gene expression (Col2a1, Acan, ColXa1) and adipocyte gene expression (Pparg, Plin1, Ucp1) were measured to check for cell lineage commitment and specific differentiation of the C3H10T1/2. All measurements were acquired at day 8. The +BMP differentiation media groups contained significantly less DNA content and more protein content than the –BMP differentiation media groups (both p<0.0001). There was also a significant interaction between phosphate and ascorbic acid treatment (p=0.0296), with 25% Pi +AA groups producing significantly more protein than 100% Pi +AA groups. Hydroxyproline production was not different in 100% Pi or 25% Pi conditions (p=0.2951). AA presence in culture media led to greater HP production than culture media lacking AA (p=0.0035) There was a trend of an interaction between phosphate content and AA availability (p=0.0744). 100% Pi ±AA groups produced significantly different amounts of HP while 25% Pi ±AA groups did not produce significantly different amount of HP. Col2a1, Acan, and ColXa1 expression were all increased in +BMP groups. Ascorbic acid treatment groups expressed significantly more Col2a1and Acan than –AA groups. 100% Pi media led to greater Acan expression over 25% Pi groups (p=0.0009), whereas 25% Pi media trended to lead to greater ColXa1 expression over 100% Pi groups (p=0.0734). Pparg and Plin1 expression were increased in the 25% Pi condition. There were no significant differences in expression of Ucp1. C3H10T1/2 cells were significantly affected by phosphate concentration, BMP-2 treatment, and ascorbic acid supplementation. Phosphate deficiency hindered maturation of early chondrocytes into proliferating chondrocytes while also promoting MSC differentiation into the adipocyte cell lineage. Hypertrophic chondrocyte expression was decreased in phosphate deficient media, which may coincide with increased protein production observed in low phosphate conditions. BMP-2 promoted chondrogenesis which resulted in increased protein production. Whereas, lack of ascorbic acid in cell culture media led to decreased hydroxyproline production.

Key regulatory events take place at very early stages of human embryonic stem cell (hESC) differentiation to accommodate their ability to differentiate into different lineages; this work examines two separate regulatory events. To investigate precise mechanisms that link alterations in the cell cycle and early differentiation, we examined the initial stages of mesendodermal lineage commitment and observed a cell cycle pause that occurred concurrently with an increase in genes that regulate the G2/M transition, including WEE1. Inhibition of WEE1 prevented the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. These findings reveal a novel G2 cell cycle pause required for endodermal differentiation. A role for phenotypic transcription factors in very early differentiation is unknown. From a screen of candidate factors during early mesendodermal differentiation, we found that RUNX1 is selectively and transiently up-regulated. Transcriptome and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that RUNX1 knockdown impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion compromised TGFβ2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFβ2, but not TGFβ1. These findings identify novel roles for RUNX1-TGFβ2 signaling in mesendodermal lineage commitment.

<p>  </p> <p>The growth of land plants depends on a group of pluripotent stem cells in a tissue called the meristem. Seed plants initiate and maintain different types of meristems at the asexual sporophyte stage, and they generate sexual gametophytes, which are dependent on their sporophytes and are devoid of a meristem. In contrast, aside from forming indeterminate meristems at the sporophyte stage, seedless vascular plants, including ferns, also develop meristems in their gametophytes to drive gametophyte development and formation of sexual organs. To date, compared to the well-characterized cell behaviors and regulatory pathways in the meristems of seed plants, the molecular and cellular basis of meristem development in seedless ferns is still poorly understood. </p> <p>In several seed plants, the HAIRY MERISTEM (HAM) family transcription factors play important roles in maintaining the indeterminacy of shoot apical meristems and promoting the <em>de novo</em> formation of axillary meristems. In the first part of this dissertation, through constructing a comprehensive phylogeny, I found that HAM family members are widely present in land plants and duplicated in a common ancestor of flowering plants, leading to the formation of two distinct groups: type I and type II. In addition, HAM members from different seed plants and seedless plants are able to replace the roles of the Arabidopsis type-II <em>HAM</em> genes, maintaining established shoot apical meristems and promoting the initiation of new stem cell niches in Arabidopsis. Furthermore, preliminary functional studies of the <em>HAM </em>homolog (<em>CrHAM</em>) in the model fern<em> Ceratopteris richardii</em> suggest that CrHAM is required for maintaining the indeterminacy of multicellular meristems in Ceratopteris gametophytes. Collectively, these results indicate that HAM family members may serve as common regulators in control of meristem development in both seed plants and seedless vascular plants. </p> <p>In the remaining chapter of this dissertation, long-term time-lapse confocal imaging was performed using Ceratopteris stable transgenic plants, in which each individual cell (nucleus) was labelled with a fluorescent marker. Real-time lineage, identity, and division activity of each single cell from meristem initiation to establishment in Ceratopteris gametophytes were then determined. Additionally, cell fate and lineage alterations during <em>de novo</em> formation of new meristems were examined by mechanical perturbations. These quantitative analyses lead to the conclusion that in Ceratopteris gametophytes, initiation and proliferation of multicellular meristems relies on a few marginal cell lineages. Once established, the meristem maintains an actively dividing zone during gametophyte development. Within the meristem, cell division is independent of cell lineages and marginal cells are more actively dividing than inner cells. The meristem also triggers differentiation of adjacent cells into egg-producing archegonia in a position-dependent manner. </p> <p>In summary, this work provides insight into the evolution of key stem-cell regulators and advances the understanding of diversified meristem development in land plants. </p>

Understanding the complex mechanisms to assemble a functional brain demands sophisticated experimental designs. Drosophila melanogaster, a model organism equipped with powerful genetic tools and evolutionarily conserved developmental programs, is ideal for such mechanistic studies. Valuable insights were learned from research in Drosophila ventral nerve cord, such as spatial patterning, temporal coding, and lineage diversification. However, the blueprint of Drosophila cerebrum development remains largely unknown. Neural progenitor cells, called neuroblasts (NBs), serially and stereotypically produce neurons and glia in the Drosophila cerebrum. Neuroblasts inherit specific sets of early patterning genes, which likely determine their individual identities when neuroblasts delaminate from neuroectoderm. Unique neuroblasts may hence acquire the abilities to differentially interpret the temporal codes and deposit characteristic progeny lineages. We believe resolving this age-old speculation requires a tracing system that links patterning genes to neuroblasts and corresponding lineages, and further allows specific manipulations. Using modern transgenic systems, one can immortalize transient NB gene expressions into continual labeling of their offspring. Having a collection of knockin drivers that capture endogenous gene expression patterns would open the door for tracing specific NBs and their progenies based on the combinatorial expression of various early patterning genes. Anticipating the need for a high throughput gene targeting system, we created Golic+ (gene targeting during oogenesis with lethality inhibitor and CRISPR/Cas “plus”), which features efficient homologous recombination in cystoblasts and a lethality selection for easy targeting candidate recovery. Using Golic+, we successfully generated T2AGal4 knock-ins for 6 representative early patterning genes, including lab, unpg, hkb, vnd, ind, and msh. They faithfully recapitulated the expression patterns of the targeted genes. After preserving initial NB expressions by triggering irreversible genetic labeling, we revealed the lineages founded by the NBs expressing a particular early patterning gene. Identifying the neuroblasts and lineages that express a particular early patterning gene should elucidate the genetic origin of neuroblast diversity. We believe such an effort will lead to a deeper understanding of brain development and evolution.