No description available.

Mosquitoes are notorious vectors for multiple diseases like malaria, yellow fever and dengue fever. To manipulate gene expression in mosquito and spread desired genes among natural population for vector control, a thorough understanding of mosquito development and gene regulation is critical. Early embryogenesis is a rapid, complex yet crucial process in the very beginning of development. Previous research in other species indicated genes transcribed that early evolved fast and played essential roles. The study of mosquito early zygotic genes (EZGs) would offer unique insights into mosquito gene evolution as well as potential targets for mosquito control. In this study, I identified 61 pure EZGs (pEZGs) in mosquito Aedes aegypti. These pEZGs were enriched in architectures adapting to the rapid embryonic cell cycles and were over represented by domains or functions related to maternal zygotic transition. Phylogenetic analysis showed that pEZGs originated mainly from duplication, retrotransposition and de novo emergence. The comparison of pEZGs in Ae. aegypti with those in Drosophila revealed an interesting evolutionary paradox where the early zygotic genes turned over fast but the regulatory motif was conserved in two species. Curiously, the motif binding protein in Drosophila (zelda) seemed unable to initiate the earliest zygotic transcription in Ae. aegypti due to late temporal expression. The regulatory motif (VBRGGTA) found in Ae. aegypti pEZGs was shown necessary and sufficient for driving early zygotic gene expression by transient reporter assays and one motif-bearing promoter was tested with success in driving gene expression as early as 2-4h after egg laying in transgenic Ae. aegypti. This was the first characterized promoter with early zygotic but no maternal expression in Ae. aegypti that can be used for future genetic studies and mosquito control strategies. As important gene regulators, miRNAs also play essential roles in early embryogenesis. The genome-wide predictions and systematic analysis of miRNAs in Ae. aegypti and Anopheles stephensi were conducted in this study. The first miRNA profiling in mosquito across all developmental stages was also performed to provide basis for future functional study. Several lineage-specific miRNAs were found highly expressed in embryos, indicating their special roles in the embryogenesis of mosquitoes. / Ph. D.

Wheat (Triticum aestivum L.) zygotic embryogenesis occurs in a dynamically regulated ovular environment, and in ovulohormones regulate embryogenic processes. Levels of ABA, IAA, and the cytokinins Z, ZR, DHZ, DHZR, iP, and iPA were studied in developing wheat kernels from anthesis to maturity . High cytokinin and low IAA and ABA levels were associated with the early stage of embryo formation and active tissue histodifferentiation. Following histodifferentiation, cytokinin levels declined while IAA accumulated throughout the stage of active grain growth and then declined with grain maturity. ABA levels increased at the soft-dough developmental stage and through to grain maturity. Endogenous +ABA levels in developing wheat grains treated with fluridone, which indirectly blocks ABA synthesis, declined at the soft-dough stage. As a result, mature desiccated fluridone-treated kernels exhibited little dormancy. However, fluridone-treated kernels were not viviparous, suggesting a strong caryopsis-embryo interaction in maintaining embryogenically competent tissues. Induction of embryogenically competent wheat callus cultures was highly variable between genotypes and pre-initiation environments. Genotypic and environmental influences altered endogenous hormone levels and affected embryogenic competence. Establishment of competent embryo explants for somatic embryo induction was favored by a high cytokinin-to-auxin ratio and very low ABA levels throughout histodifferentiation (around 4 to 8 DPA). Similar to embryos forming within the caryopses, competent callus cultures had a high cytokinin (Z)-to-auxin ratio at 7 DPI. Increased frequency of embryogenic cultures was achieved when embryo explants were excised during a narrow window of low hormone levels. Wheat line and pre-initiation environment affected this window. Simulation in vitro of the in ovulo wheat kernel environment improved zygotic embryogenesis in vitro . Embryos exposed to physiologically normal ABA levels and low 0 2 tensions of 2.5 mM (7%) most closely approached morphological and physiological normalcy. The culmination of these studies clearly defines windows of embryo development for explant excision, associated roles of plant hormones in embryogenesis, and in ovulo hormone levels that vastly improve the frequency of successful embryogenesis when simulated in in vitro culture systems.

Orientador: José Maurício Barbanti Duarte / Banca: Fábio de Melo Sene / Banca: Joaquim Mansano Garcia / Resumo: O veado mateiro, Mazama americana, apresenta populações cariotipicamente diferentes (citótipos) em diferentes regiões brasileiras. Estes citótipos apresentam uma diferenciação cariotípica que originou duas linhagens evolutivas A e B. Estas linhagens apresentam uma distância genética entre elas, maior que em relação a outras espécies de Mazama. A existência de mecanismo de isolamento reprodutivo pós-zigótico, devido a distância cariotípica das linhagens, comprovaria a existência de pelo menos duas espécies dentro do que hoje denomina-se Mazama americana. Para tal foram realizados cruzamentos inter e intracitótipos obtendo prole F1 híbrida (n=6) e pura (n=3). As fêmeas F1 foram analisadas quanto aos seus parâmetros reprodutivos por meio da dosagem de metabólitos fecais de progesterona, histologia de ovário e produção de embriões in vitro. As fêmeas puras apresentaram parâmetros similares aos já descritos para fêmeas de M. americana, porém os parâmetros das fêmeas híbridas apresentaram-se distintos das puras. Duas híbridas apresentaram esterilidade com ausência de células germinativas, resposta negativa ao protocolo de superovulação e ausência de foliculos para aspiração e posterior produção de embriões in vitro. As outras híbridas apresentaram subfertilidade. Uma vez encontrado o isolamento reprodutivo entre as linhagens A e B, foi possível identificar ao menos duas espécies dentro de Mazama americana. Sugerimos que os citótipos podem ser divididos em subespécies das linhagens, porém outros estudos devem ser realizados para que esta hipótese seja comprovada / Abstract: The red brocket deer, Mazama americana, shows populations that are karyotipically different (cytotypes) distinct regions in Brasil. These karyotype differentiation led to two evolutionary lineages A and B. These lineages have a genetic distance between them greater than for other species of Mazama. Once to these differences culminate in a division of the taxon, need to require a study of reproductive isolation among lineages. For these, crossings were performed within and between cytotypes obtaining F1 hybrid (n = 6) and pure (n = 3) offspring. The F1 females were analyzed as to their reproductive parameters. It was possible to infer the reproductive capacity of these through the measurement of fecal progesterone metabolites, histology of ovary and embryo production in vitro. The pure females showed parameters similar to those described for females of M. American, but the parameters of the hybrid females were lower than those of the pure. Two hybrid presented sterility with absence of germ cells, negative response to the superovulation protocol, and absence of follicles for aspiration and subsequent production of embryos. The other hybrids showed subfertility. Once found the reproductive isolation between strains A and B, it was possible to identify at least two species within Mazama americana. We suggest that these cytotypes can be divided into subspecies strains, but other studies must be conducted so that this hypothesis is verified / Mestre

Embryonic cells of many species polarise and the cell polarity is often important for the normal developmental progression. In the mouse embryo, the prototype of epithelial cell polarity, namely apico-basal polarisation, become established at the 2.5 days' post-fertilisation, when the embryos are at the 8-cell stage. The formation of apical domain is necessary and sufficient for the first segregation of extra-embryonic and embryonic cell lineages, as well as the following up morphogenetic transitions, such as the blastocyst formation. This study aims to explore the molecular pathways triggering the first cell polarity establishment in the mouse embryo, and to reveal the mechanism that programmes the timing of this event in the mouse embryo. The results showed that cell polarity establishment during the 8-cell stage development can be divided into two major phases: in the first phase actomyosin complex became polarised to the cell-contact free surface; and in the second phase apical proteins recruited to the actomyosin enriched cell-contact free cortex, they further became centralised in the cell-contact free surface, excluding the local actomyosin meshwork, resulting in the formation of actomyosin ring. The activation and assembly of actomyosin meshwork during the first phase, but not its contractility, was essential for apical protein recruitment. Factors responsible for actin cytoskeleton reorganisation included Phospholipase C (PLC) - Protein Kinase C (PKC) pathway components, they directly activated actomyosin in the first phase through the Rho proteins such as RhoA. Further results showed that the apical protein centralisation step required a proximate transcriptional input that was induced by two transcription factors, Tfap2c and Tead4. RNAi and Genetic depletion of these two factors prevented apical protein centralisation and the final apical domain assembly. The protein expression profile indicated that Tfap2c and Tead4 expression, and therefore their activity, were induced by zygotic genome activation. Significantly, overexpression of Tfap2c, Tead4, together with constitutively activated Rho proteins were sufficient to advance the timing of apical domain formation, indicating that the timer of cell polarity establishment at the 8-cell stage is set by the Rho proteins activation, and the zygotic transcriptional accumulation of Tfap2c and Tead4. Together, these results characterised the molecular events during the cell polarity establishment at the 8-cell stage mouse embryo, and identified the timing regulation of this event.

O gênero Passiflora é o maior gênero da família Passifloraceae, que compreende mais de 500 espécies, a maioria originária de regiões neotropicais, sendo centenas distribuídas pela América Latina. Algumas dessas espécies apresentam importância econômica na produção de fruta in natura, suco concentrado, uso ornamental e medicinal com propriedades sedativas. Estudos do desenvolvimento reprodutivo e do fruto de espécies de Passiflora são fundamentais para melhor compreensão de aspectos do desenvolvimento que possam contribuir para a produção agronômica e compreensão da evolução de estruturas florais presentes em espécies desta família. A importância das sementes para a sua propagação, para estudos taxonômicos e a presença do arilo, estrutura de interesse fundamental para a comercialização de frutos e produção de suco em espécies desse gênero, estimulou a elaboração de um estudo comparativo entre duas espécies de interesse comercial, P. edulis e P alata, associando o estudo de características morfoanatômicas e moleculares. O presente projeto teve como objetivo caracterizar o desenvolvimento do embrião zigótico e arilo de Passiflora edulis Sims e Passiflora alata Curtis. Flores foram manualmente polinizadas e amostras coletadas periodicamente após a polinização, visando à obtenção de sementes em diferentes fases do desenvolvimento embrionário e do arilo. Primórdios do arilo são observados em pré-antese, quando o saco embrionário é organizado. Células epidérmicas na base do funículo sofrem divisões periclinais formando uma borda em torno da rafe. O desenvolvimento do primórdio do arilo é interrompido, observando-se a reativação de divisões celulares e o arilo recomeça o desenvolvimento em uma estrutura multicelular ao redor da semente em desenvolvimento. Aos 14 dias após a polinização o arilo já cobre dois terços da semente, crescendo rapidamente até a semente ser recoberta totalmente, desde o funículo, até o polo calazal. O endosperma é nuclear e seu desenvolvimento se inicia logo após a fertilização, através de divisões sucessivas, formando um sincício ao redor do proembrião, simultaneamente à diminuição tamanho do nucelo. A celularização do endosperma, com a deposição de paredes celulares é observada aproximadamente 20 dias após a polinização. A embriogênese se inicia com a primeira divisão do zigoto, observada aos 7 dias após a polinização. Essa primeira divisão é transversal dividindo o zigoto em duas células, assimetricamente. A célula apical sofre sucessivas divisões que levam a estádios subsequentes de desenvolvimento do embrião, tais como, 4-8 células, globular, coração e torpedo. Aproximadamente 30 dias após a polinização o embrião atinge o estádio cotiledonar e a partir de então apenas aumenta em tamanho consumindo o endosperma e ocupando seu espaço na semente. Essas observações permitiram que fossem definidos dois estádios específicos do desenvolvimento do arilo para futura captura por microdissecção a laser. A caracterização anatômica do desenvolvimento do embrião e do arilo em ambas as espécies subsidia o estabelecimento de estádios específicos do desenvolvimento que podem servir como alvos para estudos moleculares nessas espécies de Passiflora / Passiflora is the largest genus in Passifloraceae and most of the commercially used species develop an aril around the seed, which is commercially important for fresh fruit consumption, and concentrate juice. Reproductive developmental studies associating morphoanatomical and molecular characteristics are essential for a better understanding of particularities of this genus. The present project aimed to characterize the development of Passiflora edulis Sims and Passiflora alata Curtis zygotic embryo and aril. Pollination of flowers were done manually, fruits and ovaries were collected at regular intervals after pollination and processed for scanning and light microscopy, for analysis of embryos and aril in different stages of development. Aril primordium is observed in pre-anthesis when the embryo sac is organized. Epidermic cells at the base of the funiculus undergo periclinal divisions forming a rim surrounding the raphe. Aril development is arrested until after fertilization when cell divisions are reactivated and the aril resume development into a multicellular structure surrounding the developing seed from the funicle towards the chalazal end. At approximately 14 days after pollination the aril already covers two thirds of the seed, and grows rapidly until the whole seed is covered. The endosperm is nuclear and starts developing soon after fertilization through successive divisions forming a syncytium mostly at the chalazal region, and around the developing embryo, replacing the nucellus. Cell walls are formed and the endosperm begins cellularization approximately 20 days after pollination. Embryogenesis initiates with the first division of the zygote, approximately 7 days after pollination. This first cell division is transversal and asymmetrical; the apical cell undergoes successive divisions leading to the subsequent stages of embryo development such as 4- and 8-celled, globular, heart-shaped, torpedo. Approximately 30 days after pollination, the embryo reaches the cotyledonary stage and thereafter grows only in size, consuming the endosperm and occupying its space in the seed. The first division of the zygote was observed around seven days after pollination (DAP), with the mature embryo formed approximately 30 DAP. Initial development of the aril primordium is observed at the ovule basal region, before anthesis/pollination. Embryo and aril development occurs simultaneously. These observations allowed for the definition of two specific stages of aril development for laser-capture-microdissection and further molecular analysis aiming at the evaluation of the molecular basis of aril differentiation in Passiflora. The morphoanatomical characterization of embryo and aril development in these species will serve as a source of information for the definition of specific developmental stages, which can be targets for molecular studies in Passiflora

No description available.

Post-zygotic genetic variation has previously been shown in healthy individuals and linked to various disorders. The definition of post-zygotic or somatic variation is the existence of genetically distinct populations of cells in a subject derived from a single zygote. Structural changes in the human genome are a major type of inter-individual genetic variation and copy number variation (CNV), involving changes in the copy number of genes, are one of the best studied category of structural genetic changes. In paper I we reported a pair of healthy female monozygotic (MZ) twins discordant for aneuploidy of chromosomes X and Y, contributing to the delineation of the frequency of somatic variation in MZ twins. It also illustrates the plasticity of the genome for tolerating large aberrations in healthy subjects. In paper II we showed age-related accumulation of copy number variation in the nuclear genomes in vivo for both megabase- and kilobase-range variants. Using age-stratified MZ twins and single-born subjects, we detected megabase-range aberrations in 3.4% of people ≥60 years old but not in individuals younger than 55 years. Moreover, the longitudinal analysis of subjects with aberrations suggests that the aberrant cell clones are not immortalized and disappear from circulation. We also showed that sorted blood cells display different genomic profiles.  The detected recurrent rearrangements are candidates for common age-related defects in blood cells. This work might help to describe the cause of an age-related decline in the number of cell clones in the blood, which is one of the hallmarks of immunosenescence. In paper III we described a variable number tandem repeat (VNTR) ~4 kb upstream of the IFNAR1 gene, which was somatically variable.  We detected 14 alleles displaying inter- and intra-individual variation. Further analyses indicated strong clustering of transcription factor binding sites within this region, suggesting an enhancer. This putative VNTR-based enhancer might influence the transcriptional regulation of neighboring cytokine receptor genes and the pathways they are involved in. These three studies stress the importance of research on post-zygotic variation in genetics. Furthermore, they emphasize that biobanks should consider sampling of multiple tissues to better address this issue in the genetic studies.

Although cottonseed is an important source of oil and fiber, the development of cotton embryos has not been investigated as well as development of cotton fiber. The development of cotton embryos in late heart-stage and early cotyledonary stage is less well investigated than the first 10-14 days after anthesis, or the late stages of embryo development during seed-fill and desiccation. This analysis focused on cotton embryos in the late heart-stage and early cotyledonary stage of development (1.5-4.0 mm or about 13-18 DPA). In vitro analyses are important tools for studying embryos in isolation from the endosperm and fiber and when it is necessary to monitor the developing embryo continuously. The original goal of this work was to develop an in vitro culture method that would support continued development of excised zygotic embryos from the early cotyledonary stage into complete plants with true shoots, i.e. true leaves or visible buds and then to use this method to study aspects of developmental regulation during cotyledonary stage and the transition to later stages. Not all embryos were competent to develop true shoots (an apical bud or a leaf plus a bud) in culture. A number of cultural variables were tested and eliminated. Embryo maturity at the time embryos were excised and the presence or absence of light during the first 14 days of culture affected the competence of immature embryos to developed true shoots. The effect of light was verified in several large replicated experiments. Morphological changes occurring during in vivo development were examined microscopically. The transition from heart-stage to early cotyledonary stage and the development of the first leaf from initials to a large structure were identified. Embryonic shoot apices continued to grow in cultured 1-3 mm embryos. The size and shape of light-treated and dark-treated embryonic apices was compared. A germination test of mature seeds identified seedlings with a similar phenotype occurring at similar rates in seedlings and light-cultured embryos and possible causes were discussed.

Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-25Bitstream added on 2014-06-13T19:17:41Z : No. of bitstreams: 1 cursino_ms_me_jabo.pdf: 1013380 bytes, checksum: e2baa17d3b529f6efabece49dc7a6901 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O veado mateiro, Mazama americana, apresenta populações cariotipicamente diferentes (citótipos) em diferentes regiões brasileiras. Estes citótipos apresentam uma diferenciação cariotípica que originou duas linhagens evolutivas A e B. Estas linhagens apresentam uma distância genética entre elas, maior que em relação a outras espécies de Mazama. A existência de mecanismo de isolamento reprodutivo pós-zigótico, devido a distância cariotípica das linhagens, comprovaria a existência de pelo menos duas espécies dentro do que hoje denomina-se Mazama americana. Para tal foram realizados cruzamentos inter e intracitótipos obtendo prole F1 híbrida (n=6) e pura (n=3). As fêmeas F1 foram analisadas quanto aos seus parâmetros reprodutivos por meio da dosagem de metabólitos fecais de progesterona, histologia de ovário e produção de embriões in vitro. As fêmeas puras apresentaram parâmetros similares aos já descritos para fêmeas de M. americana, porém os parâmetros das fêmeas híbridas apresentaram-se distintos das puras. Duas híbridas apresentaram esterilidade com ausência de células germinativas, resposta negativa ao protocolo de superovulação e ausência de foliculos para aspiração e posterior produção de embriões in vitro. As outras híbridas apresentaram subfertilidade. Uma vez encontrado o isolamento reprodutivo entre as linhagens A e B, foi possível identificar ao menos duas espécies dentro de Mazama americana. Sugerimos que os citótipos podem ser divididos em subespécies das linhagens, porém outros estudos devem ser realizados para que esta hipótese seja comprovada / The red brocket deer, Mazama americana, shows populations that are karyotipically different (cytotypes) distinct regions in Brasil. These karyotype differentiation led to two evolutionary lineages A and B. These lineages have a genetic distance between them greater than for other species of Mazama. Once to these differences culminate in a division of the taxon, need to require a study of reproductive isolation among lineages. For these, crossings were performed within and between cytotypes obtaining F1 hybrid (n = 6) and pure (n = 3) offspring. The F1 females were analyzed as to their reproductive parameters. It was possible to infer the reproductive capacity of these through the measurement of fecal progesterone metabolites, histology of ovary and embryo production in vitro. The pure females showed parameters similar to those described for females of M. American, but the parameters of the hybrid females were lower than those of the pure. Two hybrid presented sterility with absence of germ cells, negative response to the superovulation protocol, and absence of follicles for aspiration and subsequent production of embryos. The other hybrids showed subfertility. Once found the reproductive isolation between strains A and B, it was possible to identify at least two species within Mazama americana. We suggest that these cytotypes can be divided into subspecies strains, but other studies must be conducted so that this hypothesis is verified