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

Orientador: Marcelo Carnier Dornelas / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T19:57:11Z (GMT). No. of bitstreams: 1 Cazoto_JulianaLacorte_D.pdf: 5127995 bytes, checksum: 832b6cadc45544f0ef0e0353cad06755 (MD5) Previous issue date: 2012 / Resumo: Durante o desenvolvimento vegetal, o meristema apical caulinar e o meristema apical radicular são determinados durante o processo de embriogênese, enquanto que outros órgãos como folhas, flores e frutos são produzidos durante o desenvolvimento pós-embrionário. Durante o desenvolvimento vegetal, a expressão de diferentes fatores de transcrição em um determinado local e tempo culminam com a ativação de domínios celulares responsáveis pelo estabelecimento e manutenção dos meristemas. Foi hipotetizado que os mesmos genes que são ativados durante o desenvolvimento embrionário zigótico são também ativados durante a embriogênese somática. Portanto, o padrão de expressão de genes preferencialmente expressos durante o desenvolvimento embrionário em espécies modelo nesses dois processos foi analisado e comparado. Para este propósito, o processo de embriogênese zigótica e somática em Passiflora edulis foram caracterizados anatomicamente. Posteriormente, genes preferencialmente expressos durante a embriogênese somática e zigótica foram identificados e caracterizados. Foram selecionados genes das famílias YABBY, WOX e Aux/IAA. Estes genes foram encontrados por meio de análises de bioinformática no banco de dados de etiquetas de sequências expressas (ESTs) de Passiflora, PASSIOMA. As sequências putativas de aminoácidos codificadas pelos genes estudados, juntamente com seus possíveis homólogos em Arabidopsis, foram alinhadas e analisadas. O padrão de expressão desses genes foi estudado por RT-PCR, qPCR e hibridização in situ durante diferentes estágios do desenvolvimento embrionário somático e zigótico em P. edulis. A caracterização morfo-anatômica do embrião zigótico nessa espécie resultou na elaboração de um cronograma que identifica, a partir do dia da polinização, os diferentes estágios de desenvolvimento do mesmo. Além disso, os dados de expressão gênica sugerem que os mecanismos moleculares responsáveis pelos processos de embriogênese zigótica e somática apresentaram padrão de expressão similar no início do desenvolvimento embrionário vegetal em P. edulis / Abstract: During plant development, the shoot apical meristem (SAM) and the root apical meristem (RAM) are established during the embryo formation process, while the other organs as leaves, flowers and fruits are produced during the post-zygotic development. During plant embryogenesis, the expression of different transcription factors in a given time and place culminates with the activation of cellular domains responsible for the establishment and maintenance of the meristems. We hypothesized that the same genes that are activated during zygotic embryo development are also activated during somatic embryogenesis. Therefore we analyzed and compared the expression patterns of genes preferably expressed during embryo development in these two processes. For this purpose, we characterized anatomically both somatic and zygotic embryogenesis in Passiflora edulis (passion fruit). Later, we identified and characterized genes preferably expressed during somatic and zygotic embryogenesis in this species. Among the identified genes are members from YABBY, WOX and Aux/IAA families. These genes were selected by bioinformatics analysis from the PASSIOMA database of Passiflora reproductive expressed sequence tags (ESTs). The putative protein sequences encoded by the studied genes, together with possible horologes in Arabidopsis, were analyzed by sequence alignment. The expression patterns of these genes were studied by RT-PCR, qPCR and in situ hybridization during different developmental stages of P. edulis somatic and zygotic embryogenesis. The morpho-anatomical characterization of zygotic embryos in this specie resulted in a schedule elaboration that identifies, from the first day of pollination, the different developmental stages of the embryo. Moreover, the gene expression data suggest that the molecular mechanisms responsible for the processes of zygotic and somatic embryogenesis presented similar expression pattern during the beginning of the plant embryo development in P. edulis / Doutorado / Biologia Vegetal / Doutora em Biologia Vegetal

Agrobacterium is widely considered, when suitably modified, to be the most effective vector for gene transfer into plant cells. For a long time, many cereals crops (monocotyledonous plants) were recalcitrant species to genetic modification, mainly as a result of their recalcitrance to in-vitro regeneration and their resistance to Agrobacterium infection. However, recently Agrobacterium-mediated transformation has been used to transform monocot crops such as maize (Zea mays) but with severe restrictions on genotype suitability. This study was carried out to evaluate the transformation amenability of 2 Syrian maize varieties and 2 hybrids in comparison with the hybrid line Hi II by the Agrobacterium tumefaciens-mediated transformation technique using a callus induction based system from immature zygotic embryos IZEs. A. tumefaciens strains EHA101, harbouring the standard binary vector pTF102, and the EHA105 containing the pBINPLUS/ARS:PpCBF1 vector were used. The effects of genotypes and the size of IZEs explants on callus induction and development were investigated. Results showed that callus induction and subsequent callus growth were significantly affected by the initial explant size. Calli induction from IZEs explants sized 1.5-2.00mm was 76%. Callus weight however decreased to 8.2g, compared with 11.7g of callus derived from IZEs >2.00mm. Callus induction ranged between 73.6-78.9% for varieties and hybrids respectively. Calli derived from varieties weighed significantly more than those initiated from the hybrids. Results demonstrated that Syrian maize genotypes were efficiently transformed via the A. tumefaciens strains but there was variation in transformation frequency. A transformation frequency of 3.7-4.2% was achieved for hybrids and varieties respectively confirming that the transformation frequency was genotype-dependent. The transformation frequency averaged between 3.2-5.6% for the EHA105 and EHA101 respectively. Fertile transgenic plants were regenerated from mature somatic embryos with an average regeneration frequency of 59.2 and 17% respectively for varieties and hybrids. Transgenic seeds of R0 and R1 progenies were produced from 74% of the outcrosses attempted and more than 98% of transgenic plants were normal in morphology. Fertile transgenic maize plants carrying the transferred gene CBF were produced using the Agrobacterium EHA105/PpCBF1 and these plants were shown to be more salt tolerant. Transient expression of the GUS gene was confirmed in transgenic calli, shoots, leaves, roots and floral parts of transgenic R0 and R1 progenies using histochemical GUS assays. The presence of the introduced bar and CBF genes in the genomic DNA of the transformants was confirmed by the PCR amplification. Further, the stable expression of the CBF and bar transgenes in the maize genome of transgenic R1 progeny was confirmed by qRT-PCR. The transformation protocol developed using an A. tumefaciens standard binary vector system was an effective and reproducible method to transform Syrian maize with an anti-stress gene in which fertile salt-resistant transgenic plants were routinely produced. This approach has great potential for development of Syrian maize breeding programmes for abiotic stress resistance for application in many areas in Syrian maize production.

The TALE factors, comprising the pbx and prep/meis gene families, are transcription factors (TFs) vital to the proper formation of anterior anatomical structures during embryonic development. Although best understood as essential cofactors for tissue-specific TFs such as the hox genes during segmentation, the TALE factors also form complexes with nuclear factor Y (NFY) in the early zygote. In zebrafish, Pbx4, Prep1, and NFY are maternally deposited and can access their DNA binding sites in compact chromatin. Our results suggest that TALE/NFY complexes have a unique role in early embryonic development which is distinct from each factor’s independent functions at later stages. To characterize these TALE/NFY complexes, we employed high-throughput transcriptomic and genomic techniques in zebrafish embryos. Using dominant negatives to disrupt the function of each factor, we find that they display similar, but not identical, loss-of-function phenotypes and co-regulate genes involved in transcription regulation and embryonic development. Independently, the TALE factors regulate homeobox genes and NFY governs cilia-related genes. ChIP-seq analysis at zygotic genome activation reveals that the TALE factors occupy DECA sites adjacent to CCAAT boxes near genes expressed early in development and involved with transcription regulation. Finally, DNA elements containing TALE and NFY binding sites drive reporter gene expression in transgenic zebrafish, and disruption of TALE/NFY binding via mutation or dominant negatives eliminates this expression. Taken together, this data suggests that the TALE factors and NFY cooperate to regulate a set of development and transcription control genes in early zygotic development but also have independent roles after gastrulation.

In basidiomycetous fungi, the viability of gametes is an important component of sexual fitness and can have implications for speciation events. Prior estimates of basidiospore germination are highly variable and the occurrence of reproduction between these lineages suggests that reproductive isolation is incomplete. Genetic incompatibilities during meiosis have been attributed to much of the offspring inviability. However, the influence of environmental factors on basidiospore germination in Cryptococcus are not well known. In this study, we used human opportunistic yeast pathogens, Cryptococcus neoformans and Cryptococcus deneoformans, as models to investigate the potential effects of selected genetic and environmental factors on basidiospore germination. We evaluated basidiospore germination of six genetic crosses by pairing a total of five strains, three intraspecific crosses and three interspecific crosses, between C. neoformans and C. deneoformans. The offspring of genetic crosses were incubated under multiple media and temperature treatments and scored for their germination ability. In general, spores from intraspecific crosses had greater germination potential than those from interspecific crosses. Growth under high temperatures was the most significant influence on basidiospore germination on these crosses. Furthermore, there were notable interaction effects between environmental factors and parental strains or strain pairs on basidiospore germination. The interaction between the sex-specific genes and environmental pressures impact reproductive barriers and blur species distinctions within the Cryptococcus neoformans species complex. And so, reduced viability of hybrid offspring can have implications for Cryptococcus speciation, ecology, and pathogenesis as hybridization events are an effective method of increasing pathogenicity, expanding species distributions and increasing tolerance to novel environments or hosts. / Thesis / Master of Science (MSc) / In basidiomycetous fungi, the viability of gametes is an important component of sexual fitness and can have implications for speciation events. The Cryptococcus neoformans species complex are a group of opportunistic pathogens, for which hybridization can be facilitated readily under laboratory conditions, creating offspring that are generally completely inviable or have low germination potential. Antagonistic genetic interactions are mostly responsible for offspring inviability, yet the impacts of environmental factors is not known. Multiple genetic crosses between Cryptococcus strains were used as a model to investigate species relationships by examining the impacts of genetic and environmental factors on offspring germination potential.

No description available.

Du fait de la complexité des processus de spéciation, l'étude des relations évolutives entre espèces nécessite une approche intégrative. Nous avons combiné croisements expérimentaux et études moléculaires (séquençage mitochondrial et RAD-seq) pour étudier les relations évolutives entre espèces indigènes et introduites du genre Ciona, en Europe. Nous avons examiné deux mécanismes post-zygotiques restant à explorer pour expliquer l'absence d'introgression de C. robusta et C. intestinalis dans leur zone de sympatrie en Manche : 1) la contre-sélection des hybrides par l'environnement (température et salinité) et 2) des incompatibilités de type Dobzhansky-Muller (DMIs). L'absence de dépression hybride (étudiée chez 725 hybrides F1) nous a conduit à rejeter le premier mécanisme. Le second a été exploré grâce à l'étude de sept familles double-hybrides ou issues de rétrocroisements, analysées par RAD-seq. Une stérilité sur la fonction mâle des hybrides F1 et d'importantes distorsions de ségrégation sont en faveur de la présence de DMIs. Nous avons étendu notre étude à d'autres espèces du genre Ciona. Les principaux résultats, notamment soutenus par une approche phylogénomique (7 taxons - 89 individus), sont : 1) la remise en cause du statut d'espèce de C. roulei par rapport à C. intestinalis (probablement deux lignées allopatriques), 2) les premières données montrant la forte divergence évolutive de C. edwardsi avec les autres Ciona spp. et 3) la présence de variabilité génétique et/ou d'introgression chez C. intestinalis type C et type D. Ces résultats ouvrent d'intéressantes perspectives de recherches dont des études populationnelles des espèces présentes en Méditerranée. / Speciation is a complex process. Evolutionary relationships between close species thus required an integrative approach. We combined experimental crosses and molecular data (mitochondrial and RAD-tag sequencing) to investigate evolutionary relationships between native and non-indigenous species of the genus Ciona nowadays found in Europe. We studied two post-zygotic mechanisms, still to be examined, to explain the lack of introgression between C. robusta and C. intestinalis in their sympatric range –i.e. the English Channel: i) selection against hybrids, by the environment (here temperature and salinity) and ii) Dobzhansky-Muller incompatibilities (DMIs). The absence of outbreeding depression (over 725 F1-hybrids) makes unlikely the first mechanism. The second has been investigated with seven families of F2-hybrids and backcrosses, all studied with RAD-tag sequences. Male sterility on F1-hybrids and many segregation distortions are in favor of presence of DMIs. We then expanded our study to other Ciona species. The main outcomes, supported by a phylogenomic approach (7 taxa - 89 individuals) are: 1) a needed reappraisal of the species status of C. roulei and C. intestinalis, which are most likely two allopatric lineages of a same species (as defined by biological and phylogenetic species concepts), 2) the first data showing that C. edwardsi is strongly evolutionary divergent from the other Ciona species and 3) the presence of unexpected genetic variability and/or introgression in C. intestinalis type C and D. All together these results open new research perspectives, in particular based on population studies of the species nowadays found in the Mediterranean Sea.

Using the known sequences for serine palmitoyltransferase (SPT) and ceramide kinase (CERK) from Arabidopsis, candidates for the corresponding genes in Loblolly pine were cloned and examined during embryogenesis. The cloned two cDNA sequences from Loblolly pine, which has similarity of 81% and 88% respectively to two subunits of SPT1 and SPT2 in Arabidopsis, were presumed as the Loblolly pine SPT1 and SPT2 (Pt-SPT1 and Pt-SPT2). A few different versions of Pt-SPT1 mRNAs (2223 nts, 756 nts, 822 nts, and 754 nts respectively), most likely the alternative splicing results, were found. Three of these mRNAs are capable of encoding proteins. The long version (2223 nts) encodes a protein with 484 amino acids (Pt-SPT1); two short versions (822 nts, 756 nts) encode a 90 a.a. protein. Another cDNA sequence of 2396 nts encodes a protein of 493 a.a. (Pt-SPT2). Both predicted Pt-SPT1 and Pt-SPT2 proteins possess highly conserved serine palmitoyltransferase functional domains (E value 5.7e-61). Their expression patterns are different between somatic and zygotic embryogenesis. Two different versions of mRNAs, with 2786 nts (long), and 2320 nts (short) respectively, of ceramide kinases in Loblolly pine (Pt-CERKs) have been cloned. The long version encodes a protein with 721 a.a.; the short version with 560 a.a. The expression patterns for these two CERK mRNAs are different during embryo development. The long version is constitutively expressed, while the short one is only expressed in some stages with much lower expression level. Overexpression Pt-CERKL, Pt-CERKS, and Pt-CERKF in E.coli and function analysis in vitro show that all Pt-CERKs appear to have the same catalytic functions as their homologs in human and Arabidopsis, but with different efficiency. The catalytic efficiency was dramatically lower in the short Pt-CERK protein compared with the long Pt-CERK protein and Pt-CERKF. The membrane system is not necessary for the catalytic reactions of these three Pt-CERKs in vitro and Pt-CERKs were less dependent on the Ca2+ ions. Thus, these studies have provided the first information about SPT- and CERK- like proteins in loblolly pine, and open new avenues of investigation for the roles of sphingolipids in embryonic development.

Title: Computer Modeling of Tissue Development Author: Vojtěch Bednář Department: Department of applied mathematics Supervisor: Doc. RNDr. Zdeněk Hedrlín, CSc. Abstract: This thesis describes hybrid individual cell-based approach to modeling of systems of biological cells. In the first part reaction-diffusion model of environment is introduced together with vax equilibrium and model of a cell based on zygotic graph and cummulative states. Further, simulations modeling three biologically motivated situations are introduced: Lumen formation, tumor growth, and cellular migration in chronic inflammation. The first model shows a scenario of hollow structure formation based on directional division and cellular migration. The second model is concerned with the growth of a progeny of a slightly damaged cell. The resulting tumor exhibits three stages of malign transformation. Further, emergence of an aggressive tumor without detectable precursor is observed on one hand and a continual transformation of a benign neoplasm into a malign one is seen on the other hand. Each of these cases is a consequence of different parametrization of the model situation. The last model analyses the role of membrane enzymatic activity in migrating cells of the immune system in chronic inflammation. In this model it is observed that...

La spéciation peut être définie comme l’ensemble des processus conduisant à l’évolution de l’isolement reproducteur entre groupes d’individus. Ces dernières décennies, de nombreuses études ont souligné l’importance de la sélection naturelle comme processus majeur dans la formation de nouvelles espèces en conditions de sympatrie, notamment dans des modèles de spéciation écologique. Le chêne sessile (Quercus petraea (Matt.) Liebl.) et le chêne pédonculé (Q. robur L.) sont deux espèces sympatriques qui présentent de fortes divergences morphologiques et écologiques, malgré des taux d’hybridation naturelle estimés relativement importants. Cependant, très peu de données sont connues sur la nature et la force des barrières reproductives chez ces espèces. L’objectif de cette thèse était donc de caractériser ces barrières aux niveaux phénotypique et génétique, pour mieux comprendre leur importance relative, leur rôle dans le processus de divergence et leur évolution au sein de ce complexe d’espèces. Une première approche basée sur la réalisation de croisements contrôlés a montré tout d’abord (i) l’existence de fortes barrières reproductives au niveau pré-zygotique et de barrières significatives bien que plus faibles au niveau de la fitness des descendants hybrides, et (ii) une variabilité importante de ces barrières liée aux génotypes des individus et à leur expression dans des micro-environnements différents. Une deuxième approche de génomique des populations sur 33 gènes candidats à l’isolement reproducteur pré-zygotique a ensuite permis d’identifier des « gènes de spéciation » soumis à de la sélection divergente, liés en particulier à la phénologie de la floraison, et d’apporter des éléments de discussion supplémentaires au modèle de colonisation de ces espèces en peuplements naturels. / Speciation can be defined as the range of processes that lead to the evolution of reproductive isolation among groups of individuals. Past decades have witnessed a renewal in the recognition of natural selection as a primary force in the formation of new species, in particular for ecological speciation models in conditions of sympatry. Sessile oak (Quercus petraea (Matt.) Liebl.) and pedunculate oak (Q. robur L.) are two sympatric species which exhibit large morphological and ecological differences, despite relatively high estimated rates of natural hybridization. However, very little is known about the nature and strength of reproductive barriers between those two species. The objective of this thesis was therefore to characterize their isolating barriers at phenotypic and genetic levels, in order to better understand their relative importance, their role in the process of divergence and their evolution in that species complex. A first approach based on controlled crosses has shown (i) the existence of strong barriers at the pre-zygotic level and that of significant although weaker ones at the post-zygotic level, based on observations from fitness of hybrid progenies, and (ii) a large variability for traits linked to barriers phenotypes, across genotypes and also for their expression in different micro-environments. A second approach of population genomics used 33 candidate genes to reproductive isolation and allowed to identify «speciation genes» submitted to divergent selection, the strongest divergence being observed for genes involved in the flowering pathway. The model of colonization of these oak species in natural stands was discussed in the light of these new results.