Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. This novel HYPA is expected to expand its applications in the analysis and screening of genetic diseases.

Orientador: Lilian Madi-Ravazzi / Coorientador: Maria Tercília Vilela de Azeredo-Oliveira / Banca: Blanche Christine Pires de Bitner-Mathé Leal / Banca: Maria Izabel Camargo Mathias / Banca: Hermione Elly Melara de Campos Bicudo / Banca: Patrícia Vilamaior / Resumo: Zaprionus indianus é um drosofilídeo nativo da região Afrotropical que colonizou o continente Sul Americano, apresentando uma ampla distribuição geográfica enquanto Z. sepsoides é restrita a algumas regiões africanas. As duas espécies diferem em relação ao tamanho dos testículos e dos espermatozoides que é maior em Z. indianus do que em Z. sepsoides. Com o intuito de conhecer aspectos da biologia e o grau de diferenciação destas espécies, o presente estudo avaliou a espermatogênese de machos de diferentes idades (1, 3, 5 e 8 dias) de ambas as espécies por meio de técnicas de coloração convencional e de ultraestrutura. A espermatogênese e ultraestrutura dos espermatozoides foram semelhantes nas espécies em que foi confirmado o número diploide de cromossomos com 2n = 12. Entretanto, foi observada uma quantidade maior de espermatozoides em machos jovens (1 a 3 dias de idade) em Z. indianus do que em Z. sepsoides, o qual apresentou maior frequência de estágios iniciais da espermatogênese nestas idades. A porção da cabeça dos espermatozoides foi fortemente marcada nas duas espécies pela coloração por prata (AgNOR), orceína lacto-acética e pela reação de Feulgen. Quando submetidos à reação de P.A.S., os testículos de Z. sepsoides e Z. indianus apresentaram grânulos de glicogênio. As espécies possuem a mesma ultraestrutura flagelar, em que o axonema mostra um arranjo de 9+9+2 microtúbulos, com a presença de dois derivados mitocondriais de diferentes tamanhos e o número de 64 espermatozoides por feixe, em ambas as espécies. A grande semelhança observada no padrão do arranjo de microtúbulos do axonema e nos derivados mitocondriais com diferentes tamanhos nas espécies de Zaprionus, comparadas com outras espécies de Drosophila, é indicativa da conservação destas estruturas na família Drosophilidae... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Zaprionus indianus is a drosophilid native to the Afrotropical region that has colonized South America. Z. indianus exhibits a wide geographical distribution, whereas Z. sepsoides is restricted to certain African regions. The two species differ in the size of their testes, which are larger in Z. indianus than in Z. sepsoides. To better understand the biology and the degree of differentiation of these species, the current study evaluated spermatogenesis in males of different ages (1, 3, 5 and 8 days old) from both species by conventional staining techniques and ultrastructural analysis. Spermatogenesis and the ultrastructure of spermatozoa were similar in the two species, for which the diploid number was confirmed to be 2n = 12 chromosomes. However, a greater number of spermatozoa were observed in young Z. indianus males (1-3 days old) than in young Z. sepsoides males, which showed a higher frequency of cells at the early stages of spermatogenesis at this age. A portion of the head of the sperm was strongly marked in both species by silver staining (AgNOR), lacto-acetic orcein and the Feulgen reaction. Additionally, when submitted to P.A.S. reaction, the testes of both Z. sepsoides and Z. indianus exhibited glycogen granules. The two species also presented the same flagellar ultrastructure, in which the axoneme includes a 9+9+2 arrangement of microtubules, two mitochondrial derivatives of different sizes are present and the number of spermatozoa per bundle is 64. The great similarity in the pattern of microtubule arrangement in the axoneme and in the mitochondrial derivatives of the species Zaprionus, as compared with other species of Drosophila, indicates that these structures are preserved in the family Drosophilidae. The differences observed between the young males of Z. indianus and Z. sepsoides, including the number and frequency of sperm... (Complete abstract click electronic access below) / Doutor

Mechanistic details on the regulation of striated muscle contraction still need to be determined, particularly the specific structural locations of the elements comprising the thick and thin filaments. Of special interest is the location of the regulatory component, troponin, on the actin filament and how its presence influences the behavior of myosin binding to the thin filament. In the present study: (1) Luminescence resonance energy transfer was used to monitor potential conformational changes in the reconstituted thin filament between the C-terminal region of troponin T and myosin subfragment 1; (2) Location of troponin in previously derived atomic models of the acto-myosin complex was mapped to visualize specific contacts; and (3) Shortened tropomyosin was engineered and protein binding and ATPase assays were performed to study the effect of myosin binding close to the troponin complex. Analysis of the results suggest the following: (1) Irrespective of calcium levels, the C-terminal region of troponin T is located close to myosin loop 3 and a few actin helices that may perturb strong acto-myosin interactions responsible for force production. (2) Atomic models indicate myosin subfragment 1 cannot attain the post- powerstroke state due to the full motion of the lever arm being sterically hindered by troponin. (3) A shortened tropomyosin with five actin binding modules (instead of the native seven in muscle cells) binds actin contiguously in a head-to-tail manner and serves to increase the periodicity of troponin complexes on the actin filament. Such behavior eliminates the structure of the actin filament being responsible for the binding location of tropomyosin. (4) Differential behavior of myosin subfragment 1 i.e. (a) binding adjacent to troponin and (b) binding further away from troponin, is apparent as tropomyosin and troponin appear to govern the regions or "target zones" where myosin can bind productively along the actin filament. Physiologically, myosins able to bind close to troponin, but not participate in force production may function as mechanical sensors to attenuate or dampen the force generated from the so-called "target zones". Therefore, this could be a pseudo-regulatory mechanism that functions to protect the contractile apparatus from damage.

The tegmentum vasculosum of the avian cochlear duct mimics the stria vascularis of the mammalian cochlear duct in both location and structure, and previous studies indicate that it may be its functional counterpart with regard to endolymph synthesis. In the present study, we report on the enzymatic activity and ultrastructural localization of the Na+,K+-ATPase in the tegmentum vasculosum of the duckling. Na+,K+-ATPase activity was determined by measuring K+-dependent, ouabain-sensitive p-nitrophenyl phosphatase (p-NPPase) activity in homogenates of dissected regions of the cochlear duct. The ultrastructural localization of the Na+,K+-ATPase was identified using K+-dependent, ouabain-sensitive, p-NPPase cytochemistry. Specific enzyme activity was localized primarily in homogenates of the tegmentum vasculosum (2.27 μmol p-nitrophenyl phosphate/mg protein/min) when compared to homogenates of the entire cochlear duct (0.69 μmol p-nitrophenyl phosphate/mg protein/min). Reaction product for p-NPPase was localized primarily along the basolateral plasma membrane folds of the dark cells. The cytochemical deposits appeared to be located exclusively on the cytoplasmic side of the plasma membrane. The light cells were devoid of reaction product. Biochemical and cytochemical localization of p-NPPase activity on the basolateral plasma membrane folds of the dark cells of the tegmentum vasculosum in conjunction with the ultrastructural morphology of these cells is compatible with a Na+,K+-ATPase-dependent ion transport function related to endolymph synthesis.

Este estudo apresenta um painel de dados hematológicos, bioquímicos, morfológicos e citoquímicos para um grupo de serpentes dos gêneros Bothrops e Crotalus mantidas em cativeiro no Instituto Butantan. Para tanto, amostras de sangue foram colhidas da veia coccígea ventral e processadas de acordo com métodos padronizados. Os resultados alcançados foram analisados para determinar diferenças interespecíficas e sexuais. A avaliação citoquímica incluiu Sudan Black B (SBB), Ácido Periódico de Schiff (PAS) e Benzidina Peroxidase (BP). Os resultados hematológicos e bioquímicos mais relevantes indicaram um número significativamente mais alto de basófilos em B. jararaca e níveis mais altos de cálcio nas fêmeas da maioria das espécies. Os azurófilos marcaram positivamente para todas as colorações. A diferenciação entre trombócitos e linfócitos foi facilmente obtida com PAS. Os basófilos marcaram positivamente apenas com PAS em Bothrops alternatus. A marcação em heterófilos variou consideravelmente entre as espécies. Ultra-estruturalmente, os leucócitos foram semelhantes ao já descrito em literatura com algumas variações no tipo dos grânulos dos basófilos e heterófilos. Os grânulos dos basófilos apresentaram-se redondos em sua maioria, com tamanho e densidade variadas, enquanto que os nos heterófilos foram heterogêneos em forma, tamanho e densidade. / This study reports a panel for hematological, biochemical, morphological and cytochemical data for a group of captive snakes belonging to the genus Bothrops and Crotalus mantained at Instituto Butantan, São Paulo, Brazil. Blood samples were collected from ventral coccigeal vein and were processed according to standard protocols. Cytochemical staining including Sudan Black B (SBB), Periodic Acid-Schiff (PAS) and Benzidine peroxidase (BP) were conducted. Blood values were evaluated to determine interspecific and sex differences. We found a significant increase of basophils in Bothrops jararaca and higher levels of calcium in females. Azurophils stained positively for all stains and a differentiation between lymphocytes and thrombocytes was easily obtained with PAS stain. Basophils stained positively only with PAS in Bothrops alternatus. Heterophils staining varied between species. The ultrastructure of leucocytes were similar within described in literature, with some variations on granules of basophils and heterophils. Basophils granules were round, with heterogeneous size and density; whereas, heterphils granules were heterogeneous in shape, size and density.

A implantação embrionária e a placentação em cobaios são caracterizadas pela presença trofoblastos que se destacam da placenta principal, semelhantes ao trofoblasto extra-viloso de humanos. Nestes animais ultrapassam os limites, e podem ser encontrados infiltrados no profundamente no endométrio e no em ambiente externo ultrapassando aos limites da parede uterina. A cobaia desenvolve uma importante estrutura fisiológica de troca materno-embrionária, denominada de placenta vitelina invertida, definidas como membrana fetal destituída parcial ou totalmente do revestimento trofoblástico que permite a exposição do endoderma extra-embrionário em contato direto com o tecido materno. Tais características denotam um mecanismo de controle da resposta imune materna distinta dos paradigmas estabelecidos na reprodução humana e de roedores, assim como ratos e camundongos. Sendo a mais intrigante, a destituição do trofoblasto como célula da interface-materno-fetal que controla a tolerância imune-materna.No presente trabalho, procurou-se estabelecer a organização da placenta vitelina de cobaios a partir da identificação das células que compõe esta membrana extra-embrionária e identificar em que momento ocorre à remoção das células trofoblásticas, e a subseqüente forma de interação das células da placenta vitelina na interface com o tecido materno. Para tanto foram utilizados cobaias fêmeas com idade gestacional conhecida, sacrificadas para coleta de segmentos uterinos nos períodos iniciais da gestação e destinados ao processamento histotógico de embebição em parafina. Na ausência de marcadores celulares específicos conhecidos para cobaios, foram realizados testes prospectivos com reações: citoquímicas de PAS e azul de toluidina (AT; um painel de lectinas biotinadas com afinidade específica para diferentes açúcares; e imunocitoquímica para citoqueratina. As reações realizadas com PAS e AT não identificaram populações celulares com marcação seletiva. Contudo dentre as lectinas tetadas, a Erytrina cristagali lectin (ECL) apresentou reação altamente seletiva para a população de trofoblasto mural (TM) que se origina do trofectoderma, mantendo esta reatividade ao longo da gestação. Esta marcação permitiu avaliar temporal e espacialmente o destino destas células que ao longo da gestação eram mantidas como monocamada de TM revestindo externamente a placenta vitelina e, portanto, não expondo as células do endoderma parietal ou visceral ao ecido materno. Pelo acompanhamento do desenvolvimento embrionário nos cortes seriados, foi constatada no interior do blastocisto a organização de duas massas celulares internas em pólos opostos desde a fase de pré-eclosão. Uma das massas celulares constituída de embrioblastos que dará origem aos os folhetos embrionários nas fases subseqüentes, enquanto a outra formada as células tronco trofoblásticas precursora do cone ectoplacentário (CE). A cavidade da blastocele que separa estas duas massas celulares tem a sua parede revestida pelo endoderma parietal em fase tardia, após a formação da cavidade amniótica. Estes achados demonstram a pecularidade da embriogênese no cobaio, diferente daquelas descritas para humanos e outros roedores, não permitem analogias diretas, o que pode ter contribuído para o equívoco na descrição clássica da organização e constituição da placenta vitelina invertida de constituição córion-amniótica. Isto é, o trofoblasto participa da organização da placenta vitelina inicial e permanece na membrana âmnion-córion-vitelina perfazendo todo o limite do embrião ao longo da gestação. Portanto a hipótese da placenta vitelina parcial ou totalmente invertida baseada na descrição clássica em cobaios é decorrente da interpretação equivocada da embriogênese destes animais. / The guinea pig embryo implantation and placentation is characterized by trophoblast cells detaching from the main placenta in a similar way of human extra-villous trophobasts that deeply intrude inside the endometrium and sometimes also found outside the uterine wall. Furthermore, this animal also develops inverted yolk sac placenta defined as fetal membrane partially or fully devoided of trophoblast sheet that allows extra-embryonic endoderma direct exposition to the maternal environment. These characteristics denote a distinct control mechanism of maternal immune response from the established paradigm for human and rodents (rat and mouse) reproduction, being most intriguing the depriving of trophoblast as cells of maternal-fetal interface regulating the maternal immune tolerance. The present work aimed to establish the organization of guinea pig yolk sac based on identification of cell populations composing this membrane and identification if, or, when the trophoblast cells are removed from and subsequent interaction way of yolk sac cell in interface with maternal tissue. It was used pregnant guinea pig sacrificed on established gestational day to collect uterine fragments on early pregnancy stage and processed by conventional paraffin embedding. Due to absence of known specific cell markers for guinea pig, was performed the prospective evaluation using PAS and toluidine blue (TB) cytochemistry and a screening using a panel of biotinylated lectin specific for different sugars and, anti-cytokeratin. The PAS and TB staining did not identify any specific cell population, however, among the lectins used, Erytrina cristagali lectin (ECL) showed high selective labeling to the trophoblast cells originated from the trophectoderm that was kept through the gestational period. This reaction pattern was useful to evaluate chronologically and topologically the fate of this cell and confirmed the constancy of these cells layering the yolk sac placenta in contact with maternal tissue and therefore, endodermal cells were not exposed to maternal environment. Evaluation of embryo development step by step in the serial sections showed the presence of two inner cell mass in opposite sites inside the pre-hatched blastocyst. One of this, was formed with embryoblast that latter will originate the embryonic sheets and the other formed with trophoblast stem cells (ST) will originate the ectoplacental-cone. The wall of blastocele cavity separate these two inner cell mass was initially covered by a single ECL positive mural trophoblast and only later after the amniotic cavity is formed the extraembyonic endodermal cells migrate from the embryonic sheets to cover internally the blastocele cavity to organize the yolk sac placenta. These findings show the peculiarity of guinea pig embryogenesis, quite different from those described for human and rodents and therefore, does not allow direct analogy and seems to contribute in the misunderstanding of classic description of inverted yolk sac placenta and its cellular organization. It means, the trophoblast cell participates in the early organization of yolk sac placenta and remains in chorioamniotic yolk sac fetal membrane constantly limiting the embryo surface in contact with maternal environment. Therefore, the hypothesis of complete or partially inverted yolk sac placenta seems to be a miss understanding of guinea pig embryogenesis.

Este estudo apresenta um painel de dados hematológicos, bioquímicos, morfológicos e citoquímicos para um grupo de serpentes dos gêneros Bothrops e Crotalus mantidas em cativeiro no Instituto Butantan. Para tanto, amostras de sangue foram colhidas da veia coccígea ventral e processadas de acordo com métodos padronizados. Os resultados alcançados foram analisados para determinar diferenças interespecíficas e sexuais. A avaliação citoquímica incluiu Sudan Black B (SBB), Ácido Periódico de Schiff (PAS) e Benzidina Peroxidase (BP). Os resultados hematológicos e bioquímicos mais relevantes indicaram um número significativamente mais alto de basófilos em B. jararaca e níveis mais altos de cálcio nas fêmeas da maioria das espécies. Os azurófilos marcaram positivamente para todas as colorações. A diferenciação entre trombócitos e linfócitos foi facilmente obtida com PAS. Os basófilos marcaram positivamente apenas com PAS em Bothrops alternatus. A marcação em heterófilos variou consideravelmente entre as espécies. Ultra-estruturalmente, os leucócitos foram semelhantes ao já descrito em literatura com algumas variações no tipo dos grânulos dos basófilos e heterófilos. Os grânulos dos basófilos apresentaram-se redondos em sua maioria, com tamanho e densidade variadas, enquanto que os nos heterófilos foram heterogêneos em forma, tamanho e densidade. / This study reports a panel for hematological, biochemical, morphological and cytochemical data for a group of captive snakes belonging to the genus Bothrops and Crotalus mantained at Instituto Butantan, São Paulo, Brazil. Blood samples were collected from ventral coccigeal vein and were processed according to standard protocols. Cytochemical staining including Sudan Black B (SBB), Periodic Acid-Schiff (PAS) and Benzidine peroxidase (BP) were conducted. Blood values were evaluated to determine interspecific and sex differences. We found a significant increase of basophils in Bothrops jararaca and higher levels of calcium in females. Azurophils stained positively for all stains and a differentiation between lymphocytes and thrombocytes was easily obtained with PAS stain. Basophils stained positively only with PAS in Bothrops alternatus. Heterophils staining varied between species. The ultrastructure of leucocytes were similar within described in literature, with some variations on granules of basophils and heterophils. Basophils granules were round, with heterogeneous size and density; whereas, heterphils granules were heterogeneous in shape, size and density.

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase is the enzyme responsible for catalyzing the first reaction in the biosynthesis of KDO. KDO is an essential component in the cell wall of Gram-negative bacteria and plants. This compound is not present in mammals; therefore the enzymes responsible for its biosynthesis are potential targets for the development of new antibiotic agents. KDO8P synthase catalyzes the condensation reaction between phosphoenol pyruvate (PEP) and D-arabinose 5-phosphate (A5P) to form KDO8P. Two types of KDO8P synthase have been identified; a metal-dependent type and a non metal-dependent type. KDO8P synthase from the organism Chlorobium tepidum (Cte) has been partially purified and partially characterized. In line with predictions based on sequence alone, the activity of this enzyme is dependent on the presence of a divalent metal ion and is sensitive to the presence of the metal chelating agent EDTA. Cte KDO8P synthase was found to have the highest activity in the presence of Mn2+ or Cd2+. KDO8P synthase from the organism Acidithiobacillus ferrooxidans (Afe) has also been cloned, purified and biochemically characterized. Afe KDO8P synthase was also found to be a metallo enzyme and the catalytic activity is highest in the presence of Mn2+ or Co2+. Afe KDO8P synthase was found to exist as a tetramer in solution and is most active within the pH range of 6.8 to 7.5 and within a temperature range of 35 ºC to 40 ºC. Sequence analysis suggests that this enzyme has characteristics conserved throughout the metallo and the non-metallo KDO8P synthases and is closely related to the metal-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthases. The role of several active-site residues of Afe KDO8P synthase has been investigated. A C21N mutant of Afe KDO8P synthase was found to retain 0.5% of wildtype activity and did not require a divalent metal ion for catalytic activity. This suggests that the metallo and non-metallo KDO8P synthases have similar catalytic mechanisms.

This work presents the development of a new highly sensitive and selective chemical assay for poly(ADP-ribose) which is routinely useful for the determination of polymer levels in vivo. This method was used to carefully measure poly(ADP-ribose) levels in normal and in DNA-damaged cells. The results of these studies strongly suggest that synthesis of poly(ADP-ribose) is involved in some aspect of DNA repair. A review of the literature is presented in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in aspects of transcription, in DNA syn thesis, and in DNA repair largely based on evidence from in vitro studies. It is apparent that current methodology has not allowed the routine quantification of poly(ADP-ribose) in vivo, hence the lack of i^n vivo data concerning the function(s) of the polymer. The body of this work presents the development of two chemical methods for the quantification of poly(ADP-ribose) and the application of one of these methods to the measurement of polymer levels in normal and DNA-damaged cells. Preliminary studies are presented on the utilization of combined gas chromatography/mass spectroscopy for the selective quantification of nucleoside derivatives. A second method makes use of the unique chemistry of the polymer for quantification. The polymer was selectively adsorbed to dihydroxyboryl-sepharose which allowed the removal of most RNA, DNA, and protein from the samples. The polymer was hydrolyzed to the unique nucleoside 2'—^-l*'-ribosyladenosine by digestion with venom phosphodiesterase and bacterial alkaline phosphatase. The 1-N^-etheno derivative of ribosyladenosine was formed by reaction with chloroacetaldehyde and this derivative was seperated from other fluorescent species by reversed phase high pressure liquid chromatography.

ALMEIDA, João Paulo Nobre de. Germinação e crescimento de plântulas de amburana cearensis (Allemão) A.C. smith em função do peso de sementes e fatores abióticos. 2014. 109 f. : Dissertação (mestrado) - Universidade Federal do Ceará, centro de Ciências Agrárias, Departamento de Fitotecnia, Curso de Pós-Graduação em Agronomia / Fitotecnia, Fortaleza-CE, 2014 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-05-17T12:18:57Z No. of bitstreams: 1 2014_dis_jpnalmeida.pdf: 1353578 bytes, checksum: cba2917425c9a0dbf90e35a88352f7ff (MD5) / Approved for entry into archive by Fabíola Bezerra (fabbezerra@yahoo.com.br) on 2016-05-17T12:24:47Z (GMT) No. of bitstreams: 1 2014_dis_jpnalmeida.pdf: 1353578 bytes, checksum: cba2917425c9a0dbf90e35a88352f7ff (MD5) / Made available in DSpace on 2016-05-17T12:24:47Z (GMT). No. of bitstreams: 1 2014_dis_jpnalmeida.pdf: 1353578 bytes, checksum: cba2917425c9a0dbf90e35a88352f7ff (MD5) Previous issue date: 2014 / Given the needs of reclamation is increasing interest in the propagation of native species, which requires basic information about their germination characteristics and ecophysiological. The Amburana cearensis (Allemão) A.C. Smith known as cumaru is a species widely used for restoration of the landscape. The aim of this study was to investigate the germination and seedling growth of cumaru depending on the weight of the seeds under conditions of light, temperature, drought stress, as well as to determine the seed imbibition curve, internal morphology, chemical composition and quantification of the coumarin. Initially seeds were individually weighed and separated into three weight classes (light, medium and heavy), these being submitted to the determination of water content, thousand seeds weight and germination tests in two light conditions (presence and absence ) and six schemes temperatures (20, 25, 30, 35, 40, and 20-30 °C). In addition to these tests, it was determined the imbibition curve of each weight class and made assessment of drought stress tolerance under different potentials(-0.2, -0.4, -0.6, -0.8 and -1,0 MPa). The experiments were arranged in a completely randomized design in four replications for each treatment. For the internal morphology of seeds were used in cytochemistry usual techniques for the identification of the main structures and substances reserves. To visualize the coumarin in the seeds by NMR spectroscopy was used. The optimum conditions for seed germination occurred at 30 °C, which are insensitive to light and vigor seeds for light and medium. The best conditions for seedling growth occurred with the light and medium seeds at 25 and 30 °C and in the presence of light, with temperatures of 35 and 40 °C harmful. Light and medium seeds showed the same pattern of water absorption, while not reach the heavy phase III of the curve. The decrease in the water potential of the substrate affect the germination and growth of seedlings from seed medium and heavy compared to the light, and from -0.6 MPa in a condition strictly limiting seedling development. The chemical constituents present in the seeds of A. cearensis are quantitatively different depending on the weight of the seeds, and the heavy characterized by a high content of lipids. The NMR spectroscopy falls greater proportion of coumarin in seed extract heavy and medium, possibly affecting ecophysiological needs of the species A. cearensis. / Diante das necessidades de recuperação de áreas degradadas é crescente o interesse na propagação de espécies florestais nativas, o que demanda informações básicas sobre as suas características germinativas e ecofisiológicas. A Amburana cearensis (Allemão) A.C. Smith conhecida como cumaru é uma espécie bastante utilizada para recomposição da paisagem. O objetivo deste trabalho foi estudar a germinação e o crescimento de plântulas de cumaru em função do peso das sementes sob diferentes condições de temperatura, luz, estresse hídrico, bem como determinar nas sementes a curva de embebição, morfologia interna, composição química e identificação da cumarina. Inicialmente as sementes foram pesadas individualmente e separadas em três classes de peso (leves, médias e pesadas), sendo estas, submetidas à determinação dos teores de água, peso de mil de sementes e a testes de germinação em duas condições de luz (presença e ausência) e seis regimes de temperatura (20, 25, 30, 35, 40 e 20-30°C). Além destes ensaios, foi determinado a curva de embebição de cada classe de peso e avaliação da tolerância ao estresse hídrico sob diferentes potenciais (-0,2, -0,4, -0,6, -0,8 e -1,0 MPa). Os experimentos foram dispostos em delineamento inteiramente casualizado com quatro repetições para cada tratamento. Para a morfologia interna das sementes foram utilizadas técnicas usuais em citoquímica para a identificação das principais estruturas e substâncias de reservas. Para a visualização da cumarina nas sementes foi utilizada a espectroscopia por RMN. As condições ótimas para a germinação das sementes ocorreu na temperatura de 30°C, sendo estas insensíveis à luz e um maior vigor para sementes leves e médias. As melhores condições para o crescimento das plântulas ocorreram com as sementes leves e médias nas temperaturas de 25 e 30°C e na presença de luz, sendo as temperaturas de 35 e 40 °C prejudiciais. Sementes leves e médias apresentam o mesmo padrão de absorção de água, enquanto as pesadas não atingem a fase III da curva. O decréscimo do potencial hídrico do substrato prejudica a germinação e o crescimento de plântulas oriundas de sementes médias e pesadas em comparação às leves, sendo a partir de -0,6 MPa uma condição estritamente limitante na formação de plântulas. Os constituintes químicos presentes nas sementes de A. cearensis são quantitativamente diferenciados em função do peso das sementes, sendo as pesadas caracterizadas por um elevado teor de lipídios. A espectroscopia por RMN releva uma maior proporção de cumarina no extrato de sementes pesadas e médias, que possivelmente afetam as necessidades ecofisiológicas da espécie A. cearensis.