Orientador: Luis Antonio Violin Dias Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T03:55:25Z (GMT). No. of bitstreams: 1 Arroteia_KelenFabiola_D.pdf: 15193315 bytes, checksum: b6b3930ea05ff1d7b858f560e01c178a (MD5) Previous issue date: 2006 / Resumo: O processo de fecundação em mamíferos depende de uma seqüência de eventos que culminam na ativação do oócito pelo espermatozóide. A diferenciação completa das células germinativas testiculares em células com capacidade fecundativa envolve testículos, epidídimos, ductos deferentes e trato reprodutor feminino. No epidídimo, a superfície dos espermatozóides pode sofrer diversas modificações, em um processo conhecido como maturação epididimária. Diversas proteínas sintetizadas e secretadas pelas células epiteliais do epidídimo serão posteriormente localizadas na superfície ou mesmo no interior da vesícula acrossômica do espermatozóide. A aquisição da capacidade de fertilização pelo spermatozóide tem sido correlacionada à esta nova organização das moléculas de membrana proporcionada durante o trânsito epididimário. A obtenção de anticorpos monoclonais que reconhecem os antígenos expressos pelas células germinativas testiculares durante seus processos contíguos de diferenciação, ou pelas células dos ductos pelos quais os espermatozóides transitam durante o processo de maturação tem constituído uma importante estratégia para permitir a geração de um mapa das moléculas que atuam na preparação do espermatozóide para a fecundação. O anticorpo monoclonal (Amc) TRA (testicular germ cells immunized to rat - monoclonal antibody) 54 reconhece um antígeno localizado em espermátides dos túbulos seminíferos de camundongos C57 BL/6 com idade superior a 24 dias pós-parto e também um antígeno expresso nas células epiteliais da cabeça do epidídimo e em espermatozóides da luz deste órgão. A molécula secretada percorre a luz do epidídimo no sentido ântero-posterior e, neste percurso, é incorporada pelos espermatozóides em trânsito. Com o intuito de contribuir para a compreensão dos complexos mecanismos que preparam o espermatozóide para a fecundação, o presente trabalho buscou identificar e reconhecer a estrutura protéica e a função biológica do antígeno reconhecido pelo Amc TRA 54 nas células epiteliais do epidídimo. No futuro, os resultados obtidos poderão contribuir com a geração de novas ferramentas clínicas para a solução de problemas relacionados à biologia da reprodução / Abstract: In mammals, fertilization depends on a sequence of events that culminates in the activation of the oocyte by the spermatozoa. The complete differentiation of the testicular germ cells in cells with fertilization ability involves the testis, epididymis, vas deferens and female reproductive organs. In the epididymis, the membrane surface of the spermatozoa may be modified, through a process known as epididymal maturation. Several proteins synthesized and secreted by the epididymal epithelial cells can be further located on the spermatozoa membrane surface or even though inside its acrosomal vesicle. The acquisition of the fertilization ability by the spermatozoa has been related with a new molecular organization of the membrane provided during the epididymal transit. The production of monoclonal antibodies (mAb) that recognizes antigens exclusively expressed by testicular germ cells or by the cells of the ducts through the spermatozoa passes during its maturation has been considered an important approach to permit the elaboration of the molecular map involved in the spermatozoa preparation. The mAb TRA (testicular germ cells immunized to rat - monoclonal antibody) 54 recognizes an antigen located in spermatids of seminiferous tubules of C57 BL/6 mice more than 24 days old and also in a specific population of epithelial cells in epididymal caput and in the luminal spermatozoa. The synthesis and release of the epididymal antigen occur in an androgen-dependent manner and independent of the testicular expression. Released antigen moves down the epididymis and is further incorporated by luminal spermatozoa. This work was performed in order to comprehend the complex mechanisms that prepare spermatozoa for the fertilization process. The molecular structure of the epididymal protein and its biological functions were investigated and solved. In a near future, the obtained results can contribute with new clinical tools for solving problems in reproductive biology / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Com o objetivo de estudar o efeito das diferentes doses de NPK no crescimento e produÃÃo da bananeira âPacovan Apodiâ, realizou-se este experimento na Ãrea da empresa FRUTACOR, no distrito de Cercado do Meio, situado no municÃpio de QuixerÃ, Estado do CearÃ. As mudas foram transplantadas em setembro de 2008, no espaÃamento de 3,5 m entre fileiras duplas, 1,9 m entre fileiras simples e 1,6 m entre plantas na linha. As parcelas foram constituÃdas de 8 plantas, considerando como parcela Ãtil as 4 plantas centrais. Cinco doses de nitrogÃnio (70, 490, 700, 910 e 1329 kg N ha-1), cinco de fÃsforo (17, 122, 174, 227 e 332 kg P2O5 ha-1) e cinco de potÃssio (44, 310, 443, 576 e 842 kg K2O ha-1) foram combinadas conforme a matriz experimental Pan Puebla II e distribuÃdas em um delineamento em blocos casualizados, com cinco repetiÃÃes. A fonte de nitrogÃnio utilizada foi urÃia, de fÃsforo foi o fosfato monoamÃnico, e de potÃssio foi o cloreto de potÃssio. As doses de NPK foram divididas em sete aplicaÃÃes, sendo a primeira realizada em outubro de 2008 e repetida mensalmente. Foram realizadas coletas de dados biomÃtricos (comprimento e circunferÃncia do pseudocaule e nÃmero de folhas) no perÃodo de 120 atà 210 dias apÃs o transplantio (DAT). Coletaram-se folhas das plantas para a anÃlise dos teores de NPK. Aos 305 DAT colheram-se os cachos, que foram despencados, pesados e avaliados quanto ao nÃmero de palmas e nÃmero de frutos. A combinaÃÃo das doses que proporcionou melhores resultados de crescimento e produÃÃo da bananeira âPacovan Apodiâ foi de 490, 227 e 576 kg ha-1 ciclo-1. O incremento nas doses de nitrogÃnio influenciou negativamente o comprimento do pseudocaule e o nÃmero de palmas. A adubaÃÃo fosfatada proporcionou resposta positiva para nÃmero de folhas, peso do cacho e produtividade, e nÃo houve resposta da bananeira à adubaÃÃo potÃssica nas caracterÃsticas avaliadas. Os teores foliares de N, P e K nÃo foram significativamente influenciados pelas doses de N, P2O5 e K2O. / This research aimed to study the effect of different NPK rates related to growth and production of banana tree âPacovan Apodiâ, carrying out an experiment on the experimental field of enterprise FRUTACOR, in the district of Cercado do Meio, located in the municipality of QuixerÃ, Cearà State (Northeast Brazil). The experiment was conducted in September 2008, using seedlings of banana tree âPacovan Apodiâ, developed from tissue culture and placed in a 3,5 m planting space in double rows, 1,9 m in single rows and 1,6 m between simple plant lines. Experimental plots consisted of 8 plants, considering 4 centralized plants as useful. Five doses of nitrogen (70, 490, 700, 910 and 1329 kg N ha-1), five of phosphorus (17, 122, 174, 227 and 332 kg P2O5 ha-1) and five of potassium (44, 310, 443, 576 and 842 kg K2O ha-1) were arranged according to experimental matrix Pan Puebla II and distributed using the experimental design of randomized blocks, with five replications. The nitrogen source used was urea, phosphorus was the mono-ammonium phosphate, and potassium was potassium chloride. NPK doses were divided in seven applications, being the first one conducted in October 2008 and the other monthly. Biometric data (pseudostem length, pseudostem diameter and number of leaves) was collected from 120 to 210 days after transplanting (DAT). Leaves were collected from each plot for analysis of NPK nutrient content. At 305 DAT grapes were harvested, tumbled down, weighed, evaluated and related to the number of palms and also the number of fruits. The combination of doses which promoted the best growth results and production of banana tree âPacovan Apodiâ was composed by 490, 227 and 576 kg ha-1 cycle-1. Increase in N rates affected pseudostem length and number of palms. Phosphate fertlization reached positive results facing the number of leaves, weight of bunch and productivity, and banana tree âPacovan Apodiâ presented no answer to potassium fertilization considering characteristics evaluated on the experiment. There was no significant effect of N, P2O5 and K2O rates on foliar nutrient concentration.

Busca-se, no campo da fitotecnia, métodos que possam aumentar a produtividade dos canaviais atentando-se ao menor uso de adubos minerais, visando redução de custos e sustentabilidade da produção no setor sucroalcooleiro. Sob esse princípio, tem sido muito discutido o uso de condicionadores organominerais no solo, para promoção de alterações das propriedades físicas, químicas e biológicas, visando aumentar o teor de matéria orgânica, aumentar a diversidade e atividade da microbiota, incrementar a capacidade de troca catiônica e aniônica, desfavorecer a manutenção de íons metálicos na solução, ocasionando assim a diminuição da toxidez desses elementos. Assim, foi avaliado o uso do produto organomineral comercial a base de ácido húmico Agrolmin® na cultura da cana-de-açúcar, objetivando-se analisar sua influência na produtividade da cultura, a interação com a adubação mineral, adequar metodologia de aplicação, fixar diretriz quanto a doses de aplicação e resgatar a credibilidade dos condicionadores orgânicos como um fator de melhoria do ambiente de produção. Os experimentos foram instalados em área da Usina Santa Cândida, município Bocaina – SP, na variedade PO88-62, como cana-planta, e RB867515, como cana-soca. O delineamento experimental foi parcelas subdivididas, analisando-se as doses 0, 800, 1.600 e 2.400 kg/ha do adubo de formulação 2,5-10-10 (parcelas) com as doses 0 e 350 L/ha do produto comercial (sub-parcelas) para cana-planta, e as doses 0, 800, 1.600 e 2.400 kg/ha do adubo de formulação 09-00-09 (parcelas) com as doses 0, 300 e 600 L/ha do produto comercial (sub-parcelas) para cana-soca. Os resultados indicaram que o uso do produto comercial tem efeito significativo na produção da cultura, tanto para cana-planta como para cana-soca, equivalendo ao uso de aproximadamente 1.200 e 1.100 kg/ha de adubo mineral respectivamente, sendo que em cana-soca, a dose de 300 L/ha e 600 L/ha não mostraram diferença significativa. Em ambos os casos, nota-se que a resposta de produtividade da cultura ao uso do produto comercial é maior quanto menor a dose de adubo utilizada. Porém, tanto em cana-planta como em cana-soca, não foi observada estatisticamente interação significativa do produto comercial com a adubação mineral. Isso mostra que o uso de condicionadores de solo organominerais pode ser viável em se tratando de incremento de produção aliado a redução de adubação mineral. / Aims have been made to find ways to increase de productivity of sugar-cane crops using less mineral fertilizers, attending to a cost reduction and sustainability. Based on these principles, the use of organic-minerals products to promote physical, chemical and biological alterations in the soil system, seeking to increase the organic matter content, the biological diversity and activity, the cationic/anionic exchange capacity, disfavor the amount of metallic ions in the solution resulting in a decrease of the toxicity, caused when these metallic elements are present. The use of a commercial humic acid based soil conditioner product, named Agrolmin®, in a sugar cane crop was evaluated, in order to measure its influence in crop production, to check its interaction with the mineral fertilization, to adjust the application methodology, to define proportions procedures and to ransom the credibility of the organic conditioners as an environmental production improvement factor. The experiment was carried out on Santa Candida Sugarmill, located in Bocaina, São Paulo state, using the sugar cane varieties PO88-62 (plant cane), and RB867515 (ratoon). The experiment was laid out in split-plot, using 0, 800, 1.600 e 2.400 kg/ha of the mineral fertilizer 2,5-10-10 (plot) with 0 and 350 liters/ha of the commercial product (sub-plot) at the crop planting, and 0, 800, 1.600 e 2.400 kg/ha of the mineral fertilizer 09-00-09 (plot) with 0, 300 e 600 liters/ha of the commercial product (sub-plot), at first ratoon. Results shown that the use of the commercial product causes an significative increasing in the crop production, either in plant-cane and first ratoon, equivalent the use of 1.200 e 1.100 kg/ha of the mineral fertilizer. In both plant and ratoon, the response to productivity related to the use of the commercial product is greater as the mineral fertilizer's dose is smaller. However, either at planting or first ratoon, a significative interaction between Agrolmin® and the mineral fertilization was not observed. This shows that organic-mineral soil conditioners may be a good choice on a productivity improvement allied to a reducing mineral fertilization.

M.Sc. / The family lridaceae includes genera such as Sparaxis, Freesia and Babiana which are popular flowering plants. The purpose of this study was the propagation in tissue culture of Sparaxis plants. After the applicable techniques have been formulated, the aim was to test the efficiency of the techniques on the other genera. The techniques can then be extended to endangered genera. Apical meristems as well as axillary meristems were dissected aseptically and grown on the medium of Heinz and Mee (1969). It is difficult to acquire aseptic explant material of Sparaxis because of the net-like fibres surrounding the corm. Growth of aseptic meristem explants occurred within a week under 16h light and 25 ± 1 °C. Proliferation of meristems occurred and gave rise to more than one plant from the same explant. The study was extended to seeds as an explant source, as well as the influences of various factors such as pH, carbon source and gelling agents, on the growth and proliferation of the plants. Rooting of the plants was investigated and hardening done for transfer of the plants to the greenhouse.

Abstract The aim of this population-based cohort study was to evaluate prenatal and child outcome and costs resulting from prenatal and neonatal care after in vitro fertilization (IVF) in comparison to those after natural conception using a cohort of 304 IVF exposed children born between 1990–1995 in Northern Finland, and two cohorts of unexposed control children (I: n = 569, representing general population in proportion of multiple births; II: n = 103, matched for plurality). The control children were randomly chosen from the Finnish Medical Birth Register (FMBR) and matched for sex, year of birth, area of residence, parity, maternal age and socioeconomic status. Analyses were performed by comparing the whole IVF population with controls representing general population as well as stratifying by singleton or twin status. IVF mothers carried a higher risk for vaginal bleeding, threatened preterm birth and intrahepatic cholestasis of pregnancy than control mothers, and they used specialized antenatal care more than others. Neonatal outcome was also poorer after IVF in terms of gestational age, birthweight, morbidity and intensive care treatment. The prevalence of congenital heart malformations (septal defects) was 4-fold for IVF children in comparison to controls. The three year follow-up showed delayed growth and increased morbidity for IVF children, but their psychomotor development was similar to that of the control children. Health care costs were 1.3-fold for IVF singletons in comparison to control singletons, but for twins the costs were equal. Multiple births increased the costs ∼3-fold when compared to singleton births. IVF increased the health risks for the pregnancies and the offspring, seen mostly in the comparison between the whole IVF population and controls representing natural proportion of multiple births, indicating that multiple birth is the strongest determinant of medical outcome after IVF. The effects of fertility therapy and maternal characteristics related to infertility cannot be ruled out at this point. The increased health care costs after IVF were mostly due to the high proportion of multiple births. In order to improve the outcomes and to reduce the health care costs after IVF, the amount of multiple births should be limited to a minimum by using single embryo transfer when possible.

La fécondation est la rencontre de deux gamètes. Bien que centrale chez les espèces sexuées, les mécanismes membranaires et moléculaires ne sont pas encore établis. La communauté scientifique bute toujours sur la question centrale : Comment le spermatozoïde fusionne-t-il avec l’ovule ? Si des études ont identifié trois protéines essentielles : Izumo1, Juno et CD9, elles montrent aussi que ces acteurs ne sont pas suffisants. Notre étude a eu pour but d’identifier d’autres paramètres potentiels impliqués dans cette machinerie de fusion. Nous nous sommes donc focalisés sur la contribution des lipides spermatiques et sur celle du battement du flagelle. Nous avons développé deux méthodes expérimentales originales. Avec la première, qualifiée de « Bottom-up », nous avons tenté de déterminer la machinerie spermatique minimale pour induire la fusion avec l’ovocyte. L’idée a été de reconstituer pas à pas la membrane de la tête du spermatozoïde, d’abord avec les lipides identifiés lors d’analyses, puis en y incorporant Izumo1. Pour la seconde approche, appelée « Top-down », nous avons développé un outil microfluidique pour guider le spermatozoïde jusqu’à l’ovocyte afin de suivre la rencontre avec le « meilleur » point de vue, dans des conditions in-vitro aussi physiologiques que possible. Nous avons découvert que contrairement à ce que nous pensions, le battement du flagelle ne sert pas uniquement à atteindre l'ovocyte, mais aussi à déclencher la fécondation. En effet, les contraintes mécaniques induisent une réorganisation de la membrane ovocytaire incluant la protéine CD9. Ainsi, la chronologie des événements a pu être obtenue avec une résolution temporelle inégalée. / Fertilization is the encounter of two gametes. Although this process is crucial for sexual organisms, the timeline of the molecular events is not yet established. The researchers cannot explain: how the spermatozoon fuses with the oocyte? One of the reasons is the lack of experimental methods available. Indeed, the gametes need a specific environment to fertilize. Nevertheless, the scientific community identified three essential proteins: Izumo1 on the spermatozoon, Juno (its receptor) and CD9 on the oocyte membrane. For our part, we tried to determine if the none-proteins environment of Izumo1 and CD9 could influence the gametic interaction. To do so, we were focused on the role of the lipids composition of the sperm membranes and on the influence of the forces developed by the flagellum beating on the oocyte. We designed two original experimental methods to offer a better understanding of the mechanisms inside the gamete contact area. With the first one, we tried to identify the minimal machinery to induce fusion. We started to reconstitute step by step the membrane of the spermatozoon head. We tested first the identified lipids alone, and then we coupled these molecules with Izumo1. With the second one, we developed a microfluidic tool to observe the gametic encounter with the “best” viewpoint in the most physiological in-vitro conditions. We observed that the flagellum beating is not only involved in the crossing of the female genital tract but also in the initiation of the fusion step. Indeed, the mechanical constraints induce membrane reorganization with CD9 recruitment. So we succeed to establish the kinetic of the events with an unequaled resolution.

PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

Statistical modelling of data from the embryo transfer process of In-Vitro Fertilization (IVF) treatments is motivated by the need to perform statistical inference for potential factors and to develop predictive models for these treatments. The biggest issue arising when modelling these treatments is that a number of embryos are transferred but unless all of the embryos get implanted or fail to implant then it is not possible to identify which of the embryos implanted. Little work has been done to address this partial observability of the outcome as it arises in this context. We adopt an Embryo-Uterus (EU) framework where a patient response has distinct uterine and embryo components. This framework is used to construct statistical models, expand them to allow for clustering effects and develop a package that will enable the fitting and prediction of these models in STATA. The capabilities of this package are demonstrated in two real datasets, aimed in investigating the effect of a new embryo prognostic variable and the effect of patient clustering in these treatments. In a simulation study EU models are shown to be capable of identifying a patient covariate either as a predictor of uterine receptivity or embryo viability. However a simulation case study shows that a considerable amount of information about the embryo covariate is lost due to the partial observability of the outcome. Further simulation work evaluating the performance of a number of proposed alternatives to the EU model shows that these alternatives are either biased or conservative. The partially observed cycles are finally considered as a missing data problem and two novel modelling approaches are developed which are able to handle the structure of these treatments. These novel models, based on multiple imputation and probability weighting, are compared to the EU model using simulation in terms of predictive accuracy and are found to have similar predictive accuracy to the EU model.

M.Sc. (Botany) / Please refer to full text to view abstract

Made available in DSpace on 2016-05-02T13:54:07Z (GMT). No. of bitstreams: 1 PedroBavusoNeto-Dissertacao.pdf: 177979 bytes, checksum: 24a47b891925286484beb0e96a13da9e (MD5) Previous issue date: 2010-01-26 / The experiment was carried out at the University José do Rosário Vellano in areas of the Department of Olericulture and Experimentation, in Alfenas, MG, Brazil, from March, 2, 2009 to June, 11, 2009. The purpose was to study the viability of potato fertirrigation techniques. The design was in randomized blocks in a 2 x 3 x 3 factorial scheme, with a total of 18 treatments and three replications. The factors studied were: fertirrigation (two types: surface and subsurface); nitrogen (three doses: 75, 150, and 225 kg ha-1); potassium (theree doses: 210, 310, and 410 kg ha-1). The characteristics studied were: Full Commercial Production in tha-1 and Average Weight per Tuber in grams. For average weight of tubers two significant interactions were observed only between the nitrogen and potassium factors. Significant effects were observed only for the treatments nitrogen plus potassium, without any interaction with fertirrigtation (surface and subsurface). For Commercial Production no significant interactions were observed between the factors nitrogen and potassium treatments. It was concluded that it is possible to produce potato fertirrigated with the of a localized system, reagardless of being surface or subsurface. / O experimento foi implantado e conduzido no período de 02/03/2009 a 11/06/2009, nas dependências da Universidade José do Rosário Vellano em áreas do Setor de Olericultura e Experimentação, localizado no município de Alfenas (MG). O objetivo foi estudar a viabilidade de técnicas de fertirrigação na cultura da batata. O delineamento experimental foi em blocos ao acaso em esquema fatorial 2 x 3 x 3, totalizando 18 tratamentos com três repetições. Os fatores estudados foram: Fertirrigação (duas formas: superficial e subsuperficial); Nitrogênio (três doses: 75, 150 e 225 kgha-1); Potássio (três doses: 210, 310 e 410 kgha-1). As características estudadas foram: Produção Total Comercial em tha-1 e Peso Médio do Tubérculo em gramas. Para peso médio dos tubérculos, foram observadas interações significativas apenas entre os fatores nitrogênio e potássio. Os efeitos significativos foram observados apenas para os tratamentos doses de nitrogênio mais doses de potássio sem interação com as formas de irrigação. Não houve diferenças significativas para os tipos de fertirrigação (superficial e subsuperficial). Para a Produção Comercial não foram observadas interações significativas entre os fatores irrigação, nitrogênio e potássio. Os efeitos significativos foram observados apenas para os tratamentos doses de nitrogênio e doses de potássio. Concluiu-se que é possível produzir batata fertirrigada com uso de sistema de irrigação localizada independente de ser superficial ou subsuperficial.