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.

ABSTRACT The purpose of this project was to investigate monocarboxylate (i.e. pyruvate and lactate) transport in the preimplantation stage of embryo development. Much progress has been made over the last 15 years towards understanding preimplantation and peri-implantation embryo physiology, including metabolic preferences during this period. It is known that as the cells (blastomeres) of an embryo compact via tight junctions and the embryo differentiates into a blastocyst, a metabolic “switch” occurs to allow the blastocyst to take up glucose at a rapid rate, obtaining energy derived from glycolysis. Glucose transporter molecules have been identified and characterized during this period of development and a paradigm for glucose transport has been described. However, during the early cleavage stages (days 1-3 post-fertilization), the embryo preferentially derives its metabolic energy from the monocarboxylate pyruvate. Evidence for the expression of pyruvate transporter molecules (a family of proton-coupled monocarboxylate co-transporters, MCT) has only been indicated via some kinetic studies on pH homeostasis and PCR analysis for MCT expression, and results have been conflicting (Gibb et al., 1997, Harding et al., 1999, Herubel et al., 2002). This project aimed to clarify discrepancies in reports for mRNA expression of MCT and to enhance the understanding of monocarboxylate transport processes during preimplantation development by pioneering investigations into protein expression for various MCT isoforms. Transport kinetics for monocarboxylate, DL-lactate, were examined by measuring the uptake of radioactive [3H]-DL-lactate from the medium by two-cell embryos and blastocysts. It was discovered that blastocysts demonstrate significantly higher affinity for DL-lactate compared to zygotes (Km 20 + 10 v 87 + 35 mM lactate; p=0.03), which suggested that alterations in the expression of various MCT isoforms might be expected as the embryo developed to a blastocyst. The rate of transport showed a trend towards a decrease from the zygote to blastocyst stages, although this could not be confirmed as significant within the limitations of this experiment. Mouse embryos, both in vivo and in vitro-derived, were collected and pooled at the zygote, two-cell, morula and blastocyst stages of development. RNA purification, reverse-transcription and PCR were used to analyze the expression of the four best-characterized MCT isoforms. MCT1, MCT2 and MCT4 were all found to be expressed in oocytes and mouse embryos from the zygote through to the preimplantation blastocyst. MCT3, an isoform uniquely expressed in the retina, was not detected at any stage in embryos. Since glucose has been implicated in regulatory processes involving glucose transporter expression in mouse embryos (Pantaleon et al., 2005, Pantaleon et al., 2001), mRNA expression was examined in the presence or absence of glucose in the culture media to determine whether the same phenomena applied to MCT. It was discovered that MCT1 and MCT4 isoforms were responsive to glucose-deprivation as evidenced by a reduction in mRNA expression in compacted morula cultured from the zygote stage without glucose. When glucose-deprived embryos were exposed to a brief high concentration of glucose during the 4-cell stage of development and continued in culture without glucose, the expression of mRNA for MCT1 and MCT4 persisted post-compaction, demonstrating that glucose exposure is necessary for the continued expression of these two isoforms in the mouse blastocyst. MCT2 mRNA did not respond to the absence of glucose in this way, and mRNA expression persisted in either the presence or absence of glucose. To follow these analyses of MCT gene transcription during early embryo development, confocal laser scanning immunofluorescence and western blotting were used to identify the expression of MCT proteins at various stages of development. Culture in the presence or absence of glucose was again employed to determine whether the changes seen in mRNA expression were conveyed at the protein level. All three proteins were identified throughout preimplantation development, though their locations were uniquely different. MCT1 was notably absent from plasma membranes at all stages, and was detected diffusely within the cytoplasm. In expanding blastocysts MCT1 tended to concentrate in the cortical cytoplasm of blastomeres and staining was more intense in the polar trophectoderm. In this cytoplasmic location its function is unclear. MCT1 does not appear to be a key transporter of monocarboxylates into and out of the embryo, but it may have a role in shuttling pyruvate and lactate within the cytoplasm to maintain metabolic and redox homeostasis. In embryos cultured without glucose, the immunostaining intensity for MCT1 gradually decreased as morulae degenerated and died. Protein loss occurred from the morula stage onwards, whilst mRNA was already undetectable at this stage. This would indicate that glucose signals which maintain mRNA expression most likely operate at the level of gene activation/transcription with latent effects on protein expression. MCT4 appeared to be located on the plasma membranes of oocytes and 2-cell embryos and nuclear staining was evident throughout preimplantation development, however plasma membrane expression was not apparent in morulae and blastocysts. This is consistent with earlier kinetic evidence of a low affinity lactate transporter (Km 87 + 35 mM lactate) operating at the early preimplantation stages. MCT4 has the lowest affinity for lactate of all the characterized MCT to date. Kinetic data also suggests that a change might occur in MCT protein expression as the embryo progresses to a blastocyst with a higher affinity lactate transporter taking precedence, and the loss of MCT4 from the plasma membrane at these later stages supports this view. Similarly to MCT1, MCT4 mRNA expression was also found to be dependent on glucose exposure during the early preimplantation period, and embryos cultured entirely without glucose demonstrated a loss of MCT4 mRNA expression at the morula stage. MCT4 typically exists as a lactate exporter in glycolytic tissues and it most likely exports lactate from the embryo for pH and redox homeostasis during this period of development. Protein localization studies found MCT2 to be located on the plasma membranes of oocytes, zygotes, 2-cell embryos, and polarized to the surface of the outer blastomeres of morulae and blastocyst trophectodermal cells. Throughout preimplantation development, MCT2 protein co-localized with peroxisomal catalase in peroxisome-sized granules throughout the cells. Known to be a high affinity pyruvate transporter, given its location in embryos it was proposed here that MCT2 most likely imports pyruvate to fuel early embryos, and later works as a bifunctional pyruvate/lactate importer/exporter on the transporting epithelium (trophectoderm) of blastocysts to maintain the pH, redox and metabolic status of the embryo. MCT2 was an enigma to the other MCT. Its expression in the absence of glucose behaved in an opposite way to that of MCT1 and MCT4, with mRNA expression persisting in the absence of glucose. In fact, MCT2 and catalase proteins demonstrated a quantitative increase in embryos lacking glucose, and the increase in staining was noticed as an increase in the density of peroxisome-like structures (or peroxisome proliferation) within the embryo. As such, it was decided to investigate the possibility that peroxisome proliferators (Peroxisome Proliferator Activated Receptors, PPARs) were involved in the control of MCT expression in the same way that they are known to control the expression of catalase and other peroxisomal proteins. At this stage, no MCT isoforms had been identified as being under the control of PPARs, although it was known that their expression was most likely controlled at the level of transcription, with no translational or post-translational controlling elements. PPARα, one of three isoforms (α, γ and β/δ) was selected as a likely candidate given that it controls peroxisomal proliferation and fatty acid β-oxidation processes at the level of transcription in other tissues, and it was known to be upregulated in conditions of starvation and oxidative stress. PPARα mRNA was shown to be expressed in early cleavage preimplantation mouse embryos, but its expression was reduced in morulae and blastocysts. Further, lack of glucose led to persistence of PPARα mRNA expression at the morula stage. PPARα protein was also demonstrated to stain more brightly in early preimplantation embryos compared to later stages. Further experimentation demonstrated that the phenomenon of increased catalase and MCT2 expression in embryos cultured without glucose could be mimicked in the presence of glucose by treating these embryos with the PPARα-selective agonist, WY14,643. The timing and quantitative nature of this upregulation were very similar, suggesting that PPARα was in some way involved in the glucose-deprived upregulation pathway for catalase and MCT2. To further investigate this pathway, oxidative stress was investigated in embryos cultured in the presence and absence of glucose to test whether the generation of reactive oxygen species contributed to the PPARα/MCT2 phenomenon. It was demonstrated that within 2 h of culture in the absence of glucose, hydrogen peroxide levels were significantly elevated in zygotes. Amelioration of increased peroxide generation in glucose-deprived embryos using a non-selective flavoenzyme inhibitor diphenyleneiodonium (DPI) eliminated any increases in PPARα and MCT2 protein expression that were earlier noted in the absence of glucose. To summarize, MCT1, MCT2 and MCT4 mRNA and protein expression were successfully demonstrated in mouse preimplantation embryos and all were confirmed to be in some way regulated by glucose in the culture medium. In the absence of glucose, mRNA expression for MCT1 and MCT4 were reduced to undetectable levels in morulae indicating that their expression was glucose-dependent. Paradoxically, glucose deprivation caused an increase in PPARα, catalase and MCT2 protein expression. PPARα-selective agonism in the presence of glucose induced similar timing and effects on catalase and MCT2 upregulation, implicating PPARα in this pathway. Hydrogen peroxide levels were significantly elevated within 2 h of culture in the absence of glucose. This peroxide elevation could be quenched to control levels by treating these embryos with DPI, and reducing hydrogen peroxide to control levels also eliminated the upregulation of PPARα and MCT2, implicating oxidative stress as an important component in the glucose-deprivation induced upregulation of MCT2. The experimental data presented in this thesis demonstrate that from its very conception, the embryo interacts with, adapts to, and is indeed affected by the external environment in which it develops. Even components like glucose, once considered simply as metabolic substrates, have profound effects on gene transcription and protein expression within the embryo which may impact on later its developmental competence, a reality we need to consider more deeply in light of the implementation of artificial reproductive technologies widely used today in zoology, agriculture and clinically, in humans.

Made available in DSpace on 2014-06-11T19:22:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-09Bitstream added on 2014-06-13T20:09:28Z : No. of bitstreams: 1 valentin_fn_me_jabo.pdf: 2224903 bytes, checksum: 63d93391228c3e9a48eb572d59e71cf8 (MD5) / Universidade Estadual Paulista (UNESP) / A tilápia é uma das espécies mais significativas para a piscicultura atual. Todo o conhecimento relativo à qualidade do ovo, pode melhorar as técnicas de reversão sexual, da qual depende o seu cultivo. Assim, a identificação estrutural dos eventos que ocorrem na reprodução induzida, fertilização e desenvolvimento embrionário, numa faixa de tempo conhecida, contribuirá para uma melhor compreensão da reprodução da tilápia do Nilo e será útil tanto no manejo como no controle da espécie. O objetivo deste trabalho foi analisar o efeito da idade de matrizes sobre as taxas de fecundidade, de fertilidade e de eclosão, bem como sobre a morfometria, a morfologia estrutural e a ultra-estrutural dos ovos da tilápia do Nilo (Oreochromis niloticus), desde as primeiras clivagens até a eclosão. A reprodução induzida foi realizada em dois tratamentos: grupo 1 (2 a 3 anos) e grupo 2 (3 ½ a 4 anos), os exemplares eram pertencentes ao setor de Tilapicultura do Centro de Aqüicultura da Unesp. As amostras dos ovos foram coletadas nos tempos de extrusão, fertilização, 1 minuto, 45 minutos, 60 minutos, de 30 em 30 minutos até completar seis horas, depois de hora em hora até completar 24 horas. A partir daí, as coletas foram realizadas de seis em seis horas até a eclosão. Para observações em estereomicroscópio, morfometria dos ovos e microscopia eletrônica de varredura, as amostras foram fixadas em solução de Karnovsky, lavadas em tampão Cacodilato de Sódio e conservadas em álcool 70 %. Nos dois grupos estudados, o período embrionário se estendeu de 60 a 84 horas, desde a fertilização até a eclosão, a uma temperatura média de 30,4 ± 0,75 ºC. Em relação ao tamanho dos ovos, não houve diferença significativa entre os dois grupos, apresentando diâmetro médio de 2,58mm e 2,57mm para os ovos do grupo 1 e 2, respectivamente... / Tilapia is one of the most significant species for the fish crops nowadays. All the knowledge related to egg quality can increase the sexual reversion techniques, in which its cultivation is needed. Thus, the structural identification of the events that occur on the inducted reproduction, fertilization, and embryo development, in a known time range, will contribute to a better comprehension of the reproduction of the Nile tilapia and will be useful either in the management as well as in the species control. The goal of this research was to analyze the effect of the matrix age on the fecundity, fertility and eclosion rates, as well as about the morphometry, structural and extra-structural morphology of the Nile tilapia (Oreochromis niloticus) eggs, since the first cleavage until the eclosion. The inducted reproduction was performed in two treatments: group 1 (2 to 3 years old) and group 2 (3 ½ to 4 years old), the samples pertaining to the sector of Tilapia crop of the Center of Water Crops from Unesp (State University of São Paulo). The egg samples were collected in the periods of extrusion, fertilization, 1 minute, 45 minutes, 60 minutes, every 30 minutes until complete six hours, and after every one hour until complete 24 hours. From then, the collects were performed every six hours until eclosion. For the observation in stereomicroscopy, eggs morphometry, and scanning electronic microscopy the samples were fixed in a solution of Karnovsky, washed in cacodilate of sodium buffer and conserved in alcohol 70%. In both studied groups, the embryo period has extended from 60 to 84 hours, since fertilization until eclosion, in an average temperature of 30.4 ± 0.75ºC. When it comes to the eggs size, no significant differences between both groups were presented, showing an average diameter of 2.58mm and 2.57mm for the eggs in group 1 and 2, respectively ...(Complete abstract, click electronic access below)

Orientador: Laura Satiko Okada Nakaghi / Banca: Heid Sueli Leme dos Santos / Banca: Teresa Cristina Ribeiro Dias Koberstein / Resumo: A tilápia é uma das espécies mais significativas para a piscicultura atual. Todo o conhecimento relativo à qualidade do ovo, pode melhorar as técnicas de reversão sexual, da qual depende o seu cultivo. Assim, a identificação estrutural dos eventos que ocorrem na reprodução induzida, fertilização e desenvolvimento embrionário, numa faixa de tempo conhecida, contribuirá para uma melhor compreensão da reprodução da tilápia do Nilo e será útil tanto no manejo como no controle da espécie. O objetivo deste trabalho foi analisar o efeito da idade de matrizes sobre as taxas de fecundidade, de fertilidade e de eclosão, bem como sobre a morfometria, a morfologia estrutural e a ultra-estrutural dos ovos da tilápia do Nilo (Oreochromis niloticus), desde as primeiras clivagens até a eclosão. A reprodução induzida foi realizada em dois tratamentos: grupo 1 (2 a 3 anos) e grupo 2 (3 ½ a 4 anos), os exemplares eram pertencentes ao setor de Tilapicultura do Centro de Aqüicultura da Unesp. As amostras dos ovos foram coletadas nos tempos de extrusão, fertilização, 1 minuto, 45 minutos, 60 minutos, de 30 em 30 minutos até completar seis horas, depois de hora em hora até completar 24 horas. A partir daí, as coletas foram realizadas de seis em seis horas até a eclosão. Para observações em estereomicroscópio, morfometria dos ovos e microscopia eletrônica de varredura, as amostras foram fixadas em solução de Karnovsky, lavadas em tampão Cacodilato de Sódio e conservadas em álcool 70 %. Nos dois grupos estudados, o período embrionário se estendeu de 60 a 84 horas, desde a fertilização até a eclosão, a uma temperatura média de 30,4 ± 0,75 ºC. Em relação ao tamanho dos ovos, não houve diferença significativa entre os dois grupos, apresentando diâmetro médio de 2,58mm e 2,57mm para os ovos do grupo 1 e 2, respectivamente ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tilapia is one of the most significant species for the fish crops nowadays. All the knowledge related to egg quality can increase the sexual reversion techniques, in which its cultivation is needed. Thus, the structural identification of the events that occur on the inducted reproduction, fertilization, and embryo development, in a known time range, will contribute to a better comprehension of the reproduction of the Nile tilapia and will be useful either in the management as well as in the species control. The goal of this research was to analyze the effect of the matrix age on the fecundity, fertility and eclosion rates, as well as about the morphometry, structural and extra-structural morphology of the Nile tilapia (Oreochromis niloticus) eggs, since the first cleavage until the eclosion. The inducted reproduction was performed in two treatments: group 1 (2 to 3 years old) and group 2 (3 ½ to 4 years old), the samples pertaining to the sector of Tilapia crop of the Center of Water Crops from Unesp (State University of São Paulo). The egg samples were collected in the periods of extrusion, fertilization, 1 minute, 45 minutes, 60 minutes, every 30 minutes until complete six hours, and after every one hour until complete 24 hours. From then, the collects were performed every six hours until eclosion. For the observation in stereomicroscopy, eggs morphometry, and scanning electronic microscopy the samples were fixed in a solution of Karnovsky, washed in cacodilate of sodium buffer and conserved in alcohol 70%. In both studied groups, the embryo period has extended from 60 to 84 hours, since fertilization until eclosion, in an average temperature of 30.4 ± 0.75ºC. When it comes to the eggs size, no significant differences between both groups were presented, showing an average diameter of 2.58mm and 2.57mm for the eggs in group 1 and 2, respectively ...(Complete abstract, click electronic access below) / Mestre

No intuito de descrever a evolução do desenvolvimento do concepto bubalino entre 10 e 60 dias de gestação, esse estudo utilizou a metodologia de mensuração por Crow rump para revelar a idade estimada de 96 embriões e fetos coletados no Matadouro municipal de Macapá, no estado do Amapá entre os anos de 2006 a 2008. Os parâmetros utilizados consistiram em medidas de comprimento (crânio caudal) e peso utilizando-se balança eletrônica de precisão. Para visualização macroscópica foram feitas fotografias, e a microscopia eletrônica de varredura auxiliou na identificação de inúmeras estruturas externas dos embriões. Foram realizados cortes histológicos de 5µm os quais foram corados em HE e picrosirius, e submetidos a técnicas de imunohistoquimica para detecção de Oct4, PCNA e vimentina. Nossos resultados revelam que embriões de mamíferos até a 5° semana de gestação são muito semelhantes aos de outras espécies de mamíferos já estudados. Similaridades entre bovinos e bubalinos persistem com exceção dos estágios fetais onde aparentemente búfalos se desenvolvem mais rapidamente que bovinos. Em conclusão, o estudo indica características importantes que podem ser utilizadas para verificar a viabilidade de embriões de búfalos e auxiliar avaliações em exames complementares como os de ultrassonografia, e também indicam a presença de importantes sítios de células pluripotentes em regiões diferentes do embrião dependendo do estágio de desenvolvimento em que o mesmo se encontra. / The aim of this study was to describe the developmental changes in the bubaline conceptus from 10-60 days of gestation using the Crown rump methodology to diagnostic the estimated age of those 96 embryos and fetuses obtained at Matadouro Municipal de Macapá, on Amapá state, from 2006 until 2008. Parameters used were cranio-caudal length (CR) and weight. For macroscopic view photographies were done, Scanning electron microscopy helps to identify inumerous external structures of the embryos. Histotogic section of 5µm were done and stained using Hematoxilin-Eosin, picrosirius, and also subjected to immunohistochemistry for Oct4, PCNA and vimentin were evaluated. Transmission electron microscopy was used in fetal membranes to describe it better. The obtained results revealed that mammal embryos until 5th weeks of gestation has to much similar characteristics to others studied species. Similarities between bovine and bubaline persist; except on fetal stages that buffalos seems develop faster than bovine ones. In conclusion, the overall data indicated the important characteristics that can be evaluated to verify the viability of buffalo embryos and help the evaluations of ultrasonographic exams, also identify the presence of important regions with pluripotente cells that changes according to the stage of the embryo development.

Dissertação(mestrado)-Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2008. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-07-22T21:55:11Z No. of bitstreams: 1 dissertaao shei.pdf: 955699 bytes, checksum: 8f9882265e94129eca93eb60bbe7cdf7 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-07-27T19:09:22Z (GMT) No. of bitstreams: 1 dissertaao shei.pdf: 955699 bytes, checksum: 8f9882265e94129eca93eb60bbe7cdf7 (MD5) / Made available in DSpace on 2012-07-27T19:09:22Z (GMT). No. of bitstreams: 1 dissertaao shei.pdf: 955699 bytes, checksum: 8f9882265e94129eca93eb60bbe7cdf7 (MD5) Previous issue date: 2008 / O “neon goby” Elacatinus figaro é endêmico da costa brasileira e foi uma das espécies mais importantes no comércio de peixes ornamentais marinhos do país. Atualmente, encontra-se na lista de espécies ameaçadas de extinção e resguardadas do extrativismo. Este trabalho teve como intuito descrever a reprodução, o desenvolvimento embrionário e a larvicultura do “neon goby” em laboratório. As primeiras desovas naturais foram observadas a partir do 24° dia após a formação dos casais, sendo o intervalo entre as desovas de 8 a 10 dias a 26°C. A fecundidade variou de 430 a 1.020 ovos por desova com média de 648 ± 183 ovos (média ± desvio padrão1, com taxa de eclosão média de 69 ± 16% Os ovos são elípticos, medem 1,81 ± 0,1 mm de comprimento e 0,61 ± 0,03 mm de diâmetro, eles apresentam filamentos adesivos na parte basal e contêm cinco protuberâncias na parte distal. O tempo para eclosão das larvas é de 7 a 8 dias. As larvas recém eclodidas medem 3,15 ± 0,07 mm, apresentam fototaxia positiva, olhos pigmentados, boca aberta e vesícula gasosa inflada. As larvas foram alimentadas com rotíferos Brachionus plicatilis em sistema de água verde com a microalga Nannochloropsis oculata do 1° ao 20° dia após a eclosão. Náuplios de Artemia foram oferecidos a partir do 15 ° dia em conjunto com rotíferos e, exclusivamente, a partir do 20° dia. O assentamento ocorreu a partir do 28° dia, quando as larvas do “neon goby” atingiram comprimento de 8,50 ± 0,18 mm com sobrevivência entre 2 e 20%. / The “neon goby” Elacatinus figaro is endemic of the Brazilian coast and was one of the most important species in the marine ornamental fish trade in the country. Today it is on the list of endangered species and it can no longer be collected in the wild. This work was carried out in order to describe natural breeding, embryonic development and larviculture of neon goby in laboratory. The first natural spawning was observed 24 days after the pairs were formed 8 to 10 days at 26°C. Mean fecundity was 648 ± 183 (± SD) eggs per clutch and ranged from 430 to 1020 eggs, the hatching rate was 69 ± 16%. Eggs are elliptical in shape, they present adhesive filaments at the proximal end and five protuberances at the distal end. Egg length averaged 1.81 ± 0.1 mm and maximum width was 0.61 ± 0.03 mm. The embryonic developmental period lasts between 7 and 8 days. The newly hatched larvae measure 3.15 ± 0.07 mm, showing positive phototaxis, pigmented eyes, mouth opened and insufflated gas bladder. Larvae were fed with rotifers Brachionus plicatilis in green water system along with the microalgae Nannochloropsis oculata from day 1 to day 20 day after hatching. Artemia nauplii were added to the diet on day 15, and 5 days later rotifers were no longer offered. Larvae began to settle to the bottom on day 28, when length of neon goby was 8.50 ± 0.18 mm, the final survival rate ranged between 2 and 20%.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-22T11:15:04Z No. of bitstreams: 2 Tese - Mariana Alves Mesquita - 2017.pdf: 2201375 bytes, checksum: 148d30a33a0e8c8b215e89ab657bd8d2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-22T11:15:40Z (GMT) No. of bitstreams: 2 Tese - Mariana Alves Mesquita - 2017.pdf: 2201375 bytes, checksum: 148d30a33a0e8c8b215e89ab657bd8d2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-22T11:15:40Z (GMT). No. of bitstreams: 2 Tese - Mariana Alves Mesquita - 2017.pdf: 2201375 bytes, checksum: 148d30a33a0e8c8b215e89ab657bd8d2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-06-01 / Two experiments were conducted at North Carolina State University to elucidate the hypothesis that exposing broiler embryos to monochromatic lights during incubation period may influence embryonic growth and development, improving incubation traits and posthatching period. In the first experiment, intermittent green light, continuous green light, intermittent red light and dark were evaluated. In addition, the influence of egg position in the tray was also tested. In the second experiment, monochromatic green light exposure was evaluated during different phases of incubation period. Treatments were: 1) 21d of incubation in the dark; 2) 21d of incubation with green light; 3) dark until E5 and continuous green light until hatch; 4) continuous green light until E5 and dark until hatch; 5) dark until E18 and continuous green light until hatch; 6) continuous green light until E18 and dark until hatch. Machine temperatures were adjusted daily in order to obtain optimal eggshell temperature (99,5 a 100,4°F) during the whole incubation period. In the first trial, embryo mortality in intermediate phase was higher in the group exposed to intermittent green light. Relative proventriculus weight was influenced by light stimuli, and the group that was not exposed to light showed the greatest organ development. Feed intake was also influenced by light treatments at 21 d of age. In the second trial, exposing embryos to continuous green light at different moments during incubation period affected the percentage of hatch and hatch of fertile. In addition, dead pipped was higher in the group exposed to light until E18. Liver weight was higher in chicks incubated in the dark during 21 d. Feed conversion ratio at 35 and 42 days was also influenced by light treatments, and the group that was not exposed to light during the whole incubation period had the lower feed conversion ratio. Significant interaction was observed between light stimuli and sex for breast yield. Male chicks that were light stimulated until E18 had the lowest breast yield. Comparing sexes, the highest breast yield was observed for females that were light stimulated until E18 during incubation. In conclusion, with incubation conditions that were established during the experiments, exposing broiler embryos to monochromatic lights did not promote benefits in incubation traits and also did not influenced chick growth and development in posthatching period. / Foram realizados dois ensaios experimentais na North Carolina State University com o objetivo de elucidar a hipótese de que a exposição de embriões de frangos de corte à luz monocromática durante o período de incubação pode influenciar o crescimento e desenvolvimento embrionário promovendo melhorias no rendimento da incubação e no período pós-eclosão. No primeiro experimento avaliou-se a estimulação com luz monocromática verde intermitente, verde contínua, vermelha intermitente e ausência de luz. Foi testado também a influência da posição dos ovos no interior da bandeja em relação a fonte de luz. No segundo experimento, avaliou-se a exposição deovos à luz verde monocromática durante diferentes fases do período de incubação. Os tratamentos estudados foram: 1) 21 dias de incubação no escuro; 2) 21 dias de incubação com luz verde contínua; 3) escuro até o quinto dia de desenvolvimento embrionário (E5) e luz verde contínua até o nascimento; 4) luz verde contínua até E5 e escuro até o nascimento; 5) escuro até o décimo oitavo dia de desenvolvimento embrionário (E18) e luz verde contínua até o nascimento; 6) luz verde contínua até E18 e escuro até o nascimento. As temperaturas das máquinas, em ambos os experimentos foram reguladas diariamente com o objetivo de se obter uma temperatura ótima da casca do ovo (99,5 a 100,4°F) durante todo o período de desenvolvimento embrionário. No primeiro experimento, o percentual de mortalidade embrionária na fase intermediária foi superior no tratamento exposto à luz verde intermitente. O peso relativo do proventrículo foi influenciado pelos estímulos luminosos, sendo que o grupo que não foi exposto a luz, o que apresentou maior desenvolvimento do órgão. O consumo de ração também foi influenciado pelos tratamentos estudados aos 21dias de idade. No segundo experimento, a exposição à luz verde contínua em diferentes momentos do período de incubação prejudicou os percentuais de eclosão e eclosão sobre férteis. Além disso, o percentual de bicados mortos foi superior no tratamento exposto à luz verde contínua até E18. O peso relativo do fígado foi superior nas aves que permaneceram no escuro durante os 21 dias de incubação. A conversão alimentar aos 35 e 42 dias de idade também foi influenciada pelos tratamentos estudados, sendo que o grupo que não foi exposto à luz durante todo o período de incubação o que apresentou melhor conversão alimentar. Houve interação entre estímulo luminoso e sexo para rendimento de peito, sendo que para machos o menor rendimento de peito foi observado no grupo que recebeu estímulo luminoso até E18. Comparando-se os sexos, constatou-se maior rendimento para fêmeas oriundas de incubação com estímulo luminoso até E18. Concluiu-se que nas condições de incubação estabelecidas durante a realização dos experimentos, a exposição de embriões de frangos de corte à luz monocromática não promoveu benefícios no rendimento da incubação e também não favoreceu o crescimento e desenvolvimento das aves no período pós-eclosão.

São preocupantes as doenças que tem o Aedes aegypti como vetor. A elevada competência vetorial deste mosquito, bem como sua antropofilia e adaptação ao ambiente urbano, geram habitats favoráveis e torna sua prevenção uma árdua tarefa considerando os recursos disponíveis. O controle atual enfatiza o mosquito, mas ainda há espaço para o aprimoramento da sua efetividade e sucesso, o que torna fundamental o maior conhecimento sobre este inseto. O Brasil apresenta condições favoráveis para a disseminação do mosquito. Diante do exposto este estudo visa estudar a vigilância entomológica do Aedes aegypti no Município de São Paulo no ano de 2013, bem como descrever as características morfológicas, morfométricas da fase de ovo e seu desenvolvimento embrionário. A análise da vigilância entomológica no Município de São Paulo, foi realizada a partir de dados secundários oriundos da Secretaria de Saúde do município. Já a etapa envolvendo a descrição morfológica, morfometrica e o desenvolvimento embrionário foi realizada a partir do material biológico cedido pelo Instituto de Ciências Biomédicas-USP utilizando recursos, tais como a Microscopia Eletrônica de Varredura, Microscopia Eletrônica deTransmissão e Microscopia Confocal. Os indicadores entomológicos Índice de Breteau (IB), Índice de Infestação Predial (IP) e Índice por Tipos de Recipientes Positivos (ITR), tiveram comportamento similares entre si nos anos estudados. Já o ITR revelou que o principal tipo de recipiente positivo para presença do vetor foi o prato/pingadeira para planta, seguido do vaso de planta, ou seja, os principais recipientes positivos encontram-se no ambiente domiciliar. Na avaliação do padrão morfológico e da mensuração acerca do comprimento, largura, razão comprimento/largura (índice) e diâmetro da micrópila constatou-se diferenças significantes entre as duas populações de ovos estudados, para cada um dos atributos morfométricos avaliados. Já a morfologia e o desenvolvimento embrionário apontam exocórion de grande resistência e de difícil permeabilidade, dificultando o acesso ao embrião. Considera-se fundamental a avaliação integrada dos dados oriundos do levantamento entomológico e de pesquisas quanto a características deste vetor nas diversas fases do ciclo biológico, pois somente desta maneira é possível investir em áreas específicas e promissoras para elaboração de políticas públicas efetivas e com poder transformador da realidade. / The diseases that have Aedes aegypti as a vector are worrisome. The high vectorial competence of this mosquito, as well as its anthropophilia and adaptation to the urban environment, generate favorable habitats and makes its prevention an arduous task considering the available resources. The current control emphasizes the mosquito, but its effectiveness and success in containing its dispersion still needs to improve, which makes fundamental the greater knowledge about this insect. Brazil presents highly favorable conditions for the spread of the mosquito. In view of the above, this study aims to study the entomological surveillance of Aedes aegypti in the city of São Paulo in 2013, as well as the morphological, morphometric characteristics of the egg phase and its embryonic development. The analysis of entomological surveillance was based on secondary data from health secretariats. The morphology, morphometry and embryonic development stages were performed using the biological material provided by the Institute of Biomedical Sciences of the University of São Paulo using resources such as Scanning Electron Microscopy (SEM), Transmission and Confocal. The entomological indicators Breteau Index (IB), Infestation Index (PI) and Index by Types of Positive Containers (ITR), had similar behavior among themselves in the years studied, since the ITR revealed that the main type of positive container for presence of the vector was dish / drip tray for plant followed by plant pot, the main positive containers are in the home environment. In the evaluation of the morphological pattern and measurement on length, width, length / width ratio (index) and diameter of the micropyle, it was observed significant differences between the two egg populations for each of the evaluated morphometric attributes. The morphology and embryonic development point to exochorion of big resistance and of difficult permeability, making difficult the access to the embryo. It is considered fundamental the integrated evaluation of data from the entomological survey and research on the characteristics of this vector in the various phases of the biological cycle, since only in this way is it possible to invest in specific and promising areas for the elaboration of effective public policies with transformative power of reality.

Infertility is a widespread problem around the world, although the available treatment options constantly improve through research and methodological development. A common treatment option mainly for biologically caused infertility is in-vitro fertilization, where in a menstrual cycle multiple oocytes are stimulated to mature and are then fertilized in a laboratory. This often results in multiple good quality embryos which can be cryopreserved through vitrification and used in a later cycle to increase the success chance of the treatment. The purpose of this pilot study was to evaluate vitrification and warming kits from Kitazato and Irvine Scientific regarding results and procedures. Human cleavage stage embryos (n=76) were thawed and cultured to the blastocyst stage. The blastocysts were scored according to Gardner and cryopreserved with vitrification and warming kits from either Kitazato (n=20) or Irvine Scientific (n=20). The warmed blastocysts were controlled after 2 and 4 hours for re-expansion and freeze injuries. The data was analysed with Fisher’s exact test and considered statistically significant if two-tailed p-value <0.05. The results showed no significant difference between the kits after 2 respectively 4 hours regarding re-expansion (p=0.432; p=0.492) or freeze injury (p=1.000; p=0.476). A significant difference was observed between group AB (with higher Gardner-score) and group C (with lower Gardner-score) in the degree of freeze injury (p=0.048; p=0.034), regardless of vitrification kit used. The details of the procedures differed somewhat between the kits, both having pros and cons, although overall procedures were equivalent. Further evaluation is needed before a change in method can be conducted.

L’autophagie est un processus vésiculaire des organismes eucaryotes permettant de véhiculer au sein d’autophagosomes des protéines dysfonctionnelles et/ou des organites défectueux qui sont apportés à la vacuole pour y être dégradés. Les acides aminés et les squelettes carbonés ainsi générés pourront ensuite être exportés vers le cytosol et recyclés. Ce travail de thèse a consisté à identifier les rôles que joue l’autophagie au cours du développement et de la germination de la graine d’Arabidopsis thaliana. Dans une première partie, les graines du mutant d’autophagie atg5 ont été caractérisées d’un point de vue morphologique et leur remplissage en molécules de réserve a été étudié. Il a notamment été montré que la graine mutante présente une maturation accélérée et accumule plus de protéines que la graine sauvage. Dans une seconde partie, l’expression de gènes ATG8 a été mise en évidence au cours du développement de l’embryon, dans le phloème de la silique et du funicule, dans les téguments externes et internes, ainsi que dans l’endosperme de la graine. L’activité autophagique a été visualisée par l’observation en microscopie de structures autophagiques dans l’embryon en développement. Enfin, dans une dernière partie, les rôles de l’autophagie au cours de la germination ont été étudiés via le suivi de la mobilisation des molécules de réserve chez le mutant atg5, comparativement à des graines sauvages. Il a ainsi été montré que la graine mutante présente un défaut de mobilisation des protéines. Les résultats obtenus montrent donc que l’autophagie jouerait différents rôles dans la graine, notamment dans sa maturation et son vieillissement, dans l’apport des nutriments depuis la plante mère jusqu’à l’embryon, et encore dans la constitution des réserves au cours du développement, puis leur mobilisation après la germination. / Autophagy is a vesicular process of eukaryotic organisms, which consists of the transport of dysfunctional proteins and/or defective organelles within auto phagosomes toward the vacuole in order to be degraded. The generated amino acids and carbon skeletons are transported to the cytosol and recycled. The aim of this thesis work was to identify the roles of autophagy during seed development and germination in Arabidopsis thaliana. In the first part, seeds of the atg5 autophagy mutant have been morphologically characterized in order to study the accumulation of storage molecules. We demonstrated that atg5 mutant seeds are affected by an accelerated maturation and accumulate more proteins than wild type seeds. In a second part, the expression of ATG8 genes has been exhibited during the embryo development, into the phloem of silique and funiculus, in the outer and the inner integument, and in the seed endosperm. Autophagic activity has been visualized by microscopy observation of autophagic structures in the developing embryo. Finally, in the last part, the roles of autophagy during germination have been studied by monitoring the mobilization of storage molecules in the atg5 mutant seeds and compared with the wild type. We thus established that mutant seeds are affected by a defect in protein mobilization. These results show that autophagy may play several roles in seeds, for instance in the ageing and maturation processes, in the transport of nutrients from the mother plant to the embryo, or in the constitution of storage compounds during seed development and their mobilization after germination.