Orientador: Carmen Silvia Fernandes Boaro / Banca: João Domingos Rodrigues / Banca: Giuseppina Pace P. Lima / Banca: Marcos Roberto Furlan / Banca: Antonio Natal Gonçalves / Resumo: Pertencente à família Meliaceae, a espécie Trichilia catigua A. Juss é conhecida popularmente como catigua, cataguá, argelim-rosa e mangalto-catingam. Sua casca apresenta propriedades adstringente, inseticida, purgativa, tônica, bactericida, antiinflamatória e antidepressiva. Com o objetivo de propagar a espécie T. catigua foram desenvolvidos experimentos testando o enraizamento de estacas e a micropropagação com explantes de matrizes e sementes. Os experimentos de enraizamento de estacas foram realizados na primavera 2004, verão 2004/2005, outono, inverno e primavera 2005 e primavera 2006. Em todos os experimentos, estacas com aproximadamente 15 cm de comprimento foram coletadas de árvores adultas e preparadas da parte apical e mediana dos ramos. A seguir, foram submetidas aos reguladores vegetais IBA (ácido indolbutírico), NAA (ácido naftalenoacético) e IAA (ácido 3-indolacético), variando as dosagens. Para a avaliação dos experimentos determinou-se a percentagem de estacas enraizadas, não enraizadas e mortas e quando enraizadas, seu comprimento e diâmetro. No experimento primavera de 2004 foram testadas as concentrações de 1000 e 2000 mg L-1 dos reguladores vegetais IBA, NAA e IAA. As avaliações aos 90 dias após sua instalação revelaram maiores percentagens de enraizamento e iguais a 33,33, 25,00, 22,91 e 23,43 % para estacas submetidas a IBA 1000, 2000 mg L-1 e NAA 1000 e 2000 mg L-1, respectivamente. No verão 2004/2005, outono, inverno e primavera 2005 os experimentos foram conduzidos com as concentrações dos reguladores IBA, NAA e IAA iguais a 1000, 2000 e 3000 mg L-1 e as avaliações foram realizadas após 120 dias. Não houve enraizamento no outono e inverno. A análise conjunta dos resultados obtidos na primavera e no verão mostrou percentagem de enraizamento superior na primavera. A maior percentagem de enraizamento, igual a 19,17%... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Trichilia catigua A. Juss from the Meliaceae family is popularly known as catigua, cataguá, argelim-rose and mangalto-catingam. Its bark has astringent, insecticide, purgativa, tonic, bactericide, anti-inflammatory and antidepressant properties. With the aim of propagate T. catigua, experiments of rooting with stem cuttings and of micropropagation with explants of trees and seeds were carried out. In all the rooting experiments the stem cuttings with approximately 15 cm of length were collected from adult trees and prepared from the apical and intermediate parts. The cuttings were immersed in the vegetable regulators IBA (Indole-3- butyric acid), ANA (Naphthalene acetic acid) and IAA (Indole-3 acetic acid). The rooted stem cutting and not rooted stem cutting percentage and, when rooted, the length and diameter of roots, were evaluated. In the experiment spring 2004 the concentrations of 1000 and 2000 mg L-1 of IBA, ANA and IAA were tested, with evaluations 90 days after installation. The highest rooting percentage were 33,33, 25,00, 22,91 and 23,43% for IBA 1000, 2000 mg L-1 and ANA 1000 and 2000 mg L-1, respectively. In the summer of 2004/2005, autumn, winter and spring of 2005 IBA, ANA and AIA, with concentration of 1000, 2000 and 3000 mg L-1, were tested. The evaluation was carried out at 120 days. No rooting was observed in autumn and winter. The analysis of data from summer and spring showed higher rooting percentage in spring. The highest rooting percentage was obtained with IBA 3000 mg L-1 (19,17%). In the spring 2006 IBA (1000, 2000, 3000, 4000 and 5000 mg L-1) and ANA (1000, 2000, 3000 mg L-1) were tested. The highest rooting percentage (41,67%) was obtained with IBA 5000 mg L-1. In the in vitro cultivation, explantes obtained from trees were submitted to asepsis treatments with HgCl2, CaOCl2 and NaOCl and inoculated in Murashige & Skoog culture medium (MS) with 25%... (Complete abstract click electronic access below) / Doutor

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No. of bitstreams: 1 nunes_gb_me_jabo.pdf: 1276204 bytes, checksum: 3aba64a51e863966cadeab46cfa01561 (MD5) Previous issue date: 2018-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este estudo foi desenvolvido com o objetivo de avaliar o efeito do precursor do peptídeo natriurético tipo C (NPPC) durante o cultivo de pré-maturação in vitro (pré-MIV) de oócitos bovinos sobre: 1) a progressão da maturação nuclear; 2) a expressão gênica das células do cumulus e 3) a aquisição da competência do oócito para o desenvolvimento embrionário in vitro. Os complexos cumulus-oócito (CCOs) foram pré-MIV com 100 nM NPPC por 8 horas (grupo NPPC) e, ao final do período, foram lavados para completa remoção do NPPC e submetidos à MIV por 22h. Após 8h de pré-MIV e após 8h de pré-MIV seguidas de 22h de MIV (duração total do cultivo = 30h) foram avaliadas a progressão da maturação nuclear e a expressão relativa de mRNA nas células do cumulus. O grupo controle (C) foi maturado na ausência de NPPC por até 30h, e as mesmas avaliações anteriores foram realizadas imediatamente após a remoção do ambiente folicular (C0), após 8h de cultivo (C8), após 22h de cultivo (C22) e após 30h de cultivo (C30). Em outro experimento, cujos tratamentos foram idênticos aos supramencionados, os oócitos foram fecundados ao término da MIV e foi avaliado o desenvolvimento embrionário até a fase de blastocistos. Após 8h de cultivo de pré-MIV, a análise da progressão da meiose demonstrou que o grupo C0 apresentou 58,9% de estruturas com configuração de GV1, enquanto que o grupo C8 apresentou apenas 13,9% de estruturas nesta configuração (P<0,05). A proporção de GV1 no grupo NPPC foi semelhante a ambos estes grupos (22,8%; P>0,05). Por outro lado, o grupo C8 apresentou maior taxa de GV3 em relação ao C0 (53,1% vs. 3,62%; P<0,05), sem diferenças com o grupo NPPC (38,7%; P>0,05). Ao final do cultivo de MIV, não foi observada diferença entre os grupos (C22 vs. NPPC vs. C30) com relação à maturação nuclear, todavia, houve maior taxa de oócitos degenerados no C30 em comparação com C22 (11,4% vs. 2,8%; P<0,05). A análise da expressão relativa de genes das células do cumulus após 8h de pré-MIV com NPPC evidenciou aumento (P<0,05) na expressão de genes relacionados à expansão destas células (GREM1, PTGS2/COX2, PTX3, TNFAIP6 e VCAN), à maturação oocitária (BDNF, EGFR, NOS3, PDE5A, PRKCD e STAT3) e ao desenvolvimento embrionário (IGF1R, KRT8 e LUM). Ao final do cultivo de MIV, observou-se no grupo NPPC que o gene PTX3, relacionado à expansão das células do cumulus, além dos genes AREG e BDNF, relacionados à maturação oocitária, e o gene LUM, relacionado ao desenvolvimento embrionário estavam mais expressos em comparação com o grupo C30 (P<0,05). Com relação ao desenvolvimento embrionário, os grupos experimentais não apresentaram diferença (P>0,05) quanto à taxa de clivagem (média de 73,22%). Embora o grupo NPPC não tenha diferido (P>0,05) de C22 e C30 quanto à taxa de blastocistos, houve diferença entre C22 e C30 (69,3 vs. 37,4; P<0,05). Todavia, não houve diferença entre os grupos com relação ao número de células totais (blastômeros) e apoptóticas (P>0,05). Em conclusão, o cultivo de pré-MIV de oócitos bovinos por 8h com 100nM NPPC não bloqueou a retomada da meiose, mas a progressão da meiose ocorreu de forma mais lenta e impediu o envelhecimento e degeneração dos oócitos. O cultivo de oócitos por tempo prolongado (30h) na ausência de NPPC foi prejudicial para o desenvolvimento embrionário, mas o tratamento com NPPC (8h pré-MIV+22h MIV = duração total de 30h) reverteu parcialmente este índice. / The aim of this study was to evaluate the effect of the C-type natriuretic peptide precursor (NPPC) during pre in vitro maturation culture (pre-IVM) of bovine oocytes on: 1) nuclear maturation progress; 2) gene expression in cumulus cells and 3) acquisition of competence for in vitro embryo development. Cumulus oocyte complexes (COCs) were pre-IVM with 100 nM NPPC for 8 hours (NPPC group) and then were washed for the complete removal of NPPC and submitted to IVM for 22h. After 8h pre-IVM followed by 22h IVM (total culture time = 30h) oocytes were evaluated for nuclear maturation progress and cumulus cells for relative mRNA expression. Control group (C) was IVM in the absence of NPPC for up to 30h and the same evaluations were made immediately after follicle removal (C0) and after 8h (C8), 22h (C22) and after 30h of culture (C30). In another experiment with the same treatment, the oocytes were fertilized at the end of IVM and embryo development to the blastocyst stage was evaluated. After 8 hours of pre-IVM culture, meiosis progression analysis showed 58.9% of oocytes in GV1 configuration in C0, while C8 had only 13.9% (P<0.05). The GV1 rates in NPPC did not differ from any group (22.8%; P>0.05). On the other hand, C8 showed higher rates of GV3 in comparison with C0 (53.1% vs. 3.62%; p<0.05) and no differences compared to NPPC (38.7%; P<0.05). At the end of IVM culture, no differences between groups (C22 vs. NPPC vs. C30) were observed in nuclear maturation, however, higher rates of degenerated oocytes were observed in C30 in comparison with C22 (11.4% vs. 2.8%; P<0.05). The relative gene expression analysis in cumulus cells after 8h pre-IVM with NPPC showed an up-regulation in genes related to cumulus cells expansion (GREM1, PTGS2/COX2, PTX3, TNFAIP6 and VCAN), oocyte maturation (BDNF, EGFR, NOS3, PDE5A, PRKCD e STAT3) and embryo development (IGF1R, KRT8 and LUM). At the end of IVM culture, the cumulus cells expansion related gene, PTX3, the oocyte maturation genes, AREG and BDNF, and the embryo development gene, LUM, were up-regulated in NPPC in comparison with C30 (p<0.05). Regarding embryo development, the cleavage rates did not differ in experimental groups (mean around 73,22%). Besides, blastocyst rates did not differ between NPPC (P>0.05) and the other groups, but there was a difference between C22 and C30 (69.3 vs. 37.4%; P<0.05). There was no difference between the groups in total cell number (blastomeres) and apoptotic cells (P<0.05). In conclusion, the pre-IVM culture of bovine oocytes for 8h with 100nM NPPC did not block meiosis resumption, but meiosis progression occurred more slowly and prevented aging and degeneration of the oocytes. The prolonged time of oocyte culture (30h) in the absence of NPPC was detrimental to embryo development, but NPPC treatment (8h pre-IVM + 22h IVM = total duration of 30h) partially reversed this effect.

Orientador: Fernanda da Cruz Landim-Alvarenga / Coorientador: Anthony César de Souza Castilho / Banca: Mariana Fernandes Machado / Banca: Ester Siqueira Caixeta / Resumo: O fator de crescimento semelhante à insulina-1 recombinante-3 (IGF-LongR3), um análogo sintético do IGF-1 de maior biodisponibilidade, ainda não foi utilizado no meio de maturação in vitro (MIV) de complexos cumulus-oócito (CCOs). Portanto, o objetivo deste estudo foi avaliar e comparar os efeitos da adição de IGF-LongR3 e do fator de crescimento semelhante à insulina-1 (IGF-1) na MIV de CCOs bovinos, sobre a progressão meiótica, apoptose e expressão de genes nos oócitos (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGBFP4 e IGFBP5) e respectivas células do cumulus (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 e IGFBP5). Ovários bovinos foram coletados em abatedouro, sendo selecionados 739 CCOs após aspiração de folículos de 2-8mm de diâmetro. A MIV foi realizada em meio base de maturação contendo IGF-1 (100ng/mL), IGF-LongR3 (100ng/mL), e dois grupos controles: 0,1% de álcool polivinílico (PVA) ou 10% de soro fetal bovino (SFB), durante 22-24 horas em estufa a 38,5ºC e 5% de CO2. Posteriormente os oócitos foram desnudados e preparados para a técnica de TUNEL, coloração Hoechst 33342 e RT-qPCR, intencionando-se avaliar a apoptose, maturação nuclear e a expressão gênica, respectivamente. A análise estatística foi realizada por um modelo linear de efeitos mistos, o qual relacionou a mudança de estádio de metáfase 1 para metáfase 2 e a ausência de apoptose entre os grupos experimentais, pelo programa lmer4. Os testes ANOVA e Tukey foram utilizados para análise dos resultados obtidos pelo RT-qPCR. Ao final de dez réplicas de MIV foram avaliados 339 (n= 5 réplicas) oócitos quanto à progressão meiótica e apoptose e 400 (n= 5 réplicas) quanto à expressão gênica. Não foi observada diferença significativa (P<0,05) entre os grupos experimentais com relação à progressão meiótica e apoptose. Foi possível detectar a expressão de mRNA para todos os genes avaliados nos oócitos e respectivas células... / Abstract: The insuline like growth factor-1 recombinant-3 (LongR3-IGF-1) a synthetic analogue of IGF-1 with greater bioavailability has not yet been used in in vitro maturation medium of cumulus-oocyte complexes (COCs). Therefore the aim of this study was to evaluate and compare the effects of LongR3-IGF-1 and insulin-like growth factor-1 (IGF-1) addition in the IVM of bovine oocytes on meiotic progression, apoptosis, and genic expression COCs (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGBFP4 e IGFBP5) and their respectively cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 e IGFBP5). Bovine ovaries were collected in slaughterhouse being selected 739 oocytes after aspiration of follicles from 2-8mm diameter. In vitro maturation (IVM) was performed on basic maturation medium containing IGF-1 (100 ng/ml), LongR3-IGF-1 (100ng/ml), and two control groups: 0.1% polyvinyl alcohol (PVA) or 10% fetal bovine serum (FBS), for 22-24 hours in an incubator at 38.5°C and 5% CO2. Subsequently oocytes were denuded and prepared for TUNEL technique, staining Hoechst 33342 and RT-qPCR, intending to evaluate apoptosis, nuclear maturation and gene expression, respectively. Statistical analysis was performed using a linear mixed effects model, which correlated the change in metaphase stage 1 to 2 and the absence of apoptosis among the experimental groups. ANOVA and Tukey tests were used to analyze the results obtained by RTqPCR. After ten replicas of IVM, 339 oocytes (n=5 pools) were evaluated for meiotic progression and apoptosis and 400 (n=5 pools) for gene expression. There was no statistical difference (P>0,05) between the experimental groups with respect to meiotic progression and apoptosis. It was possible to detect mRNA expression of all evaluated genes in the oocyte and its cumulus cells in all experimental groups. There was statistical difference between the group 10% FBS and IGF-1 and LongR3-IGF-1 groups for the expression of gene IGFBP4 in ... / Mestre

Successful efficient whole cauliflower plant regeneration via somatic embryogenesis from root derived callus tissue was achieved. The research confirmed for the first time the capability of mass production of cauliflower somatic embryos through the indirect pathway. The best callus induction and proliferation was on semi solid Murashige and Skoog (MS) medium supplemented with 2, 4-D at 0.15 mg L-1 and Kinetin at 0.1 mg L-1 and 3% sucrose. The response of different explant types (cotyledon, hypocotyls and root) through callus induction and subsequent culture was determined. The best period for subsequent callus culture was 21 days. Continuous immersion in agitated liquid medium technique was subsequently used for primary somatic embryo production. The culture requirements were empirically optimized including: explants source and size of callus tissue, blending duration, plant growth regulator combinations and concentrations as well as carbohydrate type and concentration. The highest mean number of somatic embryos (30.9) per explant was achieved using root derived embryogenic callus tissue on MS medium provided with IAA 0.05 mgL-1 and Kinetin at 0.5 mgL-1 and 2% sucrose. Somatic embryos were developed and matured on this medium and germinated with the highest percentage (60%) on semi-solid MS medium devoid of growth regulators. The culture conditions that led to the formation of secondary somatic embryos were identified. The presence of activated charcoal in the culture medium had an effect on this process but some abnormality of secondary somatic embryos was observed. Artificial seeds were produced by encapsulating the somatic embryos with a sodium alginate gel (2%) and complexing with calcium chloride (100 mM) for 20 min. The ability of these artificial seed for germination was evaluated using various combinations of plant growth regulators that were either incorporated in the artificial matrix or in the germination semi-solid culture medium. It was confirmed that cauliflower root derived embryogenic callus tissue can be cryopreserved following a preculture-dehydration technique. Following cryopreservation, embryogenic cultures can proliferate in agitated liquid medium, and somatic embryos at the globular stage were formed. Also cold storage at 5 °C in the dark was used successfully to store cauliflower callus tissue for three months without diminution of the competence for somatic embryos formation. This ability for cold storage could have a positive effect in reducing costs and efforts that result from subsequent sub-culture. The encapsulation-dehydration technique was assessed for cryopreservation of somatic embryos but failed to lead to survival of any embryos. Somatic embryos that were produced in this study were able to be well acclimated using a reliable weaning procedure that achieved high rates of survival of plantlets and their subsequent growth to normal plants in the field was assessed. Morphological characteristics of somatic plants compared favourably with zygotic plants but although there was phenotypic similarity, some differences in plant height, curd size and time for curd maturity were observed.

Organic polymers S2218600, S2429901 and S2219200 (referred to as Polymer 1, Polymer 2 and Polymer 3, respectively) of varying toxic potential, designed for use in cosmetic aerosols, were used as model substances to predict inflammatory potential. In vivo inflammogenic potential was evaluated by assessment of inflammatory cell profile (alveolar macrophage (AM), polymorphonuclear neutrophil (PMN)) of broncho-alveolar lavage fluid (BAL) 24hrs after a single instillation of either 0.5 mg or 2 mg polymer in Sprague Dawley rats. Pro-inflammatory Minusil particles and non-inflammatory titanium dioxide (TiO2) particles were used as controls. For comparison, cultured rat NR8383 AM-like cells, human THP-1 monocyte cells or human monocyte derived macrophages were treated with polymer for 24 h and supernatants analysed for indicators of cytotoxicity and inflammatory mediator release. In addition, after 6 h treatment, gene changes in the rat lung tissue and also in the rat NR8383 alveolar macrophage cell line were assessed using microarray to analyse the entire rat genome. The in vivo studies showed that Polymer 1, Polymer 3 and Minusil caused significant PMN influx into BAL. Polymer 3 and Minusil caused a significant increase in AM number in BAL. Polymer 2 and TiO2 had no effect on BAL cell profile. BAL tumour necrosis factor-α (TNFα) and macrophage inflammatory protein-2 (MIP-2) levels were significantly increased following instillation of Polymer 3 and Minusil. Thus the polymers and particles were ranked for potential to cause pulmonary inflammation: Polymer 3 > Minusil > Polymer 1 > Polymer 2 > TiO2. In vitro studies using cultured rat NR8383 AM-like cells showed that the polymers and particles could be ranked similarly for cytotoxic potential and their ability to stimulate the release of both TNFα and MIP-2. Cultured human monocyte derived macrophages detected the pro-inflammatory abilities of Polymer 3, as measured by cytotoxic potential and ability to stimulate TNFα, interleukin-8 (IL-8) and macrophage inflammatory protein-1α (MIP-1α), however, did not detect the pro-inflammatory abilities of Polymer 1. Cultured human THP-1 cells predicted the pro-inflammatory effects of Polymer 3 in rat lungs using the cytotoxicity assay and by changes in IL-1β, MIP-1α and IL-10 levels. The human THP-1 cell line did not predict the pro-inflammatory effects of Polymer 1 that were observed the rat lungs. Electron spin resonance (ESR) detected free radicals produced by the pro-inflammatory polymers and particles which had the ability to break bonds in super-coiled DNA and deplete intracellular glutathione (GSH). Microarray analysis of the canonical pathways activated by the pro-inflammatory polymers, Polymer 1 and Polymer 3, showed that 3 similar pathways were significantly activated in the instilled rats and the rat NR8383 AM-like cells following treatment. ‘Xenobiotic metabolism’, ‘IL-10 signalling’ and ‘leukocyte extravasation signalling’ pathways were significantly changed by the pro-inflammatory polymers. Use of these cell model alternatives in an industrial setting will refine and reduce in vivo testing and as these models are further developed and used alongside future new alternatives they will provide a substantial contribution towards the replacement of animal testing.

published_or_final_version / abstract / Obstetrics and Gynaecology / Master / Master of Philosophy

No description available.

No description available.

One of the major limitations on research into the control of eukaryotic DNA replication has been the lack of any cell-free system that initiates DNA replication in vitro. The first part of the disseration describes the establishment of a eukaryotic system, derived from the activated eggs of the South African clawed toad, Xenopus laevis, that efficiently initiates and completes DNA replication in vitro. Using a variety of biochemical techniques I show that DNA added to the extract in the form of sperm nuclei is efficiently replicated over a period of 4 - 6 hours. Replication of nuclear DNA represents a single round of semiconservative, semidiscon-tinuous replication. The extract will also replicate naked DNA incubated in it, regardless of sequence, though less efficiently than nuclear templates. This is probably related to the unusual ability of the egg extract to assemble apparently normal interphase nuclei from any DNA molecule incubated in it Evidence is presented that initiation, rather than chain elongation, is the rate-limiting step for replication in vitro. In this and in other ways the cell-free system behaves as though it were an early embryo blocked in a single cell cycle. The second part of the dissertation describes experiments that examine the control of DNA replication in the extract The first set of experiments suggest that on replication, DNA is marked in some way so that it can no longer act as a substrate for further initiation. This provides a mechanism by which the template DNA is replicated precisely once per incubation in vitro (or per cell cycle in vivo). The second set of experiments investigate the relationship between nuclear assembly and the initiation of DNA replication in vitro. A novel method for quantifying DNA replication in intact nuclei using the nucleotide analogue biotin-11-dUTP is described. This technique reveals that although they are in the common cytoplasm of the egg extract, different nuclei start to replicate at different times. Entry into S-phase is characterised by a burst of many synchronous or near-synchronous initiations within individual nuclei. This means that nuclei act as independent and integrated units of replication in the cell-free system, and suggests a fundamental role for nuclear assembly in controlling DNA replication in vitro.

No description available.