Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-03-07T12:56:11Z No. of bitstreams: 1 MagdaLTS_DISSERT.pdf: 1454861 bytes, checksum: 4612469437c8dd924c24af03a052758d (MD5) / Made available in DSpace on 2017-03-07T12:56:11Z (GMT). No. of bitstreams: 1 MagdaLTS_DISSERT.pdf: 1454861 bytes, checksum: 4612469437c8dd924c24af03a052758d (MD5) Previous issue date: 2016-03-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The maintenance of metabolic activities during the in vitro culture of somatic cells of wild animals, especially collared peccary (Pecari tajacu), is an interesting step in the conservation of these cells for use in nuclear transfer (cloning). In this context, it is necessary to optimize the in vitro culture conditions of somatic cell by establishment of some appropriate supplementations to the media, in order to ensure the maximum preservation of the viable cell characteristics. Therefore, this study aimed to optimize the composition of the culture means of somatic cell derived from ear tissue of collared peccaries, evaluating two concentrations of fetal bovine serum (E1: FBS; 10% vs. 20%) and epidermal growth factor (E2: EGF, 5 ng/mL vs. 10 ng/mL). Thus, tissue fragments from 18 adult animals were submitted to primary culture and subcultures for 40 days until the fourth passage and the resulting cells were analyzed for morphology, adhesion, subconfluence, proliferative activity for developing growth curve for seven days and determining the population doubling time (PDT), viability by trypan blue and functional/metabolic activity by assay 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Moreover, in the E1, comparisons as cell adhesion were performed with cells cultured in the presence of bovine serum albumin (BSA, 0.5% and 1.0%). All data were analyzed by ANOVA followed by post hoc test. In the E1, no difference (P>0.05) was observed between the concentrations of FBS for the number of adhered [SFB10: 39/39 vs. SB20: 35/39] and subconfluent fragments [SFB10: 39/39 vs. SB20: 35/39], day all adhered [SFB10: 3.5 vs. SFB20: 3.0], with growth [SFB10: 7.4 vs. SFB20: 7.2] and subconfluent samples [SFB10: 11.8 vs. SFB20: 11.8]. However, significant values were observed in cells cultured in the presence of 20% FBS for viability [SFB10: 85.6% vs. SFB20: 98.2%], PDT [SFB10: 155.4 h vs.77.25 h] and MTT assay [SFB10: 0.57-0.57 vs. SFB20: 0.82-0.99 (D5-D7)]. Additionally, comparisons of supplementation of BSA and FBS confirm the potential FBS cell adhesion. Thus, 20% FBS was used in the following experiment. In the evaluation of the presence of EGF in culture, no difference was observed in the evaluated parameters for the number of attached [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] and subconfluent fragments [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] day all adhered [EGF0: 4.9 vs. EGF5: 7.0 vs. EGF10: 3.5] growth [EGF0: 7.2 vs. EGF5: 8.2 vs. EGF10: 7.9] and subconfluent samples [0 EGF: 12.6 vs. EGF5: 16.6 vs. EGF10:12.6], viability [EGF0: 84.3% vs. EGF5: 88.8% vs. EGF10: 87.0%], PDT [EGF0: 69.6 h vs. EGF5: 64.8 h vs. EGF10: 65.3 h] and MTT assay [EGF0: 1.26-1.38 vs. EGF5: 1.06-1.14 vs. EGF10: 1.13-1.16 (D5-D7)]. In all experiments, the growth curves showed clear log and lag phases of development. In conclusion, 20% FBS is suitable for the recovery of somatic cells in vitro; however, EGF does not improve the quality of growing these cells / A manutenção das atividades metabólicas durante o cultivo in vitro de células somáticas de animais silvestres, especialmente catetos (Pecari tajacu), representa uma etapa interessante na conservação dessas células para aplicação na transferência nuclear (clonagem). Nesse contexto, faz-se necessário a otimização das condições de cultivo in vitro de células somáticas pelo estabelecimento de algumas suplementações adequadas aos meios, visando garantir a máxima preservação das características celulares viáveis. Portanto, o presente trabalho teve como objetivo otimizar a composição do meio de cultivo de células somáticas derivadas de tecido auricular de catetos, avaliando duas concentrações de soro fetal bovino (E1: SFB; 10% vs. 20%) e fator de crescimento epidermal (E2: EGF; 5 ng/mL vs. 10 ng/mL). Para tanto, fragmentos teciduais de 18 animais adultos foram submetidos ao cultivo primário e subcultivos por 40 dias, até a quarta passagem e as células resultantes foram analisadas quanto à morfologia, aderência, subconfluência, atividade proliferativa pela elaboração de curva de crescimento por sete dias e determinação do tempo de duplicação da população (PDT), viabilidade por azul de tripano e atividade funcional/metabólica pelo ensaio de brometo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difeniltetrazólio (MTT). Além disso, no E1, comparações quanto à adesão celular foram realizadas com células cultivadas na presença de albumina sérica bovina (BSA, 0,5% e 1,0%). Todos os dados foram analisados por ANOVA seguido por teste post-hoc. No E1, nenhuma diferença (P>0,05) foi observada entre as concentrações de SFB para o número de fragmentos aderidos [SFB10: 39/39 vs. SB20: 35/39] e subconfluentes [SFB10: 39/39 vs. SB20: 35/39], dia de todas as amostras aderidas [SFB10: 3,5 vs. SFB20: 3,0], com crescimento [SFB10: 7,4 vs. SFB20: 7,2] e subconfluentes [SFB10: 11,8 vs. SFB20: 11,8]. Contudo, valores significativos foram observados em células cultivadas na presença de SFB a 20% quanto à viabilidade [SFB10: 85,6% vs. SFB20: 98,2%], PDT [SFB10: 155,4 h vs.77,2 h] e ensaio de MTT [SFB10: 0,57-0,57 vs. SFB20: 0,82-0,99 (D5-D7)]. Adicionalmente, comparações da suplementação do BSA e SFB confirmaram o potencial de adesão celular do soro. Assim, SFB a 20% foi empregado no experimento seguinte. Já na avaliação da presença de EGF no cultivo, nenhuma diferença foi observada nos parâmetros avaliados para o número de fragmentos aderidos [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31] e subconfluentes [EGF0: 31/31 vs. EGF5: 31/31 vs. EGF10: 31/31], dia de todas as amostras aderidas [EGF0: 4,9 vs. EGF5: 7,0 vs. EGF10: 3,5], em crescimento [EGF0: 7,2 vs. EGF5: 8,2 vs. EGF10: 7,9] e subconfluentes [EGF0: 12,6 vs. EGF5: 16,6 vs. EGF10: 12,6], viabilidade [EGF0: 84,3% vs. EGF5: 88,8% vs. EGF10: 87,0%], PDT [EGF0: 69,6 h vs. EGF5: 64,8 h vs. EGF10: 65,3 h] e ensaio de MTT [EGF0: 1,26-1,38 vs. EGF5: 1,06-1,14 vs. EGF10: 1,13-1,16 (D5-D7)]. Em todos os experimentos, as curvas de crescimento apresentaram nítidas fases log e lag de desenvolvimento. Em conclusão, o SFB a 20% é adequado para a recuperação de células somáticas in vitro; contudo, o EGF não melhora a qualidade do cultivo dessas células / 2017-03-07

Submitted by Lara Oliveira (lara@ufersa.edu.br) on 2017-08-08T21:28:55Z No. of bitstreams: 1 EdigleyceLC_DISSERT.pdf: 1495589 bytes, checksum: 331e814fbf35fd0903ce95ef191653df (MD5) / Approved for entry into archive by Vanessa Christiane (referencia@ufersa.edu.br) on 2017-08-17T22:39:03Z (GMT) No. of bitstreams: 1 EdigleyceLC_DISSERT.pdf: 1495589 bytes, checksum: 331e814fbf35fd0903ce95ef191653df (MD5) / Approved for entry into archive by Vanessa Christiane (referencia@ufersa.edu.br) on 2017-08-17T22:41:07Z (GMT) No. of bitstreams: 1 EdigleyceLC_DISSERT.pdf: 1495589 bytes, checksum: 331e814fbf35fd0903ce95ef191653df (MD5) / Made available in DSpace on 2017-08-17T22:41:14Z (GMT). No. of bitstreams: 1 EdigleyceLC_DISSERT.pdf: 1495589 bytes, checksum: 331e814fbf35fd0903ce95ef191653df (MD5) Previous issue date: 2015-02-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Aspidosperma pyrifolium Mart species. it is used in feed for farm ani-mals. However, there are few studies on the toxicity of this plant. This study aimed to iden-tify the existence of toxic effect, cytotoxic and mutagenic leaves of A. pyrifolium the test system Allium cepa and mice. It was made growth analysis of roots, inhibition relative and mitotic index with the Allium cepa test. For the LD50 they were used of groups mice were observed for 14 days to determine the number of dead and surviving animals. For the mi-cronucleus test and binucleated cells was done extracting bone marrow of animals. Bioas-says performed with the Allium cepa revealed that the aqueous extract of dried leaves showed no toxic effects, however, stimulated cell division in a concentration of 50 mg/L, is indicative of formation of tumor cells. The fresh leaves were toxic at the concentration of 300 mg/L and had a effect toxic sublethal on concentration of 5 mg/L.. In mice, the median lethal dose was 750.63 mg/kg with the aqueous extract of fresh leaves, and lethal concen-trations tested from the concentration 900 mg/kg in males and females. The evaluated ani-mals showed behavioral changes in the nervous system and change in average total weight. The concentrations tested indicated are not mutagenic or not cytotoxic in the overall analy-sis of micronucleus frequency and binucleated cells, respectively. But, this work provides data toxicity; cytotoxicity and mutagenicity still have not investigated for the species A. pyrifolium / A espécie Aspidosperma pyrifolium Mart. é utilizada na alimentação por animais de produção. No entanto, são escassos os estudos sobre a toxicidade dessa planta. Este trabalho objetivou identificar a existência de efeito tóxico, citotóxico e mutagênico de folhas de A. pyrifolium no sistema teste Allium cepa e em camundongos. Foi feita a análise de crescimento de raízes, inibição relativa e índice mitótico com o teste Allium cepa. Para a DL50 foram utilizados grupos de camundongos, observados por 14 dias para determinar a quantidade de mortos, doentes e sobreviventes. Para o teste de micronúcleo e células binucleadas foi feita a extração da medula óssea dos animais. Os bioensaios com o Allium cepa realizados revelaram que o extrato aquoso das folhas secas não mostrou efeito tóxico, contudo, estimulou a divisão celular na concentração de 50 mg/L, sendo indicativo de formação de células tumorais. As folhas frescas foram tóxicas na concentração de 300 mg/L e teve efeito tóxico subletal na concentração de 5 mg/L. Nos camundongos, a dose letal mediana foi de 750,63 mg/Kg com o extrato aquoso de folhas frescas, sendo letal nas concentrações testadas a partir da concentração de 900 mg/Kg em machos e fêmeas. Os animais avaliados apresentaram alterações comportamentais no sistema nervoso e alteração no peso médio total. As concentrações testadas indicaram não serem mutagênicas e nem citotóxicas na análise geral das frequências de micronúcleo e células binucleadas, respectivamente. Mas, este trabalho fornece dados de toxicidade, citotoxicidade e mutagenicidade ainda não investigados para a espécie A. pyrifolium / 2017-08-08

The overall objective of this study was to identify novel proteins of the nuclear matrix in order to contribute to a better understanding of nuclear structure and organization. To accomplish this, a monoclonal antibody specific for the nuclear matrix was used to screen a human λgt11 expression library. Several cDNAs were isolated, cloned, sequenced, and shown to represent NuMA, the nuclear mitotic spindle apparatus protein. Further characterization of the gene and RNA was undertaken in an effort to obtain information about NuMA. The NuMA gene was present at a single site on human chromosome 11q13. Northern and PCR analysis of NuMA mRNA showed a major 7.2 kb transcript and minor forms of 8.0 and 3.0 kb. The minor forms were shown to be alternatively spliced although their functional significance is not yet understood. Immunofluorescence microscopy demonstrated that NuMA oscillates between the nucleus and the microtubule spindle apparatus during the mitotic cell cycle. NuMA appeared as a 200-275 kDa protein detectable in all mammalian cells except human neutrophils. To determine whether NuMA's changes in intracellular distribution correlated with post-translational modifications, the protein's phosphorylation state was examined through the cell cycle using highly synchronized cells. NuMA was a phosphoprotein in interphase and underwent additional phosphorylation events in mitosis. The mitotic phosphorylation events occurred with similar timing to lamin B (G2/M transition) and were concomitant with NuMA's release from the nucleus and its association with the mitotic spindle. However, the mitotic phosphorylation occurred in the absence of spindle formation. Dephosphorylation of NuMA did not correlate with reassociation with the nuclear matrix but occurred in two distinct steps after nuclear reformation. Based on the timing of these events, phosphorylation may playa role in nuclear processes. In conclusion, the work in this dissertation identified NuMA, a nuclear matrix protein and showed that it is phosphorylated during the cell cycle and may be important for nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.

Cordia trichotoma (Vell.) Arráb. ex Steud., known as louro-pardo, belongs to the Boraginaceae family and is widely distributed in Brazil. In Rio Grande do Sul (RS), is found in the Alto Uruguai and Depressão Central. This is a large species, reaching 35 meters in height. It is appreciated for the quality of its wood, which has scenic value, and can be employed in the recovery of degraded areas; its leaves have medicinal value, which are used to prepare teas. These, in turn, are used to treat kidney diseases, rheumatism, arthritis and rickets. The Allium cepa test is used as a bioindicator of genotoxic and antiproliferative capacity of aqueous extracts (teas) prepared with different plant materials such as leaves, flowers, fruit and bark. This study aimed to evaluate the antiproliferative potential of infusions of four populations of C. trichotoma on the cell cycle of A. cepa, as well as to analyze the phenolic compounds in infusions. The leaves and fruits of louro-pardo, used in the assembly of the experiments, were collected in three municipalities in the RS. For experiment 1, teas (infusions ) leaves were used , and for the experiment 2, the fruits were used. Six groups of four onion bulbs rooting in water were used, which consisted of six treatments and four replications for each of the populations of C. trichotoma in both experiments. The treatments were: T1 - negative control in distilled water ; T2- infusion of 5 g.L-1 ; T3- infusion of 20 g.L-1 ; T4- infusion of 5 g.L-1 ; T5- infusion of 20 g.L-1 ; T6- positive control in glyphosate 2% . After treatments, the roots of A. cepa were fixed, conserved and then slides prepared by squashing technique, stained with 2% acetic orcein. Therefore, it were subsequently analyzed under a microscope 40x. It were counted 500 cells by bulb, totaling 2000 cells for each treatment and for each of the populations. The mitotic index was calculated as well as the verification of possible irregularities in the cell cycle. Phenolic compounds were determined by the technique of high performance liquid chromatography (HPLC). The results showed a statistically significant difference for the increased inhibition of cell division as the concentration of infusions of louro-pardo in both experiments, no genotoxicity was observed in extracts. Compounds that appeared in larger quantities were caffeic acid, rosmarinic acid and ellagic acid. C. trichotoma infusions prepared in different concentrations exhibit antiproliferative potential and have no genotoxic activity on the cell cycle of A. cepa. The results obtained in the area of genotoxicity as well as the phenolic found in C. trichotoma infusions constitute new information for this species. / Cordia trichotoma (Vell.) Arráb. ex Steud., conhecida como louro-pardo, pertence à família Boraginaceae e é de ampla distribuição geográfica no Brasil. No Rio Grande do Sul (RS), é encontrada nas regiões do Alto Uruguai e Depressão Central. Trata-se de uma espécie de grande porte, podendo chegar a 35 metros de altura. É apreciada pela qualidade de sua madeira, a qual possui valor paisagístico, e pode ser empregada na recuperação de áreas degradadas; suas folhas têm valor medicinal, as quais são utilizadas para o preparo de chás. Estes, por sua vez, são empregados no tratamento de doenças renais, reumatismo, artrite e raquitismo. O teste de Allium cepa é utilizado como bioindicador da capacidade antiproliferativa e genotóxica de extratos aquosos (chás) preparados com materiais vegetais diversos como folhas, flores, frutos e cascas. Este estudo teve como objetivos verificar o potencial antiproliferativo das infusões de quatro populações de C. trichotoma sobre o ciclo celular de A. cepa, bem como analisar os compostos fenólicos presentes nas infusões. As folhas e frutos de louro-pardo, utilizados na montagem dos experimentos, foram coletados em três municípios do RS. Para o experimento 1, foram usados chás (infusões) das folhas, e para o experimento 2, utilizaram-se os frutos. Foram usados seis grupos de quatro bulbos de cebola enraizando em água, os quais constituíram seis tratamentos com quatro repetições para cada uma das populações de C. trichotoma, em ambos os experimentos. Os tratamentos foram: T1- controle negativo em água destilada; T2- infusão de 5 g.L-1; T3- infusão de 20 g.L-1; T4- infusão de 5 g.L-1; T5- infusão de 20 g.L-1; T6- controle positivo em glifosato 2%. Após os tratamentos, as raízes de A. cepa foram fixadas, conservadas e então preparadas lâminas pela técnica de esmagamento, coradas com orceína acética 2%. Posteriormente, foram analisadas no microscópio com aumento de 40x. Foi realizada a contagem de 500 células por bulbo, totalizando 2000 células para cada um dos tratamentos e para cada uma das populações. Foi realizado o cálculo do índice mitótico, bem como a verificação de possíveis irregularidades no ciclo celular. Os compostos fenólicos foram determinados por meio da técnica de cromatografia líquida de alta eficiência (CLAE). Os resultados mostraram diferença estatística significativa para o aumento da inibição da divisão celular conforme o aumento da concentração das infusões de louro-pardo em ambos os experimentos, não tendo sido observada genotoxicidade nos extratos. Os compostos que apareceram em maior quantidade foram o ácido cafeico, o ácido rosmarínico e o ácido elágico. As infusões de C. trichotoma preparadas nas diferentes concentrações apresentam potencial antiproliferativo e não possuem atividade genotóxica sobre o ciclo celular de A. cepa. Os resultados obtidos na área de genotoxicidade, bem como os fenólicos encontrados nas infusões de C. trichotoma configuram informações inéditas para esta espécie.

Made available in DSpace on 2014-08-20T13:25:41Z (GMT). No. of bitstreams: 1 Tese_ Maribel_da_Silva_Mendes.pdf: 1278110 bytes, checksum: cd3b4973dbfd97e88a02745cadf7c130 (MD5) Previous issue date: 2008-08-29 / The environmental pollution with trace-elements (ETs) is an increasing problem in the modern society, having extreme importance the evaluation of these environmental risks. The use of seeds of superior plants is ideal for such tests since they are efficient, quick and of easy execution. The objectives of this work were evaluate the toxic effects on the germination and cytotoxics on the meristematics cells of Allium cepa L. and Lactuca sativa L. roots, under different concentrations of the trace-elements cadmium (0,5; 1,0; 3,0; 5,0; 7,0 and 9,0mg. L-1), arsenic (2,5; 5,0; 7,5; 10; 15; 20mg. L-1), lead (50, 100, 150, 200, 250 and 300mg. L-1), chromium (50, 100, 150, 200, 250 and 300mg. L-1) and mercury (0,25; 0,5; 1,0; 1,5; 2,0 and 2,5mg. L-1), after 168 and 48h of exhibition, respectively. In lettuce, de maximum number and classes of nucleolus per interphasic cell were observed. The results showed toxic effect of the traceelements on the germination of the seeds and Mitotic Index (IM), besides the induction of chromosomic aberrations in the meristematic cells of A. cepa L. The degree of toxicity and the different anomalies increased with the increase of ET concentration. In L. sativa it was evident that both the percentage of germination and the IM decreased with the increase of ETs concentration. The cytotoxicity caused by these ETs was demonstrated in the different chromosomic anomalies caused to the meristematic cells of this vegetable species. The presence of chrome, even in the lowest concentrations used in this study, impaired the events sequence of the germination process of lettuce seeds. Regarding the number of nucleolus, the studies demonstrated that, the lettuce has, at most, six (6) nucleolus per interphasic cell, even in the presence of the studied trace-elements. / A poluição ambiental com elementos-traço (ETs) é um problema cada vez maior da sociedade moderna, sendo a avaliação destes riscos ambientais de extrema importância. O uso de sementes de plantas superiores é ideal para tais ensaios porque são eficientes, rápidos e de fácil execução. Os objetivos deste trabalho foram avaliar os efeitos tóxicos na germinação e citotóxicos nas células meristemáticas das raízes de Allium cepa L. e de Lactuca sativa L. sob diferentes concentrações dos elementos-traço cádmio (0,5; 1,0; 3,0; 5,0; 7,0 e 9,0mg.L-1), arsênio (2,5; 5,0; 7,5; 10; 15; 20mg.L-1), chumbo (50, 100, 150, 200, 250 e 300mg.L-1), cromo (50, 100, 150, 200, 250 e 300mg.L-1) e mercúrio (0,25; 0,5; 1,0; 1,5; 2,0 e 2,5mg.L-1), após 168 e 48h de exposição, respectivamente. Observouse, em alface, o número máximo e as classes de nucléolos por célula interfásica. Os resultados mostraram efeito tóxico dos elementos-traço sobre a germinação das sementes e no Índice Mitótico (IM), além da indução de aberrações cromossômicas nas células meristemáticas de A. cepa. O grau de toxicidade e as diferentes anomalias aumentam com o aumento da concentração do ET. Em L. sativa ficou evidenciado que tanto a percentagem de germinação, como o IM decresceram com o aumento da concentração dos ETs. A citotoxicidade causada por esses ETs ficou demonstrada nas diferentes anomalias cromossômicas causadas às células meristemáticas dessa espécie vegetal. A presença de cromo, mesmo nas concentrações mais baixas utilizadas neste estudo, inviabilizou a seqüência dos eventos do processo de germinação das sementes de alface. Em relação ao número de nucléolos, os estudos demonstraram que, a alface possui, no máximo, seis (6) nucléolos por célula interfásica, mesmo na presença dos elementos-traço estudados.

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / Paspalum notatum, native grass biome Pampa, Rio Grande do Sul, has high forage value, but has difficulty germinating seeds with low viability. Inserted in this context are the promoters of germination and biomass, biostimulants stimulate® and bioagent Trichoderma spp. by mechanisms that influence the metabolism of action of the species. The present study investigated the promotion of seed germination and development of P. notatum. Through the Allium cepa test in ten treatments (four replicates per treatment), eight with metabolites of Trichoderma spp. and two controls in the absence of metabolites were selected isolates of trichoderma biological agent. The test of sanity, the identification of fungi associated with seeds used the methods of filter paper and plating on solid agar PDA (potato dextrose agar). With the in vitro technique of direct confrontation of the antagonist action on three fungal contaminants of highest incidence was observed. In a preliminary test, pre-treatment is selected to germination of P. notatum. Seed germination in greenhouse consisted of 16 treatments with four replicates, the control treatments, the three isolates, the stimulate®, the combination of each of the three isolates stimulate®, all with and without pretreatment, analysis of the data was through the Genes program. For reviews of morphogenesis, four were randomly chosen tillers per treatment, they monitored for the appearance and elongation and leaf senescence. This monitoring was performed two times per week. The top three trichoderma isolates selected in test A. cepa strain were 2B2, C1, 2B12. The removal of the seed coat structures of P. notatum was the pre-treatment showed the best performance with 34.5% twinned seeds. In the test of sanity the fungal genera Aspergillus, Curvularia, and Fusarium Geniculosporium were identified, isolated and 2B2 of Trichoderma spp. efficient for direct to these fungal genera confrontation. For the IVE (index emergency speed), the combination treatments of biological powder 2B12 isolated from trichoderma and biostimulating Stimulate® and only Stimulate® biostimulating with pretreatment and without pretreatment combination of biological powder isolated 2B2 Trichoderma with biostimulating Stimulate®, achieved the best results, with 14.48, 13.93 and 12.75, respectively. The biostimulating stimulate® promotes the rate of speed of emergence of seeds of P. notatum and the production of dry matter, the initial rate of elongation of the sheet (TAIF) positively interfere in the dry weight of shoots. / Paspalum notatum, gramínea nativa do bioma Pampa, Rio Grande do Sul, possui alto valor forrageiro, porém apresenta dificuldade de germinação de sementes, com baixa viabilidade. Inseridos neste contexto estão os promotores de germinação e biomassa, bioestimulante Stimulate® e o bioagente Trichoderma spp. que através de mecanismos de ação influenciam no metabolismo da espécie. O presente trabalho objetivou estudar a promoção da germinação das sementes e desenvolvimento de P. notatum. Através do teste de Allium cepa em dez tratamentos (quatro repetições por tratamento), oito com metabólitos de isolados de Trichoderma spp. e dois controles na ausência de metabólitos foram selecionados isolados do agente biológico trichoderma. O teste da sanidade, a identificação dos fungos associados às sementes utilizaram-se os métodos do papel de filtro e do plaqueamento em meio ágar sólido BDA (batata dextrose ágar). Com a técnica in vitro de confrontação direta foi observada a ação do antagonista sobre três fungos contaminantes de maior incidência. Em teste preliminar, selecionou-se o pré-tratamento para a germinação das sementes de P. notatum. A germinação das sementes em casa de vegetação constou de 16 tratamentos, com quatro repetições, os tratamentos controle, os três isolados, o stimulate®, a combinação de cada um dos três isolados mais stimulate®, todos com e sem o pré-tratamento, análise dos dados foi através do programa Genes. Para as avaliações das características morfogênicas, foram escolhidos aleatoriamente quatro perfilhos por tratamento, sendo os mesmos monitorados quanto ao aparecimento e alongamento de folhas e senescência. Este monitoramento era realizado duas vez por semana. Os três melhores isolados de trichoderma selecionado no teste de A. cepa foram o 2B2, C1, 2B12. A retirada das estruturas de revestimento das sementes de P. notatum foi o pré-tratamento que apresentou o melhor desempenho com 34,5% das sementes geminadas. No teste da sanidade foram identificados os gêneros de fungos Aspergillus, Curvularia, Geniculosporium e Fusarium, e o isolado 2B2 de Trichoderma spp. mostrou eficiente na confrontação direta a estes gêneros de fungos. Para o IVE (índice de velocidade emergência), os tratamentos da combinação do pó biológico do isolado 2B12 de trichoderma e o bioestimulante Stimulate® e somente o bioestimulante Stimulate® com pré-tratamento, e sem pré-tratamento a combinação do pó biológico do isolado 2B2 de trichoderma com bioestimulante Stimulate®, obtiveram os melhores resultados, com 14.48, 13.93 e 12.75, respectivamente. O bioestimulante stimulate®, promove o índice de velocidade de emergência das sementes de P. notatum e a produção de massa seca, a taxa de alongamento inicial da folha (TAIF) interfere positivamente, na massa seca da parte aérea.

During embryogenesis, a single cell gives rise to a multi-cellular embryo through successive rounds of cell division. As cells become smaller, cellular organelles adapt their sizes accordingly. The size of the mitotic spindle—the microtubule-based structure controlling these divisions—is particularly important as it determines the distance over which chromosomes are segregated. To perform its function properly, spindle size scales with cell size. However, we still lack a mechanistic understanding of the underlying microtubule-based processes that regulate spindle scaling. In this thesis, I combined quantitative microscopy and laser ablation in zebrafish embryos and Xenopus laevis egg extract encapsulated in oil droplets. My measurements revealed the influence of microtubule length dynamics, transport, and nucleation on cell size-dependent spindle scaling. Strikingly, I discovered a hierarchical regulation of spindle size. In large cells, microtubule nucleation exclusively scales spindle size relative to cell size by changing the number of microtubules within the spindle. In small cells, microtubule dynamics fine-tune spindle size by modulating microtubule length. To understand the mechanism of spindle scaling, I proposed a theoretical model based on a limiting number of microtubule nucleators and microtubule-associated proteins that regulate microtubule length. The transition from nucleation- to dynamics-based scaling requires that microtubule number and the number of microtubule-associated proteins that promote microtubule growth scale differently with cell size. This can be achieved by sequestering an inhibitor of microtubule nucleation to the cell membrane, which is consistent with my measurements of microtubule nucleation. The differential regimes of spindle scaling modulated by microtubule nucleation and dynamics imply a gradual change in spindle architecture, which may ensure faithful chromosome segregation by spindles of all sizes.

The proper transmission of genetic materials into progeny cells is crucial for maintenance of genetic integrity in eukaryotes and fundamental for reproduction of organisms. To achieve this goal, chromosomes must be attached to microtubules emanating from opposite poles in a bi-oriented manner at metaphase, and then should be separated equally through proper spindle elongation in anaphase. Failure to do so leads to aneuploidy, which is often associated with cancer. Despite the presence of a safety device called the spindle assembly checkpoint (SAC) to monitor chromosome bi-orientation, mammalian cells frequently possess merotelic kinetochore orientation, in which a single kinetochore binds microtubules emanating from both poles. Merotelically attached kinetochores escape from the surveillance mechanism of the SAC and when cells proceed to anaphase cause lagging chromosomes, which are a leading cause of aneuploidy in mammalian tissue cultured cells. The fission yeast monopolin complex functions in prevention of mal-orientation of kinetochores including merotelic attachments during mitosis. Despite the known importance of Cdk1 activity during mitosis, it has been unclear how oscillations in Cdk1 activity drive the dramatic changes in chromosome behavior and spindle dynamics that occur at the metaphase/anaphase transition. In two separate studies, we show how dynamic Cdk1 phosphorylation regulates chromosome segregation. First, we demonstrate that sequential phosphorylation and dephosphorylation of monopolin by Cdk1 and Cdc14 phosphatase respectively helps ensure the orderly execution of two discrete steps in mitosis, namely sister kinetochore bi-orientation at metaphase and spindle elongation in anaphase. Second, we show that elevated Cdk1 activity is crucial for correction of merotelic kinetochores produced in monopolin and heterochromatin mutants.

DNA damage induces mitotic exit delays through a process that requires the spindle assembly checkpoint (SAC), which blocks the metaphase to anaphase transition in the presence of unaligned chromosomes. Using time-lapse confocal microscopy in syncytial Drosophila embryos, we show that DNA damage leads to arrest during prometaphase and anaphase. In addition, functional GFP fusions to the SAC components MAD2 and Mps1, and the SAC target Cdc20 relocalize to kinetochore through anaphase arrest, and a null mad2mutation blocks damage induced prometaphase and anaphase arrest. We also show that the DNA damage signaling kinase Chk2 is required for damage induced metaphase and anaphase arrest, and that a functional GFP-Chk2 fusion localizes to kinetochores and centrosomes through mitosis. In addition, in the absence of Chk2, we find that DNA damage sufficient to fragment centromere DNA does not delay mitotic exit. We conclude that DNA damage signaling through Chk2 triggers Mad2-dependent delays in mitotic progression, both before or after the metaphase-anaphase transition.