The new field of protein-based nano-technology takes advantage of the complex interactions between proteins to form unique structures with properties that cannot be achieved with traditional components. Microtubules (MTs), self assembled proteinaceous hollow filaments, offer promise in the development of MT-based nano-systems. The compelling need for the controlled assembly of 3D MT arrays is the fundamental motivation for the first part of this research. We report on the morphology of MTs grown in a crowded environment in the form of high viscosity fluids containing agarose and a novel process that enables the assembly of MTs supported by gel-based 3D scaffolds. Our research on MTs and their interaction with other molecules lead us to discover extraordinary spherulitic structures that changed the course of the project. The novel subject situate us into a complicated dilemma that question the nature of MT asters reported in experiments carried out in cells. The second part of this research is focused in the crystallization ofTaxol, a MT stabilizing molecule used as anti-cancer drug. It was confirmed via fluorescent and differential interference contrast microscopy that Taxol crystals can be decorated with fluorescent proteins and fluorochromes without perturbing their morphology. We used theoretical calculations to further investigate Taxol-fluorescent agent interactions. Furthermore, the crystallization of Taxol was studied in pure water, aqueous solutions containing tubulin proteins and tubulin-containing agarose gels. We demonstrated that tubulin is able to heterogeneously nucleate Taxol spherulites. To explain the formation of tubulin-Taxol nuclei a new, secondary Taxol-binding site within the tubulin heterodimer is suggested. Results presented in this work are important for in vivo and in vitro microtubule studies due to the possibility of mistaking these Taxol spherulites for microtubule asters. Thus, we are confirming the need for careful interpretation of fluorescence microscopy observations of MT structures when large concentrations of Taxol are used as stabilizing agent in cells.

Submitted by Verena Bastos (verena@uefs.br) on 2015-08-04T00:07:37Z No. of bitstreams: 1 dissertacao.RGV.2015.1.pdf: 1835460 bytes, checksum: 8d5b6650247801f1f7f07285b2eedfe0 (MD5) / Made available in DSpace on 2015-08-04T00:07:37Z (GMT). No. of bitstreams: 1 dissertacao.RGV.2015.1.pdf: 1835460 bytes, checksum: 8d5b6650247801f1f7f07285b2eedfe0 (MD5) Previous issue date: 2015-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Searches related to evaluation and selection of native forages can contribute to improvement in feeding cattle in the Brazilian semiarid region, highlighting the Stylosanthes gender as an important source of forage species with great potential. This study aimed to perform morphoagronomic and cytogenetics Stylosanthes accessions, to contribute to studies that promote the inclusion of this legume in animal feed, seeking to characterize and select superior genotypes, and also select important in the characterization of germplasm of the species. To characterize morphoagronomic were evaluated 19 descriptors and of these, four were immediately discarded for not having variability among accessions, with the statistical analysis performed in the remaining 15 descriptors. The data were submitted to univariate and multivariate analyzes. Analysis of variance was performed only in quantitative descriptors NRP, CHP, Lfo, Cfo, ECAA, ECAS and CP, as to the qualitative descriptors CC, AC, PC, HC, CF, AF, PF and FF, descriptive analysis was performed. For cytogenetic characterization were obtained root tips used seed access Stylosantes 17, following the method described by War and Souza (2002). Scott and Knott test was performed for the grouping of averages, the descriptors were significant at (p ? 0.01) which allowed the formation of two groups, multivariate analysis was also performed by calculating the Mahalanobis distance using the method original Tocher, there is the formation of two groups. Group 1 consisted of 42 accesses and the group 2 only through access CPAC 4955. We observed the formation of four groups where the dendrogram also be noted that only the access CPAC 4955 did not group with any other access. This access, for long leaves that too, very thick stem and lack of body hair on the sheets. The descriptors evaluated by Singh test is observed that the main contributors to the diversity of genotypes were CP (11,64%) and NRP (10,98%) followed by HC (8,62%) and FF (8, 44%). By cytogenetic analysis were observed hits with 2n = 20 and 2n = 40 chromosomes, allowing differentiation of species S. scabra of S. seabrana with interphase nucleus of semirreticulado type, symmetrical karyotype with chromosome morphology ranging from the metacentric submetac?ntrica and size Average chromosomal around 2,5 microns. Differences were observed in the average length of the chromosomes and the total length of the genome. The analysis with double staining CMA3/DAPI enabled visualization of four CMA+ blocks, two CMA + blocks in subterminal region of the short arm of a chromosome pair submetacentric, and two blocks located in the proximal region of another metacentric pair in S. scabra, and still two blocks in the subterminal region of a metacentric pair in S. seabrana access. It was also possible to visualize DAPI and CMA+ bands in access CPAC 1261 and CPAC 5205. The accessions in this study were, in general, plants with semiereto growth habit, leaves with dark green color and hairiness, stem mostly quite branched. The differential staining of chromosomes, together with other cytogenetic markers, assists in characterizing germplasm collections access Stylosanthes spp. It was observed that there is genetic variability among the accessions studied the collection of Stylosanthes Embrapa Semi-Arid. / Pesquisas relacionadas a avalia??o e sele??o de forrageiras nativas podem contribuir para melhoria na alimenta??o dos rebanhos do Semi?rido brasileiro, destacando-se o g?nero Stylosanthes como uma importante fonte de esp?cies com grande potencial forrageiro. O presente trabalho teve por objetivo realizar caracteriza??o morfoagron?mica e citogen?tica de acessos de Stylosanthes, visando contribuir com estudos que promovam a inser??o desta leguminosa na alimenta??o animal, buscando caracterizar e selecionar gen?tipos superiores, e tamb?m selecionar descritores de import?ncia na caracteriza??o de germoplasma da esp?cie. Para caracteriza??o morfoagron?mica, foram avaliados 19 descritores e destes, quatro foram descartados imediatamente por n?o apresentarem variabilidade entre os acessos, sendo a an?lise estat?stica realizada nos 15 descritores restantes.Os dados foram submetidos ? an?lisesunivariadas e multivariadas. Foi realizada a an?lise de vari?ncia apenas nos descritores quantitativos NRP, CHP, Lfo, Cfo, ECAA, ECAS e CP, j? para os descritores qualitativos CC, AC, PC, HC, CF, AF, PF e FF, foi realizada analise descritiva. Para a caracteriza??o citogen?tica foram utilizadas pontas de ra?zes obtidas de sementes de 17 acessos de Stylosanthes, seguindo a metodologia descrita por Guerra e Souza (2002). O teste de Scott e Knott foi realizado para o agrupamento de m?dias, os descritores foram significativos a (p ? 0,01) o que possibilitou a forma??o de dois grupos, tamb?m foi realizada analise multivariadas pelo c?lculo da dist?ncia de Mahalanobis,utilizando o m?todo de Tocheroriginal,observa-se a forma??o de dois grupos.O grupo 1foi formado por 42 acessos e o grupo 2 apenas pelo acesso CPAC 4955. Observou-se a forma??o de quatro grupos onde no dendogramaobservar-se tamb?m que apenas o acesso CPAC 4955n?o agrupou com nenhum outro acesso.Este acesso apresentou folhas mais compridas que os demais, caule muito espesso e falta de pilosidade nas folhas. Dos descritores avaliados pelo teste de Singh observa-se que os que mais contribu?ram para a diversidade dos gen?tipos foram CP (11,64%) e NRP (10,98%) seguidos por HC (8,62%) e FF (8,44%). Atrav?s da an?lise citogen?tica observaram-se acessos com 2n=20 e 2n=40 cromossomos, permitindo a diferencia??o das esp?cies S. scabra da S. seabrana, com n?cleo interf?sico do tipo semirreticulado, cari?tipo sim?trico com morfologia cromoss?mica variando de metac?ntrica a submetac?ntrica e tamanho cromoss?mico m?dio em torno de 2,5 ?m.Foram observadas diferen?as no comprimento m?dio dos cromossomos e no comprimento total do genoma. A an?lise com dupla colora??o CMA3/DAPI permitiu a visualiza??o de quatro blocos CMA+, sendo dois blocos CMA+ localizados na regi?o subterminal do bra?o curto de um par cromoss?mico submetac?ntrico, e dois blocos localizados na regi?o proximal de outro par metac?ntrico em S. scabra, e ainda dois blocos na regi?o subterminal de um par metac?ntrico nos acessos de S. seabrana.Tamb?m foi poss?vel a visualiza??o de bandas DAPI+ e CMA- nos acessos CPAC 1261 e CPAC 5205.Os acessos avaliados neste estudo apresentaram,em geral,plantas com h?bito de crescimento semiereto, folhas com colora??o verde escura e com pilosidade, caule em sua maioria bastante ramificado. A colora??o diferencial de cromossomos, associados a outros marcadores citogen?ticos, auxilia na caracteriza??o de acessos de cole??es de germoplasma de Stylosanthes spp.Observou-se que h? variabilidade gen?tica entre os acessos estudados da cole??o de Stylosanthes da Embrapa Semi?rido.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2016-09-28T21:50:56Z No. of bitstreams: 1 Disserta??o_Jessica_Coelho_Valerianoatual.pdf: 1739335 bytes, checksum: 6ad29bd975f2995c8c9a68ceee755a5b (MD5) / Made available in DSpace on 2016-09-28T21:50:56Z (GMT). No. of bitstreams: 1 Disserta??o_Jessica_Coelho_Valerianoatual.pdf: 1739335 bytes, checksum: 6ad29bd975f2995c8c9a68ceee755a5b (MD5) Previous issue date: 2016-02-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Schinopsis brasiliensis Engl. and Pseudobombax simplicifolium A. Robins are species widely distributed in the Brazilian semiarid. The pressure on the genetic resources from Caatinga biome, mainly medicinal and for wood, caused by subsistence or commercial use, leads to reduce genetic variability of these species. Based on that, the cytogenetic and the tissue culture can support the characterization and propagation strategies of their populations. The research goals of this study were evaluate growth regulators and culture media effects on the establishment and in vitro micropropagation for the species, and the use of fluorochromes CMA3/DAPI in the cytogenetic characterization of P. simplicifolium. For P. simplicifolium it was evaluated different DKW ingredient concentration, added or not activated charcoal and different concentration of BAP and IBA on the establishment and in vitro multiplication of the species. For S. brasiliensis, it was evaluated the micropropagation media DKW and WPM (with different concentration of BAP and IBA) and the use of activated charcoal and polyvinylpyrrolidone to reduce the oxidation during establishment of in vitro cultivation of the species. In S. brasiliensis, the addition of BAP in the micropropagation media did not promote in vitro multiplication of the species. In P. simplicifolium, the results obtained for variables studied varied depending on the regulators concentrations and among genotypes. Concentrations above 4.92 and 6.64 ?M.L-1 AIB inhibited the multiplication in vitro in both species. The double staining CMA3/DAPI allowed the visualization of 2n = 84 chromosomes and different numbers of CMA+ blocks in the genotypes of P. simplicifolium. / As esp?cies Schinopsis brasiliensis Engl. e Pseudocombax simplicifolium A. Robins encontram-se amplamente distribu?das no semi?rido brasileiro. A press?o sobre os recursos gen?ticos do bioma caatinga, principalmente, medicinal e madeireiro, seja devido ao uso para subsist?ncia ou comercial, tem levado ? perda da variabilidade gen?tica destas esp?cies. Em vista a isto, a cultura de tecidos e a citogen?tica podem auxiliar nas estrat?gias de caracteriza??o e propaga??o de suas popula??es. O objetivo deste trabalho foi avaliar o efeito de reguladores de crescimento e meios de cultura no estabelecimento e multiplica??o in vitro das duas esp?cies, bem como o uso dos fluorocromos CMA3/DAPI na caracteriza??o citogen?tica do imbiru?u. Em P. simplicifolium, avaliou-se diferentes concentra??es dos sais DKW, acrescidos ou n?o com carv?o ativado e diferentes concentra??es de BAP e AIB no estabelecimento e multiplica??o da esp?cie. Em S. brasiliensis, avaliou-se os meios DKW e WPM e o uso do carv?o ativado e da polivinilpirrolidona na redu??o da oxida??o, e o efeito de diferentes concentra??es de BAP e AIB no meio WPM no estabelecimento do cultivo in vitro da esp?cie. Em S. brasiliensis, a adi??o de BAP ao meio de cultura n?o favoreceu a multiplica??o in vitro da esp?cie. Em P. simplicifolium as respostas para as vari?veis estudadas variaram em fun??o das concentra??es dos reguladores e entre os gen?tipos da esp?cie. Concentra??es acima de 4,92 e 6,64 ?M.L-1 de AIB inibiram a multiplica??o in vitro em ambas esp?cies. A dupla colora??o CMA3/DAPI permitiu a visualiza??o de 2n= 84 cromossomos e diferentes n?meros de blocos CMA+ nos gen?tipos estudados de P. simplicifolium.

Made available in DSpace on 2017-07-10T18:13:25Z (GMT). No. of bitstreams: 1 Cassia Fernanda Yano.pdf: 1692770 bytes, checksum: 2f6626f3bb741520d86130373963a00f (MD5) Previous issue date: 2011-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cytogenetics studies were carried out in two genera of fishes from Pindorama and Lopei streams, Upper Paraná River basin, with cytossistematic and ecologic-evolutionary approaches. In Heptapterus mustelinus, the first cytogenetics characterization in the genus was carried out, verifying 54 chromosomes (26m+18sm+4st+6a), which is a diploid number not observed among the Heptapteridae species till now studied. Differently from the others members of this family, multiple Ag-NORs and centromeric heterochromatin in almost all the chromosome of the complement were observed, being these heterochromatin CMA3+/DAPI- in nine chromosome pairs, beyond that coincident with Ag-NORs. These data show a different way in the karyotypic evolution of H. mustelinus in relation to the others Heptapteridae genus. In the genus Astyanax, an interpopulation comparative analysis was carried out in Astyanax aff. paranae, Astyanax fasciatus and Astyanax altiparanae from Pindorama and Lopei streams, in way to observe the chromosomal differentiation and the evolutionary trends in the three species. It was verified 48 and 50 chromosome in Astyanax aff. paranae and A. fasciatus, respectively, with interpopulation differences in the karyotypic formulae. The diploid number obtained to A. altiparanae was 50 chromosomes, with the same karyotypic formulae in both populations. The three species presented multiple Ag-NORs, with interpopulation differences observed in Astyanax aff. paranae and A. fasciatus. In relation to the heterochromatin distribution pattern, interpopulation differences were verified. The occurrence of Ag-NORs/CMA3+ was observed in the three species, besides DAPI+ bands in A. altiparanae. The results showed that Astyanax aff. paranae and A. fasciatus presented more conspicuous interpopulation differences than A. altiparanae populations, probably due bionomic characteristic that avoid the presence of Astyanax aff. paranae and A. fasciatus to upper segments in the streams, leading to the fixation of chromosomal rearrangements in these populations. However, the absence of interpopulation karyotypic differences in A. altiparanae suggests gene flow between the two populations. By the way, the present study showed that cytogenetics is an important tool in understanding the chromosomal mechanism involved in the evolutionary process in fishes group, and that the association to ecological studies is fundamental to the comprehension of dispersion process of some group of fishes. / Estudos citogenéticos foram realizados em dois gêneros de peixes nos riachos Pindorama e Lopei, pertencentes à bacia do Alto rio Paraná, com diferentes abordagens: citossistemática e ecológico-evolutiva. Em Heptapterus mustelinus foi realizada a primeira caracterização citogenética no gênero, tendo sido verificado 54 cromossomos (26m+18sm+4st+6a), número diplóide não relatado entre os heptapterídeos até o momento estudados. Diferentemente da maioria das espécies da família, foram observadas Ag-RONs múltiplas e heterocromatina presente na região centromérica da maioria dos cromossomos do complemento, sendo estas heterocromatinas CMA3+/DAPI- em nove pares cromossômicos, além daquelas coincidentes com as Ag-RONs. Estes dados revelam um caminho diferente na evolução cariotípica de H. mustelinus em relação aos demais gêneros de Heptapteridae. No gênero Astyanax foi realizada uma análise comparativa entre populações de Astyanax aff. paranae, Astyanax fasciatus e Astyanax altiparanae coletadas nos riachos Pindorama e Lopei, verificando-se as tendências da evolução e diferenciação cromossômica nas três espécies. Nas populações de Astyanax aff. paranae e A. fasciatus foram verificados 48 e 50 cromossomos, respectivamente, mas com diferenças interpopulacionais nas fórmulas cariotípicas em ambas as espécies. O número diplóide para A. altiparanae foi de 50 cromossomos com mesma fórmula cariotípica em ambas as populações. As três espécies apresentaram Ag-RONs múltiplas, com diferenças interpopulacionais observadas em Astyanax aff. paranae e A. fasciatus. Em relação ao padrão de distribuição de heterocromatina, diferenças interpopulacionais foram verificadas. A ocorrência de Ag-RONs/CMA3+ foi observada nas três espécies, além de bandas DAPI+ em A. altiparanae. Os dados demonstraram que Astyanax aff. paranae e A. fasciatus apresentaram diferenças cariotípicas interpopulacionais mais conspícuas do que as populações de A. altiparanae, provavelmente por características bionômicas que restringem Astyanax aff. paranae e A. fasciatus a trechos superiores de riachos, com a fixação de rearranjos cromossômicos nas populações. Entretanto, a ausência de diferenças cariotípicas interpopulacionais em A. altiparanae sugerem fluxo entre as duas populações. De forma geral ficou evidente que a citogenética é uma importante ferramenta para o entendimento dos mecanismos cromossômicos envolvidos no processo evolutivo dentro dos grupos de peixes, e que a associação a estudos de ecologia é fundamental para compreender os processos envolvidos na dispersão de algumas espécies.

En aquest treball s'ha dissenyat un mètode ràpid i fiable de tinció amb fluorocroms per a l'anàlisi de la integritat i viabilitat espermàtiques a partir del marcatge de la beina mitocondrial amb MitoTracker®Green FM, de l'acrosoma amb la lectina Trypsin inhibitor from Soybean (SBTI) conjugada amb el fluorocrom Alexa Fluor®488 específic per la proacrosina i del nucli amb els fluorocroms bis-benzimida (específic per a cèl·lules viables) i iodur de propidi (específic per a cèl·lules no viables). També s'ha determinat l'efecte de la filtració de dosis seminals de mascles astentoteratonecrospèrmics en columnes de Sephadex neutre i de dosis de mascles amb baixa qualitat espermàtica per filtració en columnes de Sephadex iònic, llana de vidre i glass beads sobre la qualitat espermàtica dels diferents grups de mascles analitzats. Els resultats obtinguts han mostrat que diversos paràmetres de qualitat espermàtica milloren després de la filtració en les diferents reïnes segons la patologia que presentin. / In this work, a quick and reliable method of staining with fluorochromes was designed for the assessment of the sperm integrity and vitality by marking the mitochondrial sheath with MitoTracker®Green FM, the acrosome with the lectine Trypsin inhibitor from Soybean (SBTI) conjugated with the fluorochrome Alexa Fluor ® 488m, specific for the proacrosine, and the nucleus with the fluorochromes bis-benzimida (specific for viable cells) and propidium iodide (specific for non-viable cells).Moreover, in this work, the effects of filtration of seminal doses from asthenoteratonecrospermic boars through columns of Sephadex neuter and seminal doses from boars with low sperm quality through ionic Sephadex, glass wool and glass beads columns on sperm quality were tested. The results obtained showed that some sperm quality parameters improved after filtration through the different matrixes depending on the pathology that the males presented before filtration.