Septinas são GTPases consideradas como um novo componente do citoesqueleto. Essas proteínas interagem entre si para formar heterocomplexos filamentosos e estruturas de alta ordem que são importantes para a citocinese e uma variedade de outros processos celulares. Existem muitos aspectos mecânicos dessas proteínas que não são totalmente compreendidos, incluindo a forma como os heterocomplexos se agrupam corretamente. Em humanos, há 13 genes que codificam septinas, classificadas em quatro grupos quanto à similaridade em relação à estrutura primária. O complexo hexamérico SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 foi o melhor caracterizado, com uma estrutura cristalina resolvida à 4 Å. Segundo às Regras de Kinoshita, as septinas desse complexo podem ser substituídas nesse arranjo por outras pertencentes ao mesmo grupo. Neste trabalho utilizamos a técnica de microscopia eletrônica de transmissão com análise de partícula única para estudar dois complexos de septinas. Um dos complexos estudados neste projeto é formado por septinas humanas, para as quais atualmente não há informações estruturais disponíveis. O complexo SEPT5-SEPT6-SEPT7 foi expresso heterólogamente em E. coli e purificado em alta concentração salina para evitar a polimerização. A análise de partículas únicas de imagens por contrastação negativa mostrou a presença de partículas alongadas de aproximadamente 25 nm de comprimento, compostos por seis monômeros, como esperado. Com o objetivo de localizar a posição da SEPT5 no complexo, foi realizada uma fusão com MBP (Maltose Binding Protein) e imunomarcação com anticorpo monoclonal anti-SEPT5, concluindo que a SEPT5 está localizada na extremidade do complexo hexamérico. Porém, a SEPT5 pertence ao mesmo grupo da SEPT2, que foi relatada estar localizada no centro do hexâmero. Este resultado possibilitou uma nova discussão sobre a maneira que as septinas formam os complexos e, como a sensibilidade à concentração salina está relacionada com a fragilidade da interface NC, análogo ao observado em complexos de Saccharomyces cerevisiae. Um complexo de Ciona intestinalis incluindo a SEPT2, SEPT6, SEPT7 e SEPT9, também expresso heterólogamente em E. coli, foi preparado por contrastação negativa. A análise de partícula única das imagens coletadas mostrou um heterocomplexo aparentemente hexamérico, embora fosse esperado um octâmero devido à presença das quatro septinas diferentes, sendo uma pertencente à cada um dos quatro grupos. Os resultados deste trabalho proporcionaram um avanço na compreensão da formação de heterocomplexos de septinas e como essas proteínas interagem umas com as outras nesta montagem. / Septins are GTPases that appear to be a novel component of the cytoskeleton. These proteins interact with each other to form filamentous heterocomplexes and high order structures which are important for cytokinesis and a variety of other cellular processes. There are many mechanistic aspects of these proteins that are not fully understood, including how the heterocomplexes correctly assemble. In humans, there are 13 genes encoding septins, classified in four groups based on primary structure. The SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 hexameric complex was the best characterized, with a crystalline structure solved at 4 Å. According to Kinoshita´s Rules, the septins of this complex can be replaced in this arrangement by others belonging to the same group. In this work, we used transmission electron microscopy with single particle analysis to study two septin complexes. One of the complexes studied in this project is composed of three human septins, for which there is currently no structural information available. The SEPT5-SEPT6-SEPT7 complex was heterologously expressed in E. coli and purified at high salt concentration to avoid polymerization. Single particle analysis of negatively stained samples showed the presence of elongated particles of approximately 25 nm in length. To locate SEPT5 in the complex, a fusion with MBP (Maltose Binding Protein) and immunoblotting with anti-SEPT5 monoclonal antibody was performed, concluding that SEPT5 is located at the end of the hexameric complex. However, SEPT5 belongs to the same group as SEPT2, which was reported to be located in the center of the hexamer. This result allowed for a new discussion on the way that septins form heterocomplexes and also, on how the sensitivity of the NC interface in related to salt concentration, analogous to that observed in the heterocomplex of Saccharomyces cerevisiae. A Ciona intestinalis complex including SEPT2, SEPT6, SEPT7 and SEPT9, also expressed heterologously in E. coli, was prepared by negative staining. The single particle analysis of the collected images showed an apparently hexameric heterocomplex, although an octamer was expected due to the presence of the four different septins, one belonging to each of the four groups. The results of this work represent advances in the understanding of the formation of septin heterocomplexes and how these proteins interact with each other during assembly.

Os primatas neotropicais, ou primatas do novo mundo, são pertencentes a um grupo diverso e variado de animais, com características anatômicas, comportamentais e taxonômicas próprias, que os diferem dos primatas do velho mundo. Estima-se que representem 31% dos primatas não-humanos do planeta. Têm sofrido com as altas taxas de crescimento populacional e perturbação antrópica resultante do desflorestamento, agricultura e criação de gado. Com o intuito de reverter esta situação e maximizar o potencial reprodutivo das espécies, o desenvolvimento de biotecnologias associadas à reprodução tem sido amplamente justificado nos dias de hoje, por representarem importantes ferramentas reprodutivas, contribuindo para a preservação da biodiversidade e manutenção da diversidade genética. Assim, o objetivo do presente estudo foi padronizar a colheita de sêmen de Alouatta guariba clamitans por eletroejaculação por via retal com posterior análise do sêmen fresco, procedimentos nunca descritos para a espécie, e adaptar o método de preparo de sêmen de Leontopithecus chrysomelas, Alouatta caraya e Alouatta guariba clamitans para microscopia eletrônica de transmissão, permitindo a comparação da ultraestrutura dos espermatozoides das três espécies e apresentando resultados também inéditos na literatura científica. Para este estudo, foram utilizados quatro exemplares de L. chrysomelas, dois exemplares de A. caraya e dois exemplares de A.g. clamitans, machos adultos mantidos em cativeiro. L. chrysomelas tiveram o sêmen colhido por vibroestimulação peniana, e A. caraya e A.g. clamitans foram submetidos à eletroejaculação por via retal. Após a obtenção dos ejaculados, o sêmen foi avaliado quanto ao volume, pH, concentração, motilidade, integridade de membrana plasmática, integridade de membrana acrossomal e morfologia, e posteriormente preparado para microscopia eletrônica de transmissão. A análise dos 12 espermatozoides das três espécies estudadas revelou que todos apresentam os componentes básicos descritos para o espermatozoide humano cabeça, colo e cauda, formada pelas peças intermediária, principal e terminal com variações na forma e tamanho de cada componente. As diferenças mais significativas encontradas entre os espermatozoides comparados foram extensão de acrossomo, comprimento, largura e organização de peça intermediária. O presente estudo atingiu os objetivos propostos, apresentando resultados inéditos na literatura científica / The neotropical primates, also known as primates of the new world, are a diverse and varied group of animals, with unique anatomical and taxonomic characteristics, as well a unique behaviour, that distinguish those from the old world primates. It is estimated that these present 31% of the non-human primate population of the planet. High taxes of populational growth and anthropological disturbance have been undergoing due to deforestation, agriculture and cattle farms. In order to revert this situation and maximize the reproductive potential of the species, the development of biotechnologies associated to the reproduction are widely justified nowadays, for representing important reproductive tools, contributing to the preservation of the biodiversity and maintenance of genetic diversity. Hence, the purpose of this study was to collect Alouatta guariba clamitans semen by rectal electroejaculation then analyze the fresh semen, procedures never before described for the species, and adapt the preparation method of Leontopithecus chrysomelas, Alouatta caraya and Alouatta guariba clamitans semen for electron transmission microscope, allowing the comparison of the spermatozoa ultrastructure from the three species and also presenting new results to the scientific literature. For this study, it was used four specimens of L. chrysomelas, two specimens of A. caraya and two specimens of A.g. clamitans, adult males kept in captivity. L. chrysomelas had the semen taken by penine vibratory stimulation, and A. caraya and A.g. clamitans were submitted to rectal electroejaculation. After the semen was obtained, it was evaluated regarding the volume, pH, concentration, motility, sperm membrane integrity, acrosome integrity and morphology, and then prepared for electron transmission microscopy. The analysis of the spermatozoa of the three studied species revealed that all present the basic components described for the human spermatozoa - head, neck region and tail, formed by middle, principal and terminal pieces - with variations in 14 shape and size of each component. The most significant differences found between the compared spermatozoa were the acrosome extension and middle piece length, thickness and organization. The present study attained the proposed goals, presenting unprecedented results for the scientific literature

Fast excitatory neurotransmission is critical for learning and memory, and its dysregulation is linked to numerous neurological diseases. These include developmental diseases such as fragile X syndrome, psychiatric disorders like schizophrenia, and chronic neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. Throughout the central nervous system, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors (AMPARs) mediate the fastest excitatory neurotransmission. In response to the neurotransmitter glutamate, AMPARs open their ion channels and allow cation flux through the post-synaptic membrane. This initiates rapid depolarization and signaling in the post-synaptic neuron. Nearly all AMPARs exist as complexes with auxiliary subunits, which are regulatory proteins that modulate receptor assembly, trafficking, pharmacology and function. These auxiliary subunits determine brain region-specific AMPAR signaling, and aberrancies in complex formation or function lead to neuropathologies. Despite their importance for CNS signaling and implication in neurologic disorders, the structural bases underlying the function of AMPARs and AMPAR complexes remain ambiguous, representing a critical barrier to our understanding of excitatory neurotransmission. As a consequence, structure-based design of neuro-therapeutics is largely undeveloped: there is only a single FDA-approved drug targeting AMPARs. To address these problems, I wanted to dedicate my thesis work to study AMPAR synaptic complexes across an array of functional states and provide a new foundation for our structural understanding of AMPAR signaling. First, I designed a covalent-fusion construct approach to guarantee assembly and expression of AMPAR synaptic complexes in heterologous cells (HEK293). Then, I developed purification protocols allowing me to obtain chemically homogenous and pure complex protein. Since synaptic signaling is highly dynamic, complexes of AMPARs with auxiliary subunits are conformationally heterogeneous and are not amenable to X-ray crystallography. Cryo-electron microscopy (cryo-EM) enabled me to approach these complexes structurally, where I could collect data and parse out heterogeneity through image classification. With cryo-EM, I solved the structure of an AMPAR bound to the auxiliary subunit stargazin, which promotes AMPAR activation. This work provided the first structural information on how AMPARs form complexes with regulatory subunits. In a following study, I solved the structure of an AMPAR in complex with a functionally distinct auxiliary subunit, GSG1L. In contrast to stargazin, GSG1L promotes inactivation and desensitization of AMPARs, thus having a neuroprotective effect. To further characterize the function of these auxiliary subunits, I designed chimeras between stargazin and GSG1L and examined their function electrophysiologically. This experiment revealed that AMPAR auxiliary subunits have a modular design, where variable extracellular domain regions, supported by a conserved transmembrane α-helical bundle, distinctly regulate function of the core AMPAR. This study provided the first evidence of how brain region-specific expression patterns of similarly-structured auxiliary subunits may contribute to unique AMPAR functions. More recently, I’ve taken advantage of the modulatory effects of stargazin on AMPARs and I applied cryo-EM to an AMPAR-stargazin complex. This study determined how AMPARs are activated by the neurotransmitter glutamate, and revealed a novel mechanism by which glutamate binding induces opening of AMPAR ion channels. Our data show that two-fold symmetric kinking of ion channel helices allows cation flux into neurons, which triggers neurotransmission. Importantly, this study also provides insights into how mRNA editing and patient-derived disease mutations in the transmembrane (i.e., resulting in aberrantly firing of receptors during epilepsy) reshape AMPAR function and excitatory neurotransmission. Collectively, the findings from my thesis work provide a new paradigm for the molecular-level understanding of glutamatergic neurotransmission throughout the CNS. These studies lay the groundwork for new directions in precision-medicine design of therapeutics targeting brain region-specific AMPAR synaptic complexes in neurological diseases.

<p>Investigation of laser-matter interaction has been an important research topic which is closely related to applications in various fields including industry, military, electronics, photonics, etc. With the advent of ultrafast transmission electron microscope (UTEM), in situ investigation of the interaction between pulsed laser and nanostructured materials becomes accessible, with unprecedented spatial and temporal resolution. Here, we studied two categories of materials with the help of UTEM, namely, energetic materials and plasmonic nanostructures. The results demonstrate that UTEM provides a novel and convenient way for the investigation the structural and morphological change of energetic materials under external stimuli at nanoscale. Also, UTEM makes it possible to visualize the light-induced welding between plasmonic nanostructures at real time, which helps to reveal more details about the mechanisms involved. Furthermore, we studied the formation of some novel structures by combing different gold and silver nanostructure.</p>

In this project, we have assessed the structural tolerance of advanced refractory alloys to simulated nuclear fusion reactor environments, by using intense proton beams to mimic fusion neutron damage and analysing the proton damaged structures using in-situ/ex-situ transmission electron microscopy and nano-hardness measurements. Refractory metals such as tungsten or tantalum, and their binary alloy combinations, are considered as promising structural materials to withstand the unprecedented high heat loads and fast neutron/helium fluxes expected in future magnetically-confined fusion reactors. Tungsten is currently the frontrunner for the production of plasma-facing components for fusion reactors. The attractiveness of tungsten as structural material lies in its high resistance to plasma-induced sputtering, erosion and radiation-induced void swelling, together with its thermal conductivity and high-temperature strength. Unfortunately, the brittle nature of tungsten hampers the manufacture of reactor components and can also lead to catastrophic failure during reactor operations. We have focused on two potential routes to enhance the ductility of tungsten-containing materials, namely alloying tungsten with controlled amounts of tantalum, and using alternatively tantalum-based alloys containing specific tungsten additions, either as a full-thickness structural facing material or as a coating of first wall reactor components. The aim was to investigate the formation and evolution of radiation-induced damaged structures in these material solutions and the impact of those structures on the hardness of the material. The main results of this work are: (1) the addition of 5wt%Ta to W leads to saturation in the number density and average dimensions of the radiation-induced a/2 dislocation loops formed at 350C, whereas in W the loop length increases progressively and evolves into dislocation strings, and later into hydrogen bubbles and surface blisters, (2) the recovery behaviour of proton irradiated W5wt.%Ta alloy is characterized by dislocation loop growth at 600-900C, whereas voids form at 1000C by either vacancy absorption or loop collapse, (3) the presence of radiation-induced a loops at 590C in Ta hinders the formation and ordering of voids observed with increasing damage levels at 345C, (4) the addition of 5-10wt.%W to Ta delays the evolution of a/2 dislocation loops with increasing damage levels, and therefore the appearance of random voids. These results expand the composition palette available for the safe selection of refractory alloys for plasma facing components with enhanced, or at least predictable, tolerance to the heat-radiation flux combinations expected in future nuclear fusion plants.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Esta pesquisa estudou a influência de diferentes velocidades de corte e marcas de discos diamantados nos valores de resistência adesiva, durante a preparação dos espécimes a serem submetidos à microtração, e na integridade das amostras por meio do microscópio eletrônico de varredura (MEV). Vinte blocos da cerâmica à base de dissilicato de lítio (IPS e.max Press) foram unidos com cimento resinoso (Rely X ARC) a blocos de compósito (Z100), construídos incrementalmente. Foram seguidas as recomendações dos fabricantes no tratamento da superfície da cerâmica e aplicação do cimento resinoso. Após 24 horas em água destilada a 37C, os espécimes foram divididos em dois grupos de discos: marcas Buehler e Extec e subdivididos nas velocidades de 200rpm e 400rpm (B2; E2; B4 e E4, respectivamente). Cada espécime foi cortado em dois eixos perpendiculares para obtenção de palitos com área adesiva de 1,0mm. Para cada condição experimental, os palitos foram separados, aleatoriamente, 15 palitos para análise ao MEV e 30 palitos para serem submetidos à força de tração. As médias de resistência adesiva em MPa foram E4=20,312 ; B4= 24,2 11,3 ; B2= 25,2 9,0 e E2= 28,6 10,4. Na análise estatística, observou-se que os valores de resistência adesiva na velocidade de 200rpm foram significativamente maiores comparados a velocidade de 400rpm, independente do disco empregado. Ao MEV, observou-se melhor integridade dos palitos na velocidade de 200rpm com presença de trincas menos extensas nas bordas externas. Constatou-se também que o disco Extec na velocidade de 400rpm apresentou movimentos excêntricos ao corte e obteve-se maior número de perdas prematuras, uma diminuição significante na média da área total de união (p<0,05), além de diferença significativa nos valores de resistência comparada a velocidade de 200rpm. Concluiu-se que a utilização de diferentes velocidades e sua interação com o disco empregado interfere na integridade dos espécimes e nos valores de resistência adesiva, sendo mais acentuada ao se utilizar o disco da marca Extec. / The aim of this research was to verify the influence of different cutting speeds and brand of diamond saws during microtensile specimen preparation on the bonding strength values and on the samples microscopy integrity (SEM). Twenty lithium disilicate glass-ceramic blocks (IPS e.max Press) were bonded with dual-cure resin cement (Rely X ARCTM) to composite (Z100TM) blocks, built incrementally. The manufacturers recommendations were followed for ceramic treatment surface and placement on the resin cement, as well. After storage for 24 hours in distillated water at 37C, the specimens were divided in two groups of diamond saws, Buehler and Extec brands, which were further subdivided using 200rpm and 400rpm speeds (B2; E2; B4 and E4 respectively), and they were sectioned into two perpendicular axes obtaining sticks shaped specimens with cross-sectional area of 1.0mm. For each experimental situation the sticks were randomly separated; 15 for the analysis to the SEM and 30 to be submitted to tensile strength. Mean bond strength values (MPa) were: E4 = 20.3 12; B4 = 24.2 11.3; B2 = 25.2 9.0; and E2 = 28.6 10.4. The statistical analysis of data showed that the values of bond strength at 200 rpm speed were significantly higher as compared to 400 rpm speed, regardless of saw employed. In SEM, it was noted better integrity of sticks at 200 rpm speed with presence of less extensive cracks in external edges. It was also found that the Extec saw at 400 rpm showed eccentric movement to cutting and achieved higher number of premature losses, a significant decreased mean total area of bond (p<0.05), in addition to significant difference in strength values compared with 200 rpm speed. It was concluded that using different speeds and their interaction with the used saw interfere with the integrity of specimens and the bond strength values, being more pronounced when using the Extec saw.

Made available in DSpace on 2017-03-14T14:10:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-09-08. Added 1 bitstream(s) on 2017-03-14T14:42:46Z : No. of bitstreams: 1 000874748_20170908.pdf: 292993 bytes, checksum: 3298d7a3590d3abb3c5e413fade7e716 (MD5) Bitstreams deleted on 2017-09-11T13:56:01Z: 000874748_20170908.pdf,. Added 1 bitstream(s) on 2017-09-11T13:56:51Z : No. of bitstreams: 1 000874748.pdf: 2035422 bytes, checksum: 847aa737ff6653a0ac4fbff7fb1be39f (MD5) / O objetivo do presente estudo foi comparar os efeitos de substâncias de limpeza (etanol a 95% ou xilol) e o momento do condicionamento ácido (imediato ou após 7 dias) na obtenção de túbulos dentinários abertos e avaliar a persistência de resíduos de um cimento à base de resina epóxi sobre a superfície dentinária. Além de, avaliar a capacidade de limpeza da superfície dentinária, impregnada por um cimento endodôntico à base de resina epóxi (AH Plus), proporcionada pelo etanol 95% ou pelo xilol e condicionamento ácido e seus efeitos sobre a interface dentina/sistema adesivo dentinário, por meio da avaliação da formação da camada híbrida e da resistência de união do sistema adesivo dentinário Scotchbond Universal (3M ESPE), imediatamente ou após 7 dias da limpeza dentinária e, da avaliação da formação da camada híbrida e da resistência de união do sistema adesivo dentinário Adper Scotchbond Multi Purpose (3M ESPE), imediatamente após a limpeza da superfície dentinária impregnada com o cimento. No capítulo 1, fragmentos de dentina de incisivos bovinos foram tratados com irrigantes endodônticos, impregnados com o cimento e divididos em 4 grupos (n=10): G1, etanol 95.0% - imediato; G2, xilol - imediato; G3, etanol 95.0% - após 7 dias e G4, xilol - após 7 dias. Após a obtenção das imagens em MEV, a contagem de túbulos dentinários abertos foi realizada com o programa Photoshop CS5. Outros 40 fragmentos foram preparados e analisados em MEV (500X), para a avaliação da persistência de resíduos do cimento endodôntico na dentina. A contagem dos túbulos dentinários abertos foi analisada com ANOVA e teste de Tukey (P=0,05) e a persistência de resíduos com os testes de Kruskal Wallis e Dunn (P=0,05). O protocolo de limpeza com o xilol e condicionamento ácido após 7 dias proporcionou maior quantidade de túbulos dentinários abertos e menor ...(Resumo completo, clicar acesso eletrônico abaixo) / The aim of this study was to evaluate the cleaning capacity of the dentin surface, contaminated by a endodontic sealer containing epoxy resin (AH Plus), provided by ethanol or by xylene and its effects on the interface dentin / dentin adhesive system, by evaluating the formation of the hybrid layer and the bond strength of Scotchbond Universal (3M ESPE), immediately or after 7 days of dentin cleaning and, assessing the formation of the hybrid layer and the bond strength of Adper Scotchbond Multi Purpose (3M ESPE), immediately after cleaning the dentin surface impregnated with the sealer. Besides, compare the effects of cleaning substance (95% ethanol or xylene) and the etching time with phosphoric acid at 37% (immediately or after 7 days) to obtain open dentinal tubules and to evaluate the residues persistence of a sealer-based epoxy resin (AH Plus) on the dentin surface. In Chapter 1, dentin fragments of bovine incisors were treated with root canal irrigation, impregnated with sealer and divided into 4 groups (n = 10): G1 (EIDE), ethanol 95.0% - DE immediate; G2 (XIDE), xylene - DE immediate; G3 (EPDE), ethanol 95.0% - DE after 7 days and G4 (XPDE), xylene - DE after 7 days. After obtaining the SEM pictures (500X), the open dentinal tubules counts were performed using Photoshop CS5 program. Another 40 fragments were also prepared and also obtained SEM pictures (500X) for evaluating the persistence of sealer residues in the dentin using scores. The count of open dentinal tubules was analyzed with ANOVA and Tukey tests (P = 0.05) and the persistence of residues with the Kruskal Wallis and Dunn tests (P = 0.05). The cleaning protocol with xylol and etching after 7 days provided the greatest amount of open dentinal tubules and lowest persistence of residues in the dentin (P <0.05). There were no differences between the other groups (P> 0.05). In Chapter 2...(Complete abstract electronic access below)

Resumo: Os vírus pertencentes ao gênero Begomovirus da família Geminiviridae são transmitidos pela mosca-branca Bemisia tabaci Gennadius, atualmente constituem um dos problemas fitossanitários mais sérios em diversas culturas. A mosca-branca encontra-se disseminada em todas as principais regiões produtoras de hortaliças do Brasil. No estado de São Paulo, em 2005, plantas de pimentão mostrando sintomas de deformação dos frutos, folhas e mosaico foliar, apresentaram-se infectadas por begomovírus. Até então, no Brasil, a infecção de pimentão por vírus deste gênero havia sido verificada apenas nos estados de Pernambuco e Bahia, no ano de 1997. Amostras de plantas de pimentão foram coletadas nos campos de produção localizados nos municípios de Paranapanema, Piraju, Alvinlândia, Ubirajara, Elias Fausto, Mogi Guaçu, Paulínia e Botucatu, no período de novembro de 2004 a maio de 2006, com finalidade de detecção e verificar a variabilidade genética dos begomovírus infectando esta cultura. A detecção de begomovírus foi realizada por meio de PCR com oligonucleotídeos universais e seqüenciamento de parte da região codificadora para a proteína capsidial. Do total de 228 plantas coletadas, foram encontradas 30 amostras positivas de begomovírus, sendo que seqüências de 23 isolados apresentaram uma identidade de nucleotídeos de 98 a 100% com o Tomato severe rugose virus (ToSRV) e dois isolados, um proveniente da região de Alvinlândia e outro de Mogi Guaçu, apresentaram maior identidade (98 e 95% respectivamente) com a provável espécie Tomato yellow vein streak virus (ToYVSV). O DNA-A do isolado ToSRV[PJU] coletado de pimentão Lilac da região de Pirajú apresentou identidade de nucleotídeos de 99% com outro isolado de ToSRV (DQ207749) proveniente de pimenta e 97% com um isolado de ToSRV (AY029750) proveniente de tomate... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The viruses belonging to the genus Begomovirus, of the family Geminiviridae, are transmitted by the whitefly Bemisia tabaci Gennadius, and considered one of the most important fitossanitary problems for several crops. The whitefly is disseminated throughout the country. In São Paulo State, 2005, pepper plants showing fruits deformations, and mosaic on the leaves were infected by begomovirus. Until then in Brazil, the infection of begomovirus in pepper was only reported in 1997, in Pernambuco and Bahia States. Pepper plants were collected in Paranapanema, Piraju, Alvinlândia, Ubirajara, Elias Fausto, Mogi Guaçu, Paulínia and Botucatu, in the period of November of 2004 to May of 2006, with the aim to study the genetic variability of the begomovirus infecting this culture. The begomovirus were detected by PCR using universal oligonucleotídeos and the coat protein region was sequenced. On the total of 228 samples, they were found 30 positive samples of begomovírus, and sequences of 23 isolated presented nucleotides identies of 98 to 100% with Tomato severe rugose virus (ToSRV), while two isolates, one of Alvinlândia and another of Mogi Guaçu, showed identity highest nucleotide (98 and 95% respectively) with the tentative species Tomato yellow vein streak virus (ToYVSV). The nucleotide sequence of the DNA-A of ToSRV[PJU] from pepper cv. Lilac of Pirajú has 99% nucleotide identies with other isolate of ToSRV (DQ207749) collected from pepper and 97% with an isolate of ToSRV (AY029750) from tomato. The sequence of the DNA-B revealed identity of 98% with the DNA-B of ToRMV (AF291706) from Triângulo Mineiro, Minas Gerais and 95% with ToSRV (AY029751). Sap-transmission was verified for Nicandra physaloides and pepper ‘Magda' using infected N. benthamiana as source. By whitefly the virus was transmitted to Lycopersicum esculentum... (Complete abstract click electronic access below) / Orientador: Marcelo Agenor Pavan / Coorientador: Renate Krause Sakate / Banca: Antonio Carlos Maringoni / Banca: Murilo Francisco Zerbini Junior / Banca: Romulo Fujito Kobori / Banca: Valdir Atsushi Yuki / Doutor

Orientador: Katia Cristina Kupper / Banca: Sérgio Florentino Pascholati / Banca: João Martins Pizauro Junior / Banca: Rita de Cássia Panizzi / Banca: Sandra Regina Ceccato Antonini / Resumo: The biocontrol efficiency of Aureobasidium pullulans strain ACBL-77 was evaluated against Geotrichum citri-aurantii, the causal agent of sour rot in citrus, and their interactions were studied in vitro and in vivo. For this purpuse, were evaluated (i) the biocontrol efficiency of A. pullulans ACBL-77 against the pathogen; (ii) the effect of different culture media on the production of yeast cells; (iii) the incorporation of nutritional sources in optimizing the antagonistic activity; (iv) the competition for nutrients between the microorganisms; (v) the effect of nutrients on yeast cell and biofilm production and their relation in the biocontrol of disease; (vi) the survival of yeast in citrus fruits; (vii) the interaction between the microorganisms by scanning electron microscopy; (viii) the effect of A. pullulans applications in the citrus fruits quality; (ix) the effect of ACBL-77 in the activity of the enzymes possibly involved with resistance induction. In the results this study, were verified that in the fruits treated with A. pullulans ACBL-77, preventively, the control percentage was higher than the curative treatment. The potato-dextrose-agar medium promoted the highest amount of yeast cells. The incorporation of micronutrients, such as boric acid, cobalt chloride and ammonium molybdate (1 mM), favoured the antagonistic action of A. pullulans in vitro. Ammonium sulphate 1mM and sucrose 0.5% favoured the yeast during the competition between the microorganisms. However... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The biocontrol efficiency of Aureobasidium pullulans strain ACBL-77 was evaluated against Geotrichum citri-aurantii, the causal agent of sour rot in citrus, and their interactions were studied in vitro and in vivo. For this purpuse, were evaluated (i) the biocontrol efficiency of A. pullulans ACBL-77 against the pathogen; (ii) the effect of different culture media on the production of yeast cells; (iii) the incorporation of nutritional sources in optimizing the antagonistic activity; (iv) the competition for nutrients between the microorganisms; (v) the effect of nutrients on yeast cell and biofilm production and their relation in the biocontrol of disease; (vi) the survival of yeast in citrus fruits; (vii) the interaction between the microorganisms by scanning electron microscopy; (viii) the effect of A. pullulans applications in the citrus fruits quality; (ix) the effect of ACBL-77 in the activity of the enzymes possibly involved with resistance induction. In the results this study, were verified that in the fruits treated with A. pullulans ACBL-77, preventively, the control percentage was higher than the curative treatment. The potato-dextrose-agar medium promoted the highest amount of yeast cells. The incorporation of micronutrients, such as boric acid, cobalt chloride and ammonium molybdate (1 mM), favoured the antagonistic action of A. pullulans in vitro. Ammonium sulphate 1mM and sucrose 0.5% favoured the yeast during the competition between the microorganisms. However, the addition of ammonium sulphate at 1% in the yeast culture stimulated biofilm production and consequently increased the antagonistic activity against the pathogen in vivo. The application of A. pullulans to fruits allowed the better survival of yeast in wounded sites of citrus fruit. The yeast was found to be able to form biofilm on citrus, deforming the ... (Complete abstract click electronic access below) / Doutor

A resistência de plantas daninhas é um grande problema que afeta a produtividade das culturas em nosso país, ao passo que quando é identificada, deve ser estudada visando desenvolver estratégias de manejo que evitem a expansão e seleção em novas áreas, além de seleção de resistência múltipla em áreas onde está presente. Nesta temática o objetivo desta pesquisa foi determinar manejo alternativo ao glyphosato para as espécies Chloris elata e Digitaria insularis no período de entressafra da cultura da soja e identificar características morfológicas da superfície foliar de Chloris elata que possam interferir sobre o controle com herbicidas. Foram estudadas alternativas ao glyphosate para manejo em áreas de sistema de produção soja/milho, em dois experimentos com as plantas daninhas Chloris elata e Digitaria insularis perenizadas e com duas condições de roçada (15 e 30 cm). Foram avaliados o controle visual das plantas daninhas, redução de matéria fresca e seca. Para morfologia das folhas da espécie Chloris elata foi realizada análise histológica com recursos de microscopia eletrônica de varredura, visando caracterizar a morfologia da folha, além de determinar o índice estomático, densidade estomática e densidade de tricomas para as superfícies adaxial e abaxial das folhas, nos estádios 4 e 6 folhas totalmente expandidas. Digitaria insularis se mostrou menos suscetível as técnicas de manejo empregadas para plantas perenizadas, necessitando duas aplicações sequenciais para conter a rebrota dos tratamentos. O uso da maior dose do herbicida haloxyfop associado aos herbicidas glyphosate ou amônio-glufosinato se mostrou eficaz no controle de C. elata e D. insularis. O manejo de roçagem potencializou a ação dos herbicidas, e a planta daninha D. insularis se mostrou mais suscetível a está técnica em relação ao C. elata. Para este manejo a associação da maior dose de haloxyfop ao glufosinato foi eficaz para as duas gramíneas. As alturas de roçagem somente se diferenciaram nas primeiras avaliações. O biótipo resistente de C. elata apresentou menor densidade estomática, no estádio de 4 folhas completamente expandidas e maior densidade de tricomas no estádios de 6 folhas completamente expandidas, o que pode contribuir para a resistência ao glyphosate. Está espécie apresentou formações de cristais de cera que circundam o ostíolo, no estádio de 6 folhas completamente expandidas, podendo estar relacionada a a menor suscetibilidade ao glyphosate desta planta daninha perenizada. / Weed resistance is a major problem that affects crop productivity in our country, whereas when it is identified, it must be studied in order to develop management strategies that avoid expansion and selection in new areas, as well as multiple resistance selection in areas where it is present. In this subject the objective of this research was to determine alternative management of glyphosate for the Chloris elata and Digitaria insularis species during the off - season of the soybean crop and to identify the morphological characteristics of the leaf surface of Chloris elata that could interfere the control with herbicides. It was studied alternatives to glyphosate for management in areas of soybean / maize production system in two experiments with weeds Chloris elata and Digitaria insularis perennial and with two mowing conditions (15 and 30 cm). The visual control of weeds, fresh and dry matter reduction were evaluated. For the morphology of the leaves of the Chloris elata species, a histological analysis was carried out using scanning electron microscopy to characterize the leaf morphology, as well as to determine the stomatal index, stomatal density and trichome density for the adaxial and abaxial surfaces of the leaves, stages 4 and 6 fully expanded leaves. Digitaria insularis was less susceptible to the management techniques used for perennial plants, requiring two sequential applications to contain the regrowth of treatments. The use of the highest dose of the herbicide haloxyfop associated with the herbicides glyphosate or ammonium glufosinate proved to be effective in the control of C. elata and D. insularis. Herbicide management potentiated herbicide action, and D. insularis weed was more susceptible to this technique than C. elata. For this management, the association of the highest dose of haloxyfop to glufosinate was effective for both grasses. The cutting heights differed only in the first evaluations. The resistant C. elata biotype showed lower stomatal density at the stage of 4 fully expanded leaves and greater density of trichomes in the stages of 6 fully expanded leaves, which may contribute to resistance to glyphosate. This species presented waxy crystal formations that surround the ostrich, in the stage of 6 fully expanded leaves, and may be related to the natural tolerance to the glyphosate of this perennial weed.