Orientador: Andrea Trevas Maciel Guerra / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-22T08:11:21Z (GMT). No. of bitstreams: 1 Viguetti-Campos_NilmaLucia_D.pdf: 2337035 bytes, checksum: 32c9a2a31f7d885c1709b08e2d8df65c (MD5) Previous issue date: 1997 / Doutorado / Genetica / Doutor em Ciências Biológicas

Orientador: Fernandes Denardi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-07-27T16:22:03Z (GMT). No. of bitstreams: 1 Matheus_WagnerEduardo_M.pdf: 9690090 bytes, checksum: c360620812517ab9e47fde081043552f (MD5) Previous issue date: 2001 / Resumo: o carcinoma urotelial superficial de bexiga é uma neoplasia que apresenta altas taxas de recorrência, se um tratamento complementar não for associado à ressecção completa do tumor. Dentre as alternativas terapêuticas, o BCG intravesicaltem sido o agente mais utilizado, com a finalidade de prevenir recidivas, progressão e aumentar a taxa de sobrevida dos pacientes com tumor de bexiga. o objetivo desse trabalho foi avaliar a eficácia, a longo prazo, da imunoterapia intravesical com BCG, descrever sua toxicidade, analisar fatores de risco do Carcinoma de Células Transicionais (CCT) superficial, tais como: tamanho tumoral, multifocalidade, estadiamento inicial e presença de carcinoma in situ (CIS), e também, correlacionar esses fatores de risco com recidiva tumoral, progressão histológica e realização de cistectomia radical. Nesse estudo, foram analisados 46 pacientes com diagnóstico de CCT superficial de bexiga e que foram submetidos ao tratamento de imunoterapia intravesical com BCG, no período de junho de 1988 a janeiro de 2000. Vinte e dois pacientes (48%) estavam livres de tumor, após o primeiro ciclo de BCG, 37 pacientes (80%), após o segundo ciclo, 41 pacientes (89%), após o terceiro ciclo, e 42 pacientes (91%), após o quarto ciclo. A taxa total de progressão para doença invasiva foi de 9% (4 pacientes). Complicações severas foram observadas em 4 pacientes (9%): 2 casos de hematúria, 1 de orquiepididimite e 1 de febre por mais de 24 horas. A presença de múltiplas lesões tumorais e o tamanho do tumor não apresentaram correlação com o prognóstico desses pacientes. Os tumores que evoluíram para cistectomia radical apresentaram estadiamentos iniciais PTIG2 ou PTIG3. A presença de carcinoma in situ indicou maior chance de recidiva, progressão e evolução para cistectomia radical. A análise dos resultados permitiu as seguintes conclusões: o BCG apresenta uma boa eficácia no tratamento complementar de CCT superficial; o uso do BCG está associado a um baixo índice de complicações severas; os fatores prognósticos mais importantes, na evolução clínica do tumor de bexiga, são estadiamento, grau histológico e carcinoma in situo / Abstract: The bladder superficial urotelial carcinoma is a neoplasy tOOthas shown high recurrence rates if a complementary treatment is not associated to the thorough resection of such tumor. BCG intravesical has been the most applied agent for the prevention either of the recurrences or progression and also for prolonging life of patients suffering ftom bladder cancer. Goal: Analysing effectiveness of the intravesical immunotherapy with BCG and describing its toxicity in the long termo Also analysing the risk factor of superficial CCT, such as: tumor sÍZe, multiplicity, initial staging and presence of cis, establishing a relationship among tumoral recurrences, histological progression and radical cistectomy. Methods and Material: 46 patients with b1adder superficial CCT diagnosis OOve been analysed prospectively and submitted to intravesical uno therapy treatment with BCG, ftom june 1988 to january 2000. Results: The number of patients without a tumor, afier the fust cycIe of BCG was 22 (48%), afier second was 37 (80%), afier third was 41 (89%), afier the forth was 42 (91%) and the total rate of advancing for invasive diasease was 4 (9%). Serious complications OOvebeen observed in 4 patients (9%). Two cases ofhematuria, 1 case of orchiepididymitis and 1 case of fever for over 24 hours. The presence of multiple tumorallesions and sÍZeof tumors OOd no correlation with the prognosis. Tumors which advanced to radical cistectomy showed initial staging pTIG2 or pTIG3. The presence of carcinoma in situ has indicated a greater chance of recurrences, progression and development to radical cistectomy. Conclusion: BCG has showed a good eifectiveness as a complementary treatment for superficial CCT and also a low toxicity. The most significant prognostic factors on the clinicaldevelopment ofbladder tumor are staging, histological grade and carcinoma in situo / Mestrado / Mestre em Cirurgia

The Standard Penetration Test (SPT) is the most widely used in-situ soil test in the world. "Large Penetration Test" (LPT) is a term used to describe any scaled up version of the SPT. Several types of LPT have been developed around the world for the purpose of characterizing gravel deposits, as SPT blow counts are less reliable in gravels than in sands. Both tests suffer from the lack of a reliable means of determining transferred energy. Further, the use of LPT blow counts is generally limited to calculation of equivalent SPT blow counts using correlation factors measured in sands. Variation of LPT blow counts with grain size is assumed to be negligible. This research shows that safety hammer energies can be reliably estimated from measurements of hammer impact velocity for both SPT and LPT. This approach to determining transferred energy is relatively simple, and avoids the primary limitation of existing methods, which is the inability to calibrate the instrumentation. Transferred energies and hammer impact velocities are collected from various sources. These data are used to determine the ratio between the hammer kinetic energy and the transferred energy (energy transfer ratio, ETR), which is found to follow a roughly Normal distribution for the various hammers represented. An assessment of uncertainty is used to demonstrate that an ETR based approach could be superior to existing energy measurement methods. SPT grain size effects have primarily been characterized as the variation of an empirical relative density correlation factor, (CD)SPT, with mean grain size. In this thesis, equivalent (CD)LPT data are back-calculated from measured SPT-LPT correlation factors (CS/L). Results of a numerical study suggest that SPT and LPT grain size effects should be similar and related to the ratio of the sample size to the mean grain size. Based on this observation, trend-lines with the same shape as the (CD)SPT trend-line are established for the back-calculated (CD)LPT data. A method for generating the grain size effect trend-line for LPT is then proposed. These trend lines provide a rational approach to direct interpretation of LPT data, or to improved prediction of equivalent SPT blow counts. / Applied Science, Faculty of / Civil Engineering, Department of / Graduate

The binding of a novel GABAs receptor radioligand eH]-CGP62349 to human hippocampal control and epileptic sections was investigated using quantitative receptor autoradiography. Kinetic analyses performed on rat brain sections, to conserve the use of human tissues, demonstrate that eH]-CGP62349 associated rapidly. The same radioligand dissociated rapidly initially, then very slowly. Utilising human hippocampus it was shown that CH]-CGP62349 bound with high affinity (O.SnM) to human control hippocampal sections. The kinetics of GABAs receptor in human hippocampus using the novel compound confirmed previous studies performed in rat membrane. The localisation of GABAB receptors in human hippocampal control partially supported former studies using agonist ligands such as CH]-GABA and eH]-baclofen, despite differences have been noticed. Hippocampal slices from surgical resected specimens obtained from hippocampal sclerotic (HS)/temporal lobe epilepsy (TLE) patients were compared with neurologically normal post-mortem control subjects for neuropathology and GABAs receptor density and affinity. Neuronal loss was observed in most of the hippocampal subregions, whereas in subiculum any significant difference was detected. For GABAa receptor density (Bmax) a significant reduction was reported in CA3, CA4, and DG; the affinity was increased exclusively in DG. After the correction of Bmax value for the neuronal loss a significant increase was seen in CA3. Oligonucleotides were designed to investigate the two GABAB1 isoforms (la, and lb), and the GABAa2 subunit in human hippocampal control and HSITLE tissues, obtained as well as for the autoradiography. GABAsla, GABAB1b, and GABAB2 transcript distribution was in agreement with the receptor protein localisation, even though in the human hippocampus GABAa2 has to be yet localised and to verify if it is associated with GABAsla or GABABlbto form the dimeric active receptor. The present study suggests an involvement of GABABreceptors in HS/TLE in some not all the hippocampal subregions in terms of receptor density and affinity. Further investigations in regard to the quantitative in situ hybridisation data and the immunocytochemical results are fundamental to gain insight into the pathological role of GABAs receptors.

<p> Recrystallization of aluminum alloys, which affects their mechanical properties, was examined by a variety of experimental techniques with electrical resistivity (ER) measurements being one of them. In this work, a new method based on in-situ electrical resistivity measurements has been originated and employed for characterization of recrystallization. In contrast to traditional method of resistivity measurements at either room or liquid N2 temperature on isothermally/isochronally heat-treated samples, a new and simpler approach was proposed. In this work, resistivity of a long sample with a small cross section area (usually, 70 x 2 x 0.2 mm3 strips were employed) was continuously measured while the sample was heated from room temperature to 400°C. In this case, there are several independent contributions to electrical resistivity including contributions from lattice vibrations, second-phase particles, presence of solutes and defects, etc. </p> <p> In order to separate precipitation and recrystallization, a new annealing cycle was proposed through thermodynamic modeling. It was demonstrated that for 5xxx AI alloys only contributions from phonons, Pr, and dislocations, pd, were important. By analyzing a temperature dependence of p , it was intended to separate the effect related to a dislocation density, i.e. to a degree of recrystallization. Since Pr >> pd, this effect is elusive. It can only be detected if a robust numerical differentiation is utilized. A novel spline-based method of robust numerical differentiation was developed and employed to reveal these elusive effects from p(T) (or R(T)) profiles. It was shown that a characteristic peak of the d p (T) / dT curves points to onset and end of "events" taking place in AI alloys and that peaks' positions depend on the degree of deformation and amount of alloying additions such as Fe and Mn. The onset and extent of recrystallization seen in the resistivity measurement experiments were confirmed by optical metallography and hardness measurements. Uniformity of deformation in samples was investigated by micro-hardness measurements followed by a detailed statistical analysis to prove that deformation is uniform throughout cross-section of the materials. </p> <p> Results suggested that increasing degree of deformation and alloying additions (Fe and Mn) lower the temperature of onset of recrystallization. In addition, it was observed that kinetics of recrystallization was accelerated by the increasing degree of deformation and alloying additions whereas recrystallized grain size decreases with increasing additions of Fe and Mn in Al-3% Mg alloys. </p> / Thesis / Master of Applied Science (MASc)

The characterization of nanomaterials involved in dynamic processes are conventionally conducted using microscopy, spectroscopy and other physical/chemical methods through the pseudo-dynamic approach. In details, the dynamics processes are recorded by repeating or terminating the process multiple times. However, the above approach can lead to missing important transition information and inducing contamination for mechanistic studies. This motivates the efforts to develop real time characterization techniques which can probe the dynamic change of nanoparticles in their native operating environments. With the capability of probing structural change at the nanoscale, in situ transmission electron microscopy, has shown great potential in studies and applications of various processes. Targeting at conducting precise analysis, which has been limited by many uncertainties from electron beam effects and the miniaturized reaction cell used for TEM, the work presented herein pursues a quantitative characterization of a few electrochemical and biological processes through in situ liquid-phase transmission electron microscopy. In this work, the in situ transmission electron microscopy system is evaluated by comparing the in situ results with those from standard experiments to show its capabilities in studying dynamic processes. The in situ system is quantitatively calibrated to obtain the optimized observation conditions to avoid detectable electron beam interference, solution depletion and achieve sufficient resolution for analysis through micrometer thick liquid. These form the fundamentals for the in situ studies. Moreover, a comprehensive analysis protocol is established by incorporating multiple ex situ and post situ characterizations. Using this optimized in situ system, the mechanism of electrodeposition of gold on carbon electrode is studied. The in situ results allow quantitative analysis of the growth process. The prevailing diffusion limited three dimensional growth model is examined. A study of the effect of supporting electrolyte on the electrodeposition of palladium is also conducted. The self-limiting, surface diffusion and aggregation/recrystallization growth model is found to describe the early stage of growth, rather than the classical Volmer-Weber growth model. A further study is conducted on the structural evolution of palladium nanoparticles under electrochemical cycling. The mechanisms involved in this process, including electrodeposition, dissolution, hydrogen co-deposition and hydrogen desorption, are studied. The supporting electrolyte, HCl, is found to enhance the dissolution of deposited palladium clusters and induce movements and aggregation of the deposits during the hydrogen interaction process to form chain-like and irregular clusters, which provide direct experimental proof on the morphology formation of palladium with hydrogen involvement. Ultimately, the in situ technique is applied to the study of calcium phosphate biomineralization. Combined with multiple post situ characterization techniques, the study provides direct experimental evidence of the non-classical pre-nucleation and attachment growth of calcium phosphate structures. This demonstrates the potential of the in situ technique for studying the mechanisms involved in biological processes. / Dissertation / Doctor of Philosophy (PhD) / Nanostructured materials have been widely used in various fields. In situ transmission electron microscopy, a technique used to characterize nanomaterials involved in different dynamic processes in their operating environments, is an advanced tool over the traditional characterization methods such as ex situ microscopy and spectroscopy. However, there are several challenges in applying this in situ technique to processes occurring in liquid media. In this thesis, an in situ transmission electron microscopy system is applied to study the mechanisms of structural changes during different processes in liquids with both high spatial and temporal resolution. Protocols to evaluate and optimize the in situ system are developed to provide results comparable with those from their actual applications. Then in situ studies on the structural evolution of nanomaterials during electrochemical processes are performed and different theoretical models are applied to describe these processes. Finally, this technique is extended to investigate biomineralization to show its capabilities in future studies on biological processes.

Liquid cell electron microscopy has been proven to provide high spatial and temporal resolution for studying liquid layers and the solid-liquid interface at the micro and nano scale. The in situ environment allows for spatial and spectral characterization and quantification of the dynamics and kinetics involved with structural and chemical changes of nanostructures, which has seen application is fields of materials science, electrochemistry, corrosion, biomaterials, and nanophysics. The rapid growth of in situ liquid cell electron microscopy has motivated the fabrication of a custom liquid cell for improved control over the experimental conditions, including cell dimensions and materials. In this work, the process flow and micro-fabrication of a custom liquid cell system are proposed and executed, with proof of operation through the in situ imaging of suspended gold nanoparticles and electrochemical characterization. The in situ TEM system is improved upon by forming 1μm diameter holes through the viewing membranes, removing the background noise contribution from imaging electrons through the windows. This allows for high resolution liquid cell imaging. This improved system is used to study the oxidative etching conditions for palladium nanocrystals, which are commonly used as catalysis for hydrogen fuel cells. The dendritic etching is studied through native etching under exposure to hydrochloric acid without the presence of oxidizing species, followed by the radiolytic generation of oxidizing radicals via the microscope electron beam, and finally by the application of electrical biasing. / Thesis / Master of Applied Science (MASc) / Characterization of nanomaterials has been available for several decades and has aided in the improvement of material design, such as steel strength and corrosion resistance, electrical systems such as those involved in computers and smartphones, and biological sensing and detection. Observation of dynamic process which occur at the interface between solid and liquid phases, or purely within liquid layers, has always been a challenging topic due to the difficulty of finding a stable environment for both solids and liquids to exist at the nano scale within a measurement device. In situ liquid cell electron microscopy offers the ability to image this interface with real-time data acquisition for recording of dynamics and kinetics at the nano scale. Previous work has shown the liquid cell to provide high spatial and temporal resolution of systems in an environment which mimics their native operating conditions. This thesis addresses current developments for in situ systems and works to develop a custom liquid cell to further the applications of the liquid cell and provide improved control over experimental conditions. The work then aims to improve on current technology by increasing the spatial resolution obtainable. Finally, the technique is applied to study the structural changes of nanocrystals under various etching conditions as a demonstration of its’ capabilities.

Com o aumento na prevalência das lesões de erosão, medidas preventivas e de controle das mesmas vêm sendo propostas. Dentre elas, encontram-se os produtos fluoretados e, mais recentemente, os compostos contendo cátions metálicos polivalentes, como o tetrafluoreto de titânio (TiF4). Este estudo in situ visou avaliar os efeitos do gel de TiF4 na inibição da progressão da erosão em dentina humana erodida e abrasionada. Para tanto, foram obtidas oitenta e quatro amostras de dentina (3 x 3 x 1 mm) a partir de terceiros molares humanos hígidos. Setenta e duas amostras foram erodidas in vitro previamente a etapa in situ e divididas aleatoriamente em 6 grupos (n=12) de acordo com o tratamento e com o número de ciclos erosivos/abrasivos a serem executados durante a etapa in situ. Os grupos controle foram submetidos a apenas 1 (C1), 2 (C2) e 3 (C3) ciclos erosivos/abrasivos. Já os grupos experimentais receberam 1 (TiF4 1), 2 (TiF4 2) e 3 (TiF4 3) aplicações de gel de TiF4 (4%) seguidos de 1, 2 e 3 ciclos erosivos/abrasivos, respectivamente. Um sétimo grupo controle (n=12) foi incluído, sendo que amostras sem erosão in vitro foram submetidas a 3 ciclos erosivos/abrasivos. Cada ciclo erosivo/abrasivo correspondeu a 2 dias de desafios erosivos (ácido cítrico 0,5%, pH 2,6, 6x/dia) e abrasivos (1x/dia). Para realização da etapa in situ, as amostras foram posicionadas em dispositivos intra-orais removíveis utilizados por 12 voluntários. Os espécimes foram avaliados em perfilometria (n=12), microscopia eletrônica de varredura ambiental (MEV ambiental) (n=12) e microscopia de força atômica (AFM) (n=3) para avaliar as alterações causadas pelo fluoreto sobre a superfície da dentina. A ANOVA a dois critérios para medidas repetidas mostrou que o desgaste superficial foi afetado pelos tratamentos avaliados (p<0,001). O teste de Tukey demonstrou que o grupo TiF4 2, que não diferiu dos grupos TiF4 1 e TiF4 3, apresentou redução significativa no desgaste quando comparado aos grupos C1, C2, C3 e controle sem erosão in vitro. Os grupos TiF4 1 e TiF4 3 não revelaram diferença significativa em relação ao grupo C1, porém ambos os grupos de tratamento demonstraram desgaste significativamente menor que C2 e C3. Os maiores desgastes foram verificados para C3 e controle sem erosão in vitro. As micrografias obtidas em MEV ambiental e AFM sugeriram a manutenção do glaze sobre a superfície da dentina tratada com o gel de TiF4, mesmo após os desafios ácidos. A superfície passou a apresentar um aspecto mais liso que as amostras dos grupos controles, com redução dos diâmetros dos lúmens dos túbulos dentinários, justificando o efeito protetor do TiF4. Portanto, o TiF4 demonstrou potencial em reduzir a progressão das lesões de erosão in situ, independente do número de aplicações avaliado. / With the increase in the prevalence of erosion lesions, preventive and control measures for them have been proposed. Among them are the fluoride products and, more recently, compounds containing polyvalent metal ions such as titanium tetrafluoride (TiF4). This in situ study aimed to evaluate the effects of the TiF4 gel on the erosion progression inhibition in human eroded and abraded dentin. For this purpose, eighty-four dentin samples (3 x 3 x 1 mm) were prepared from undamaged human third molars. Seventy-two samples of previously eroded dentin in vitro were allocated into 6 groups (n=12) according to the treatment to be received during the in situ phase and the number of erosive/abrasive cycles. Control groups were subjected to 1 (C1), 2 (C2) and 3 (C3) erosive/abrasive cycles only. Experimental groups had TiF4 gel (4%) applied once (TiF41), twice (TiF42) or three times (TiF43) followed by 1, 2 and 3 erosive/abrasive cycles, respectively. A seventh group (n=12) comprised in vitro uneroded samples that were subjected to 3 erosive/abrasive cycles. Each cycle corresponded to 2 days of erosive (citric acid 0.5%, pH 2,6, 6x/day) and abrasive (1x/day) challenges. To perform the in situ stage, the samples were placed in removable intra-oral devices used by 12 volunteers Specimens were evaluated in profilometry (n=12), environmental scanning electron microscopy (ESEM) (n=12) and atomic force microscopy (AFM) (n=3) to evaluate the changes caused by fluoride on the surface of the dentin. The two-factor ANOVA for repeated measures showed that, after profilometric analysis, the surface loss was affected by the treatments evaluated (p<0.001). Tukeys test showed that TiF4 2 group, which did not differ from TiF4 1 and TiF4 3 groups showed a significant reduction in surface loss compared to C1, C2, C3 and in vitro-uneroded control. The TiF4 1 and TiF4 3 groups showed no significant difference from C1, but both treatment groups demonstrated significantly smaller surface loss than C2 and C3. The greatest losses were observed for C3 and in vitro-uneroded control. The micrographs by ESEM and AFM suggested the maintenance of the glaze on the dentin surface treated with the TiF4 gel, even after acid challenge. The surface started to show a smoother appearance than samples from control groups, with reduced diameters of the dentinal tubules lumens, explaining the protective effect of TiF4. Therefore, the TiF4 demonstrated potential to reduce the progression of erosion lesions, regardless of the number of applications evaluated.

Le travail présenté dans ce manuscrit est consacré à la restitution et à l’interprétation des profils verticaux derapports de mélange du chlorure d’hydrogène (HCl) et du formaldéhyde (H2CO) mesurés par l’instrumentsous ballon SPIRALE (SPectroscopie InfraRouge par Absorption de Lasers Embarqués), au cours de deuxvols effectués en région intertropicale depuis Teresina (Brésil, 5.1°S-42.9°S), le 22 Juin 2005 et le 9 Juin2008. Les problématiques scientifiques liées à l’étude de ces composés, les caractéristiques de la hautetroposphère et de la basse stratosphère intertropicale et enfin celles du spectromètre SPIRALE sontdécrites. L’étude de faisabilité de la mesure de H2CO a permis de définir la position et l’intensité de la raied’absorption la plus adéquate pour la mesure stratosphérique de ce composé (à 2912.1 et 1701.5 cm-1). Lesprofils verticaux de rapports de mélange de H2CO obtenus au cours de ces vols de SPIRALE sont présentéset comparés. Les mesures de HCl sont les premières à avoir été réalisées in situ dans la basse et moyennestratosphère intertropicale. Dans la couche de transition intertropicale, celles-ci sont utilisées en lien avecdes mesures récentes de composés à très courte durée de vie (VSLS), afin d’estimer la contribution deceux-ci au bilan du chlore stratosphérique. Dans la moyenne stratosphère, les signatures visibles sur lesprofils verticaux de HCl et sur ceux d’ozone acquis simultanément sont étudiées en lien avec l’oscillationquasi-biennale. En outre, le bon accord entre SPIRALE et l’instrument MLS du satellite Aura a permis deconforter la fiabilité de celui-ci pour la mesure de HCl. / The work presented in this manuscript is devoted to the retrieval and the interpretation of the mixing ratiovertical profiles of hydrogen chloride (HCl) and formaldehyde (H2CO), derived from the measurementsperformed by the SPIRALE balloon-borne instrument (SPectroscopie InfraRouge par Absorption de LasersEmbarqués) over Teresina (Brazil, 5.1°S-42.9°S), on 22 June 2005 and 9 June 2008. The scientificquestions associated with the study of these compounds, the characteristics of the tropical uppertroposphere and lower stratosphere and those of the SPIRALE spectrometer are described. The feasibilitystudy of the H2CO measurement has allowed to define the position and the intensity of the most suitableabsorption line for the stratospheric measurement of this compound (at 2912.1 and 1701.5 cm-1). The mixingratio vertical profiles of H2CO obtained during the SPIRALE flights are presented and compared. The HClmeasurements presented are the first to be made in situ in the lower and mid-stratosphere. In the tropicaltransition layer, they have been combined with recent very short-lived species (VSLS) ones in order toestimate the contribution of these compounds to the chlorine budget of the stratosphere. In the midstratosphere,the signatures present on the vertical profiles of HCl and ozone acquired simultaneously arestudied in connection with the quasi-biennal oscillation.

O Brasil atualmente ocupa a nona posição entre os maiores produtores de tomate (Solanum lycopersicum L.) do mundo. O desenvolvimento das plantas e a produção dos tomateiros, no entanto, podem ser afetados por diversos problemas fitossanitários, entre os quais aqueles causados por vírus. Atualmente, entre as viroses que mais tem se destacado estão aquelas causadas por espécies dos gêneros Begomovirus, Crinivirus e Tospovirus. Entre os begomovirus, especial atenção tem sido dada ao Tomato severe rugose virus (ToSRV), pois tem sido a espécie prevalente na maioria das regiões produtoras de tomate no país. Esse begomovirus, que até o momento foi relatado somente no Brasil, é transmitido pelo aleirodídeo (\"mosca branca\") Bemisia tabaci biótipo B, numa relação persistente circulativa. 8Estudos da relação do ToSRV com esse aleirodídeo indicaram períodos mínimos de acesso à aquisição e inoculação de 5 minutos. Esse trabalho teve como objetivos: a) identificar o menor tempo de alimentação da B. tabaci biótipo B para a aquisição do ToSRV em tomateiros e b) identificar possíveis sítios de aquisição do ToSRV em tecido foliar de tomateiro via microscopia de luz de epifluorescência por hibridização in situ. Insetos livres de vírus foram confinados em folha de tomateiro infectado com o ToSRV durante 1, 3 e 5 minutos e 24 h (controle) para a aquisição do vírus. Parte dos insetos foi utilizada para a detecção do vírus no vetor, enquanto a outra parte foi usada em testes de transmissão do ToSRV para tomateiros. A detecção do vírus no inseto e nos tomateiros foi feita por PCR. B. tabaci biótipo B foi capaz de adquirir o ToSRV nos diferentes tempos de alimentação. Os insetos foram capazes de transmitir o vírus para tomateiros, com eficiência de 33% a 100%. Análises de cortes histológicos longitudinais na região das nervuras de folhas de tomateiro infectados com o ToSRV, em microscopia de luz de epifluorescência por hibridização in situ, revelaram vários pontos de fluorescência localizados nas células do parênquima do floema, incluindo as células companheiras e nas células da epiderme. Essa fluorescência não foi constatada em tecido sadio. A localização do ToSRV em células do parênquima foliar do tomateiro, associada ao conhecimento de que esse aleirodídeo efetua picadas de prova intracelulares, de curta duração, durante o processo de penetração intercelular do estilete, podem explicar a aquisição deste begomovirus durante curtíssimos períodos de alimentação. / Brazil currently ranks ninth position among the largest tomato (Solanum lycopersicum L.) producers in the world. Several diseases, including those caused by virus, however, can affect plant growth and yield of tomatoes. Currently, among the most important virus diseases are those caused by species of the Genus Begomovirus, Crinivirus and Tospovirus. Among the begomoviruses, especial attention has been given to Tomato severe rugose virus (ToSRV), because it has been the prevalent species in most of the tomato producing regions of the country. This begomovirus, which so far has only been reported in Brazil, is transmitted by the whitefly Bemisia tabaci biotype B, in a persistent circulative relationship. Studies on the virus-vector relationship indicated five minutes as the minimum period of access for virus acquisition and inoculation by the vector. This study aimed to: a) identify the shortest feeding period of B. tabaci biotype B for acquisition of ToSRV in tomato plants, and b) identify possible ToSRV acquisition sites in infected leaf tissue via epifluorescence light microscopy by in situ hybridization. Virus free insects were confined in tomato leaf infected with ToSRV during 1, 3 and 5 minutes and 24 hours (control) for virus acquisition. Part of the insects was used for virus detection in the vector, while the other part was used on ToSRV transmission tests for tomato plants. Virus detection in both, insect tomato plants, was carried out by PCR. B. tabaci biotype B was capable to acquire the ToSRV on the different feeding times. The insects were capable to transmit the virus to the tomato plants, with efficiency of 33% to 100%. Analysis of longitudinal histological cuts of ToSRV infected leaves, in epifluorescence light microscopy by in situ hybridization, revealed several fluorescence spots located in phloem parenchyma cells, including the companion and the epidermal cells. Fluorescence was not verified on healthy tissue. The location of ToSRV in parenchyma cells of tomato leaf, associated with the knowledge that this insect performs very short intracellular feeding probes, during the process of intercellular penetration of the stylet, may explain the acquisition of this begomovirus during very short feeding periods.