Orientador: Valéria Helena Alves Cagnon Quitete / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T10:44:47Z (GMT). No. of bitstreams: 1 Montico_Fabio_M.pdf: 7570263 bytes, checksum: a775095a6141dbe3b18c6ec768585813 (MD5) Previous issue date: 2011 / Resumo: A reposição androgênica representa uma alternativa para minimizar os efeitos prejudiciais do desequilíbrio hormonal em homens senis, embora seus efeitos sobre o desenvolvimento de doenças prostáticas ainda seja assunto controvertido. Assim, o objetivo deste estudo foi caracterizar aspectos estruturais e moleculares do lobo ventral da próstata de ratos senis frente à reposição de hormônios esteróides, relacionando as alterações decorrentes da terapia hormonal a possíveis condições de lesões prostáticas. Ratos machos (Sprague- Dawley) foram divididos em um grupo Jovem (JOV) (4 meses), que recebeu óleo de amendoim (5 mL/Kg, s.c.), e grupos senis (10 meses), submetidos aos tratamentos: grupo Senil (SEN): óleo de amendoim (5 mL/Kg, s.c.); grupo Testosterona (TEST): cipionato de testosterona (5 mg/Kg, s.c.); grupo Estrógeno (EST): 17?-estradiol (25 ?g/Kg, s.c.); grupo Castrado (CAS): castração cirúrgica; grupo Castrado-Testosterona (CT): castração e após 30 dias tratamento similar ao grupo TEST; grupo Castrado-Estrógeno (CE): castração e após 30 dias tratamento similar ao grupo EST. Após 30 dias de tratamento, foram coletadas amostras de sangue, para dosagens hormonais séricas, e do lobo ventral, para análises em microscopias de luz e eletrônica de transmissão, morfométricas, imunohistoquímicas e Western Blotting. As moléculas investigadas foram: distroglicanas ? e ? (?-DG e ?-DG), receptor do fator de crescimento homólogo à insulina tipo I (IGFR-1), metaloproteinase-9 de matriz (MMP-9), fator de crescimento do endotélio vascular (VEGF) e endostatina. Redução dos níveis séricos de testosterona foi verificada na senescência, com aumento destes após a reposição hormonal no grupo TEST. O estroma do grupo SEN apresentou hipertrofia e células inflamatórias. Após a reposição hormonal na senescência ou frente à castração, verificou-se atrofia no epitélio, células epiteliais com halo citoplasmático claro ao redor do núcleo, microácinos e manutenção do estroma hipertrófico com células inflamatórias. Diminuição dos níveis das DGs foi verificada na senescência, sendo que após a terapia hormonal ocorreu aumento dos níveis protéicos dessas moléculas, especialmente nos grupos que receberam estradiol. Aumento do IGFR-1 e da MMP-9, bem como distribuição diferencial dessas moléculas no compartimento epitelial, foram observados nos grupos submetidos à reposição hormonal e no grupo CAS. O grupo SEN caracterizou acréscimo dos níveis de VEGF, sendo o inverso observado para a endostatina. A terapia hormonal e a castração levaram à elevação dos níveis de VEGF, sobretudo nos grupos EST, CAS e CE. Em oposição, a endostatina demonstrou-se aumentada especialmente nos grupos submetidos à reposição de testosterona. Os presentes resultados sugeriram que o desequilíbrio entre andrógenos e estrógenos verificado na senescência foi acentuado após a terapia hormonal e a castração, potencializando as alterações estruturais associadas a esse período e rompendo o equilíbrio das sinalizações parácrinas prostáticas, com aumento simultâneo de IGFR-1 e MMP-9 e geração de fatores pró e anti-angiogênicos em resposta ao tratamento com estrógenos e andrógenos, respectivamente. Assim, concluiu-se que a terapia hormonal, apesar de seus efeitos positivos sobre as DGs, gerou microambiente prostático reativo, caracterizado por aumento de um fator mitogênico e da remodelação tecidual bem como por desequilíbrio da angiogênese, o que possivelmente comprometeu a função do órgão e o predispôs a desordens glandulares / Abstract: Androgen replacement is an alternative to minimize the harmful effects of hormonal imbalance in elderly men, even though its influence on the development of prostatic diseases is unclear. Thus, the aim herewith was to characterize structural and molecular features of the ventral prostate of senile rats submitted to steroid hormone replacement, relating the alterations resulting from hormonal therapy to possible prostatic lesions. Male rats (Sprague-Dawley) were divided into Young group (YNG) (4 months old rats), which received peanut oil (5 mL/Kg, s.c.), and senile groups (10 months old rats), submitted to the treatments: Senile group (SEN): peanut oil (5 mL/Kg, s.c.); Testosterone group (TEST): testosterone cipionate (5 mg/Kg, s.c.); Estrogen group (EST): 17?-estradiol (25 ?g/Kg, s.c.); Castrated group (CAS): surgical castration; Castrated-Testosterone group (CT): castration and after 30 days treatment similar to TEST group; Castrated-Estrogen group (CE): castration and after 30 days treatment similar to EST group. After 30 days treatment, blood samples were collected for hormonal analysis and ventral prostate samples were processed for light and transmission electronic microscopies, morphometric, immunohistochemical and Western Blotting analysis. The investigated molecules were alfa and beta dystroglycans (?DG and ?DG), insulin-like growth factor receptor-1 (IGFR-1), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and endostatin. Decreased serum testosterone levels were verified in senescence, with an increase after hormonal replacement in the TEST group. Hypertrophied stroma with inflammatory cells was seen in the SEN group. After hormonal therapy in the senescence or following castration, atrophic and pale cytoplasmatic epithelial cells, microacini and hypertrophied stroma were observed. Decreased DG levels were verified in senescence, but there was an increase of these levels following hormonal therapy, especially in the groups treated with estradiol. Increased IGFR- 1 and MMP-9 protein levels and differential distribution of these molecules were observed in epithelial compartment in those groups which received hormone replacement and in the CAS group. SEN group showed increased VEGF levels in contrast to decreased endostatin levels. Hormonal therapy and castration led to raised VEGF levels, mainly in EST, CAS and CE groups. On the other hand, endostatin was increased especially in those groups submitted to testosterone replacement. The present results suggested that the imbalance between androgens and estrogens in senescence was enhanced after hormone replacement and castration, intensifying structural changes associated with this period. Also, there was a disruption of prostatic paracrine signaling balance, with simultaneous increase of IGFR-1 and MMP-9 and generation of pro and anti-angiogenic factors in response to treatment with estrogens and androgens, respectively. Thus, it could be concluded that despite its positive effects on DGs levels, hormonal therapy created a reactive prostatic microenvironment, characterized by increase of a mitogenic factor and tissue remodelling as well as prostatic angiogenesis imbalance, which could compromise glandular functions and lead to the emergence of prostatic lesions / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

Eukaryotic cells have to divide constantly in order to promote the growth of certain organs, to replace dying or damaged cells, or to set up an entire organism. These essential processes are called mitosis in the case of somatic cell division. Mitotic cell division is the process during which chromosomes, centrosomes, and microtubules (MTs) are involved to set up a bipolar structure called the “mitotic spindle”. This bipolar spindle is formed by MTs, which are presumably mainly organized from the centrosomes. However, more data are being published that suggest MTs nucleation can occur from other MTs or even a chromosome surface. These biopolymers are built from α/β-tubulin heterodimers and can dynamically grow and shrink to exert forces necessary for chromosome segregation. Previous studies of spindles during mitosis have allowed the identification of different MT classes based on their plus-ends interaction with different cellular target sites. One of the MT classes is the kinetochore microtubules (KMTs), which physically connect chromosomes and centrosomes (i.e. spindle pole) via a specialized protein structure termed the “kinetochore”. This kinetochore-to-spindle pole connection has been studied in many organisms. In budding yeast, this connection is established by only a single KMT. In contrast, multiple KMTs bind to each mammalian kinetochore and form an MT bundle also called “k-fiber”. The ultrastructural architecture of the mammalian k-fiber connection is not well documented. Currently, different models concerning the nature of the kinetochore-to-spindle pole connection via k-fibers are discussed in the literature, i.e. a direct, semi-direct or indirect connection. The widely accepted ‘direct’ model proposes that all k-fibers of the mammalian spindle are formed through tight bundles of up to 20 KMTs, with all MT minus ends associated with the centrosome. However, it is necessary to understand the k-fibers structure in order to interpret its role during chromosome segregation. Here the architecture of the k-fiber was studied in human HeLa, U2OS and RPE-1 cell lines, as these different types of cells have been widely used in studies of mitosis. This thesis aimed to systematically investigate the characteristics of mammalian k-fibers and their attachment to the kinetochore within mammalian metaphase spindles. For that, the ultrastructure of mitotic spindles and k-fibers were analyzed using serial-section electron tomography primarily in HeLa cells. Furthermore, the spindle ultrastructure was compared by electron tomography to metaphase spindles in both U2OS and RPE-1 cells. Electron tomographic analysis of the mitotic spindle in HeLa cells revealed that the kinetochore-to-spindle pole connection is formed by k-fibers consisting of ~9 KMTs. Moreover, the data revealed that not all KMTs in k-fibers are directly associated with one of the spindle poles. Instead, KMT ends were located along the length of k-fibers indicating strongly for a semi-direct connection between the kinetochores and the spindle poles. Unexpectedly, by correlating the k-fiber ultrastructure with its position in the mitotic spindle, it can be demonstrated that the k-fiber structure varied depending on the position on the metaphase plate. It can also be shown that k-fibers located in the center of the metaphase plate had a tendency to form straighter and more bundled k-fibers. In contrast, k-fibers associated with the periphery of the metaphase plate had a more loose and disorganized structure resembling a fusiform shape. Furthermore, additional analysis of U2OS and RPE-1 cells indicated ultrastructural differences between the different cell lines. Mainly, differences between HeLa and RPE-1 cells were observed. K-fibers observed in RPE-1 cells showed a lower curvature and overall a more bundled ultrastructure compared to HeLa or U2OS cells. However, due to the small sample size for U2OS and RPE-1 cells, the results have to be confirmed in future experiments to conclude that there are indeed functional and structural differences in the k-fiber organization in different mammalian cell lines. Taken together, this work presents the first detailed quantitative ultrastructural analysis of KMTs in whole spindles in three different human cell lines. The data revealed that the currently favored direct model of k-fiber ultrastructure is oversimplified and needs to be corrected in terms of the k-fibers interaction with the spindle pole and the surrounding MT network within the mitotic spindle. The data here will serve as a structural basis for further analyses of mutant situations and contribute to our understanding of the overall organization and function of MTs in mitotic spindles.:Summary 6 Zusammenfassung 8 List of figures 10 List of tables 13 List of abbreviations and symbols 14 1 Introduction 19 1.1 The morphology of the mitotic spindle 21 1.1.1 Centrosomes 22 1.1.2 Microtubules 23 1.2 Kinetochores, KMTs and k-fibers 28 1.2.1 A brief history of k-fiber formation in mammalian cells 30 1.2.2 Models of the k-fiber ultrastructure in mammalian cells 32 2 Aims of this thesis 35 3 Materials and methods 36 3.1 Materials 37 3.1.1 Mammalian cell lines 37 3.1.2 Chemicals 38 3.1.3 Instrumentation and materials 40 3.1.4 Solutions and buffers 44 3.1.5 Software 46 3.2 Methods 47 3.2.1 Handling of cell cultures 47 3.2.2 Custom-designed incubation chambers 49 3.2.3 Specimen preparation for electron microscopy 51 3.2.4 Quality assessment of samples, acquisition of the tomographic data, and the 3D reconstruction 59 3.2.5 Ultrastructural analysis of MTs in mitotic spindles 62 3.2.6 Ultrastructural analysis of the k-fiber organization 70 4 Results 76 4.1 Initial characterization of mammalian mitotic spindles 77 4.2 Ultrastructure of KMTs 84 4.3 Curvature and tortuosity of KMTs 91 4.4 Ultrastructure of k-fibers 98 4.5 Effect of metaphase position on the k-fiber ultrastructure 102 5 Discussion 110 5.1. Comparison of data sets from different cell lines 111 5.2. Establishing a data analysis pipeline for the analysis of KMTs 113 5.3 Ultrastructural characterization of KMTs and k-fibers in HeLa cells 114 5.3.1 K-fibers have an unexpectedly low number of KMTs 115 5.3.2 Semi-direct kinetochores-to-spindle pole connection 117 5.3.3 Shape of k-fibers 121 5.4 Positional effect on the k-fiber shape 124 5.5 Comparison of k-fiber ultrastructure in different mammalian cells 127 5.6 Outlook 130 References 133 Appendix 1 149 Appendix 2 150 Appendix 3 151 Appendix 4 152 Acknowledgments 153

West Nile virus (WNV) is a widespread emerging zoonotic neurotropic flavivirus cycling naturally between mosquitos and birds. WNV causes disease in 20% of infections in the most susceptible incidental hosts which are horses and humans. Up to 40% of affected horses and 1- to approximately 50% of affected humans develop neurological signs and/or flaccid paralysis, in some cases fatal or severely debilitating, due to variable encephalitis, meningitis and poliomyelitis. Two predominant genetic lineages exist, 1 and 2, with neurovirulent lineage 1 strains recorded in the northern and western hemispheres, the milder lineage 1 Kunjin strain in Australia, and the lineage 2 strain endemic to southern Africa and Madagascar and considered, until recently, to have mainly mildly pathogenic strains. Since 2002 investigations into South African lineage 2 WNV strains showed that they resulted in severe neurological disease in horses and humans. From 2004 lineage 2 strains were recorded for the first time in southern Europe as a cause of neurological signs and death in birds, and increasingly, in horses and humans. In 2011 the mild lineage 1 Kunjin strain mutated to an equine neurovirulent strain in New South Wales, Australia, and in 2010 the first South African case of lineage 1 WNV was reported from the western Cape in a mare which showed severe neurological signs, abortion and death. Laboratory strains of mice are extremely susceptible to WNV and have been mostly used in experimental studies since the 1937 discovery of the virus in Uganda. In the early 2000s studies in mice showed that field strains of lineage 1 and 2 WNV ranged from mildly pathogenic to highly neurovirulent, however, the associated pathology of the lineage 2 infections was not studied. In the current study, the macroscopic and microscopic pathology of a South African human-neurovirulent field strain of lineage 2 WNV (SPU93/01) and the neurovirulent lineage 1 (NY99/385) strain were investigated and compared in mice used as controls in 2 WNV vaccine studies. The clinical signs, CNS and extra-CNS pathology were indistinguishable between the lineages and some lesions were comparable to those previously reported. Lineage 1 WNV equine pathology has been well described but that of lineage 2 only briefly previously described. The pathology in 6 naturally-occurring fatal lineage 2 WNV-infected horses with severe neurological signs, was investigated and compared with that of the single South African lineage 1 WNV field infection. Diagnoses were confirmed by real-time RT-PCR. Similarities and some slight differences in lesions were found in both mouse and horse studies when compared with lineage 1 pathology cases and with previous reports, and the neurovirulence of the lineage 2 field strains was confirmed. WNV immunohistochemistry (IHC) of all mouse tissues allowed speculation as to pathogenesis of intestinal lesions, but in equine CNS lesions was mostly negative. Ultrastructure of IHC positive cells showed rare WNV particles. In the horse cases rabies, equine herpes virus, and other arboviral co-infections were excluded and similarities and implications of gross lesions of African horsesickness to those often seen in WNV infections were discussed. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Medical Virology / unrestricted

Material und Methoden: Insgesamt 40 Proben glatter und rauer Oberflächen von Titanimplantaten wurden mit Parodontalsonden aus Metall oder Kunststoff in einem Winkel von 20° oder 60° bearbeitet. Zur Bestimmung verschiedener standardisierter 2D- und 3D-Rauheitsparameter wurden die Titanoberflächen mit einem konfokalen Laser-Scanning-Mikroskop (CLSM) vor und nach der Durchführung evaluiert. Ergebnisse: Die durchschnittliche Profil- und Oberflächenrauheit (Ra und Sa) zeigten unabhängig vom Sondenmaterial keinen signifikanten Unterschied zwischen behandelten und unbehandelten Proben auf glatten und rauen Oberflächen. Auf glatten Oberflächen führten Metallsonden zu erhöhten Amplitudenrauheiten wobei nur für Rp eine Signifikanz (p = 0,007) erreicht wurde. Raue Oberflächenbereiche wurden nach dem Kontakt mit Metallsonden leicht aber nicht signifikant geglättet. Nach Verwendung von Kunststoffsonden blieb die Oberflächenrauhigkeit von glatten und rauen Bereichen nahezu unverändert. Unabhängig des Sondenmaterials zeigte sich kein Zusammenhang zum Applikationswinkel. Fazit: Das Sondieren von Titanimplantaten mit Kunststoffsonden aber auch mit Metallsonden verursacht nur geringe Veränderungen der Oberflächenrauhigkeit. Die klinische Bedeutung dieser Veränderung bleibt ungeklärt. Klinische Relevanz: Ultrastrukturelle Veränderungen an Titan-Oberflächen durch periimplantäre Untersuchungen könnten durch die Verwendung von Kunststoffsonden vermieden werden.

Indiana University-Purdue University Indianapolis (IUPUI) / Since their introduction in 1970, glass ionomer cements have been used in a wide variety of clinical situations in dentistry. The main advantages of glass ionomer cements are chemical bonding, fluoride release and uptake, excellent seal against microleakage, and biocompatibility. The main objective of this study was to compare the microleakage of two new paste-paste glass ionomer systems to their traditional RMGIC counterparts when conditioning the dentin with newly developed no-rinse conditioners or polyacrylic acid. Materials and methods: Standardized cavity preparations were made, centered on the cementoenamel junction of the buccal surface, on 96 extracted human molars divided in 8 groups (n = 12). G1 Ketac Nano with Ketac Nano Primer, G2 Ketac Nano with Ketac Conditioner, G3 Photac Fil with Ketac Nano Primer, G4 Photac Fil with Ketac Cavity Conditioner, G5 Fuji Filling LC with GC Self Conditioner, G6 Fuji Filling LC with GC Cavity Conditioner, G7 Fuji II LC with GC Self Conditioner, G8 Fuji II LC with GC Cavity Conditioner. The cavities were treated with either a no-rinse or polyacrylic acid conditioner and restored with a paste-paste RMGIC or traditional RMGIC from the same manufacturer (n =12). The teeth were then sealed to within 2 mm of the restoration margins and thermocycled. The teeth were immersed in 2.0-percent methylene blue and stored at room temperature for 24 hours. Then, the teeth were be embedded in resin and sectioned longitudinally in a buccolingual direction making 1 section (1 mm thick) per tooth. The occlusal and gingival restoration margins of each specimen were examined with a stereomicroscope at X10 magnification to determine the degree of microleakage. Results: Mixed-model ANOVA was used to test the fixed effect of the eight groups and cervical vs. occlusal location within each tooth sample on microleakage, with sample as the random effect. Both main effects and the interaction are significant, p < 0001 for both group and location effects, and p = 0.0013 for the interaction of group and location. The cervical interface showed more microleakage in all groups except group 8 where microleakage was the same as at the occlusal margin. No significant difference was observed among groups for microleakage at the occlusal interface. There was significant difference among groups at the cervical interface with Fuji II LC using GC Cavity Conditioner performing best. For the occlusal interface Group 4 performed the best and Group 2 performed the worst, although the difference was not significant among the groups. For the cervical interface, Group 8 performed the best followed by Group 3, Group 4 and Group 6, although these four groups were not significantly different. For the cervical interface, group 2 performed the worst followed by group 1. Based on these results we can conclude that, overall, traditional RMGIC with polyacrylic acid conditioning performed better than the new paste-paste RMGIC systems utilizing the no-rinse conditioners.

No description available.

No / We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

The main aim of this study was to investigate the effects of water availability and SO2 pollution, imposed separately or simultaneously, on the photosynthetic metabolism of Augea capensis Thunb., a succulent of the Namib Desert in the region of Skorpion Zinc mine, Namibia. The main driver for this investigation was the need to distinguish between the effects of water availability on plants native to a desert environment, where water availability dominates plant response, but where the possibility of anthropogenic SO2 pollution poses a new threat to the unique succulent vegetation. Fifteen measuring sites were selected in the vicinity of the mine to determine how rainfall influenced the physiological status of the vegetation. Chlorophyll a fluorescence measurements, and analysis of recorded OJlP fluorescence transients with the JIP-test, were used for this purpose. A series of laboratory experiments were also conducted on A. capensis to determine the precise physiological response that water deprivation and SO2 pollution had under controlled growth conditions. Potted plants were exposed to water deprivation or SO2 fumigation in the light or dark. Besides chlorophyll a fluorescence, photosynthetic gas exchange and Rubisco activity were also measured. Changes in fast fluorescence rise kinetics observed under field conditions suggest considerable modulation of photosystem II function by rainfall with concomitant involvement of a heat stress component as well. In both the field and laboratory experiments, one of the JIP-test parameters, the so-called performance index (PIABS), was identified as a very sensitive indicator of the physiological status of the test plants. Moreover, under laboratory conditions, a good correlation existed between the water deprivation-induced decline in CO2 assimilation rates and the decline in PIABS values. The JIP-test in general, and the PIABS in particular, shows considerable potential for application in the investigation of water availability influences on desert ecosystems. In the laboratory experiments, water deprivation caused stomatal closure but also a slight elevation in intercellular C02 concentration and inhibition of Rubisco activity, suggesting that mesophyll limitation was the dominant factor contributing to the decrease in C02 assimilation rates. Following re-watering, A. capensis showed remarkable recovery capacity. Fumigation of A. capensis with 1.2 ppm SO2 in the dark or light revealed relatively small effects on C02 assimilation. The inhibitory effects on photosynthesis were also fully reversible, indicating no permanent metabolic/structural damage. The effects on photosynthesis were more pronounced when fumigation occurred in the dark. This phenomenon might be related to diurnal differences in cellular capacity for SO2 detoxification. When long-term moderate water deprivation was combined with simultaneous SO2 fumigation, there was no additional inhibitory effect on photosynthesis. These findings suggest that water deprivation do not increase sensitivity towards SO2 pollution in A. capensis. Fumigation with SO2, singly or in combination with water deprivation also had no major effect on chloroplast ultrastructure. It appears that A. capensis is remarkably resistant to SO2 pollution even in the presence of low water availability, which is a common phenomenon in desert ecosystems. Since A, capensis seems to be highly tolerant to S02, its suitability as an indicator species for the detection of SO2 pollution effects at Skorpion Zinc mine is questionable. Because water availability dominates the physiological/biochemical response in this species, subtle SO2 pollution effects might be difficult to detect against this dominant background. The high water content of A. capensis and similar succulents might act as a substantial sink for SO2 and could convey considerable tolerance against this form of air pollution. / Thesis (M.Sc. (Botany))--North-West University, Potchefstroom Campus, 2006.

A podridão floral dos citros (PFC) é uma doença causada pelo fungo Colletotrichum acutatum responsável por causar grandes danos à produção de citros no Brasil. A doença surge apenas em botões florais com 8mm de comprimento ou maiores, levando a formação de lesões alaranjadas nas pétalas, lesões necróticas no estigma, promove a queda prematura dos frutos e a retenção do cálice e pedúnculo, sendo este último sintoma denominado estrelinha. Este trabalho tem por objetivo: observar o modo de penetração do fungo no hospedeiro Citrus sinensis Valência e os estágios posteriores da colonização, verificar se há fatores estruturais e químicos pré-formados que expliquem o porquê do fungo não conseguir infectar botões florais com menos de 8mm, caracterizar anatomicamente o sintoma estrelinha e estigmas lesionados, investigar ultraestruturalmente pétalas inoculadas, analisar se há o estabelecimento de uma infecção quiescente nos tecidos foliares, analisar grãos de pólen após a inoculação in vivo e in vitro com o fungo. Botões florais sadios, pétalas e estigmas com e sem lesões, foram submetidos às técnicas convencionais de microscopia de luz e eletrônica. Folhas e grãos de pólen foram inoculados e analisados. Foi desenvolvida uma nova técnica de coloração para tecidos vegetais infectados por fungos. A resistência dos botões florais menores que 8mm pode estar associada às barreiras químicas e estruturais pré-formadas. O ápice, nesses botões, apresenta papilas entremeadas, cristais de oxalato de cálcio no mesofilo e câmara subestomática e cavidades de óleo localizadas muito próximas umas das outras. Botões com 8mm e 12mm possuem, no ápice, papilas com arranjo frouxo, ausência de cristais e maior distanciamento entre as cavidades de óleo. No ápice da pétala, verificou-se que as células papilosas são osmóforos. No sintoma estrelinha, nota-se sob a região de abscisão do ovário a instalação de um meristema de cicatrização. A lignificação das paredes das células da medula do receptáculo e do pedúnculo floral está associada à retenção destas estruturas na planta. Nas pétalas infectadas, o C. acutatum pode penetrar intra, intercelularmente e via estômato. O fungo pode crescer de modo subcuticular e intramural e coloniza todos os tecidos da pétala. A nova técnica de coloração se mostrou muito útil nas análises histopatológicas. O fungo associa-se aos tecidos vasculares. Acérvulos ocorrem em ambas as faces das pétalas. A cutícula nos estágios mais avançados da lesão apresenta-se alterada, ou seja, ocorre a perda da ornamentação estriada e maior deposição de material lipofílico. A síntese de materiais lipofílicos envolve o retículo endoplasmático liso e rugoso e plastídios. Vesículas provenientes de dictiossomos e de corpos multivesiculares são observadas ao longo da parede celular e estão associadas ao depósito de material lipofílico na cutícula. No estigma lesionado há a formação de uma camada de proteção. O fungo apresenta quimiotropismo e cresce em direção aos grãos de pólen infectando-os 24 horas após a inoculação. Sugere-se que C. acutatum pode utilizar grãos de pólen para a sua dispersão. Após 48 horas da inoculação as folhas apresentam conídios germinados com apressórios. / The postbloom fruit drop (PFD) is a disease caused by Colletotrichum acutatum responsible for causing great damage to citrus crops in Brazil. The disease appears only in flower buds 8 mm in length or greater, leading to orange lesions in petals, necrotic lesions on the stigma, promoting the young fruit drop and the retention of the calyx and peduncle, which is called buttons. In this context, this study aimed to: observe the fungus penetration mode into the host Citrus sinensis \'Valência\' and the later stages of colonization; study the presence of preformed structural and chemical factors to explain why the fungus cannot infect floral buds with less than 8 mm in length; characterize anatomically the symptom \"buttons\" and injured stigmas; investigate the ultrastructural changes in tissues of inoculated petals; analyze whether there is the establishment of a quiescent infection in leaf tissues, analyze pollen grains after inoculation in vivo and in vitro with the fungus. Healthy buds, petals and stigmas with and without lesions, were processed and analyzed using conventional light and electron microscopy techniques. Leaves and pollen grains were also inoculated and analyzed with light microscopy. It was developed a new staining method for fungal-infected plant tissues. The resistance of flower buds smaller than 8mm may be associated with preformed structural and chemical barriers. These buttons display the apex with interspersed papillae, with crystals in mesophyll and substomatic chamber and oil cavities, which are located very close to each other on the abaxial surface. In 8mm and 12mm flower buds, the papilas in the apex become weakly interspaced, the crystals are not observed and there is the increase of the distance between the oil cavities. The papillose cells are osmophores. In the symptom \"button\", it is noted in the abscission of the ovary, an installation of wound meristem. There is also the lignification in the pith of receptacle and pedicel that can be associated with the retention of these structures in the plant. In infected petals, it was found that C. acutatum can penetrate intra and intercelullar or via stomata. The fungus may grow subcuticular and intramural and colonize all tissues of petal. The new staining technique developed has proved very useful for histopathological analysis. The fungus is closely associated with vascular tissues. The acervulli occur on both surfaces of petals. The cuticle in the later stages of the lesion is altered, i.e., there is loss of striated ornamentation and increased deposition of lipophilic material. The synthesis of lipophilic materials involves rough and smooth endoplasmic reticulum and plastids. Vesicles from dictyosomes and multivesicular bodies were observed throughout the cell wall and are associated with the deposit of lipophilic material in the cuticle. There is the formation of protective layer over the stigma damaged area. The fungus shows chemotropism and grows toward the pollen infecting it 24 hours after inoculation. It is suggested that C. acutatum can use pollen grains for dispersal. After 48 hours of inoculation, the leaves have germinated conidia with appressoria.

O presente trabalho explora os aspectos taxonomicos, ecologicos, morfologicos, biometricos e biogeograficos dos Arcellinida Kent, 1880 coletados no Parque Ecologico do Rio Tiete, Sao Paulo Brasil. Foram encontrados organismos pertencentes a cerca de 30 taxons nominais, no entanto, a revisao da literatura, novos dados morfologicos obtidos atraves do Microscopio Eletronico de Varredura e medidas biometricas realizadas com grande numero de individuos permitem afirmar que muitos destes taxons estao se referindo a mesma entidade na natureza. Logo, na presente pesquisa sao descritas, com detalhes de distribuicao geografica, morfologia ultra-estrutural, morfometria e ecologia, especies pertencentes a quatro familias e cinco generos: Difflugia corona Wallich, 1864; Difflugia gramen Penard, 1902; Difflugia lanceolata Penard, 1890; Difflugia claviformis Penard, 1899; Difflugia gigantea Chardez, 1967; Centropyxis aculeata (Ehrenberg, 1838); Netzelia wailesi (Ogden, 1980); Lesquereusia modesta Rhumbler, 1895; Lesquereusia mimetica Penard, 1911; Arcella hemisphaerica Perty, 1852; Arcella gibbosa Penard, 1890; Arcella discoides Ehrenberg, 1871 e Arcella brasiliensis Cunha, 1913. Sao discutidas inovacoes taxonomicas para que a comparacao de dados obtidos usando tecnicas atuais com aqueles reportados na literatura tradicional seja feita da maneira mais explicita possivel, de modo a delimitar melhor o conceito taxonomico de cada especie abordada. / The present survey explores the taxonomic, ecologic, morphologic, biometric and biogeographic aspects of the Arcellinida Kent, 1880 collected at the Ecological Park of the Tiete River, Sao Paolo Brazil. Around 30 nominal taxa were identified, however, a review of the literature and new morphologic data obtained via the Scanning Electron Microscope and biometric measures with a large number of individuals allow the inference that many of these taxa are referring to the same natural entity. Therefore, the present work describes species from four families and five genera, along with details about geographic distribution, ultra-structural morphology, morphometry and ecology: Difflugia corona Wallich, 1864; Difflugia gramen Penard, 1902; Difflugia lanceolata Penard, 1890; Difflugia claviformis Penard, 1899; Difflugia gigantea Chardez, 1967; Centropyxis aculeata (Ehrenberg, 1838); Netzelia wailesi (Ogden, 1980); Lesquereusia modesta Rhumbler, 1895; Lesquereusia mimetica Penard, 1911; Arcella hemisphaerica Perty, 1852; Arcella gibbosa Penard, 1890; Arcella discoides Ehrenberg, 1871 e Arcella brasiliensis Cunha, 1913. Taxonomic innovations are discussed in order to make comparison of recent data with those reported on traditional literature a more explicit practice, allowing a better understanding of each species taxonomic concept.