This project examined multiple aspects of the reproductive biology of the wetland invasive species, purple loosestrife (<i>Lythrum salicaria</i> L.), in central Saskatchewan. An examination of insect taxa visiting the three floral morphs of <i>Tristylous</i> L. <i>salicaria</i>, as well as a ranking of the pollination efficiency of individual insect species, an apparent first for L salicaria, was undertaken. Surface features of the floral nectary of L. <i>salicaria</i>, as well as floral nectar secretion dynamics, were also investigated. This project also re-visited some of the previous work done on this invasive species, including various floral organ morphometrics in relation to heterostyly, and aspects of the tristylous breeding system including self-fertilization, and fertilization potential of both illegitimate pollination and legitimate pollination.<p>The trimorphic nature of the sexual floral organs of L. <i>salicaria</i> were well defined in Saskatchewan. Significant differences in length (long-, intermediate- and short-style lengths) exist between all three floral morphs. Lengths of the staminal filaments (long, intermediate, and short) were also significantly different. Also the floral nectary in L. <i>salicaria</i> is located in a depression formed at the interface of the hypanthium and the gynoecium. Several stomata are located at regular intervals along the nectary surface, and may constitute the escape route for floral nectar. No morphological differences in nectary structure were apparent among the three floral morphs.<p>Nectar secretion dynamics of L. <i>salicaria</i> were examined between the three floral morphs throughout two summer days in 2006. Peak average nectar volumes and nectar sugar quantities were detected at 3:00 pm, and, interestingly, no significant differences were detected between floral morphs, in accordance with nectary morphology. The estimated secretion rates for L. <i>salicaria</i> ranged from 61 83 µg of nectar sugar per flower per hour.<p>Hand-pollination experiments carried out over the summers of 2006 and 2007 at three field sites in and around Saskatoon have verified the strong self-incompatibility in the breeding system of this tristylous species. Intramorph pollination, using illegitimate pollen, did not result in fertilisation, whereas legitimate hand-pollination experiments yielded multiple pollen tubes at the style base, without exception.<p><i>Lythrum salicaria</i> in central Saskatchewan was visited by several bee taxa including honeybees (<i>Apis mellifera</i> L.), bumblebees (Bombus spp.), leafcutter bees (Megachile spp.), and sweat bees (Lasioglossum spp.). A single visit by <i>Anthophora furcata</i> (Panzer) was also recorded in 2007. Generally, bee visits led to high levels of pollination success as determined by fluorescence microscopy of pollen tubes following single insect visits to previously-unvisited flowers. However, most visits by hoverflies (Syrphidae) were non-pollinating. Visits by Pieris rapae (L.), yellowjacket wasps (Vespidae) and some non-syrphid flies (Diptera) also yielded no pollen tubes at the style base.<p>A study of the ultrastructure and development of the floral nectary of the purple loosestrife cultivar Morden Gleam (<i>Lythrum virgatum</i> L. x L. alatum Pursh.) showed that starch build up in pre-secretory nectary tissues declined throughout secretion, and is virtually absent in post-secretory nectary tissues. The lack of a direct vascular supply to the floral nectary suggests that the starch breakdown products likely make up most of the floral nectar carbohydrates. Surface features of the floral nectary in Morden Gleam closely resembled those of L. salicaria, located in the valley formed between the hypanthium and gynoecium. Nectary stomata, occasionally in pairs, likely serve as outlets for nectar in this cultivar.

This project examined multiple aspects of the reproductive biology of the wetland invasive species, purple loosestrife (<i>Lythrum salicaria</i> L.), in central Saskatchewan. An examination of insect taxa visiting the three floral morphs of <i>Tristylous</i> L. <i>salicaria</i>, as well as a ranking of the pollination efficiency of individual insect species, an apparent first for L salicaria, was undertaken. Surface features of the floral nectary of L. <i>salicaria</i>, as well as floral nectar secretion dynamics, were also investigated. This project also re-visited some of the previous work done on this invasive species, including various floral organ morphometrics in relation to heterostyly, and aspects of the tristylous breeding system including self-fertilization, and fertilization potential of both illegitimate pollination and legitimate pollination.<p>The trimorphic nature of the sexual floral organs of L. <i>salicaria</i> were well defined in Saskatchewan. Significant differences in length (long-, intermediate- and short-style lengths) exist between all three floral morphs. Lengths of the staminal filaments (long, intermediate, and short) were also significantly different. Also the floral nectary in L. <i>salicaria</i> is located in a depression formed at the interface of the hypanthium and the gynoecium. Several stomata are located at regular intervals along the nectary surface, and may constitute the escape route for floral nectar. No morphological differences in nectary structure were apparent among the three floral morphs.<p>Nectar secretion dynamics of L. <i>salicaria</i> were examined between the three floral morphs throughout two summer days in 2006. Peak average nectar volumes and nectar sugar quantities were detected at 3:00 pm, and, interestingly, no significant differences were detected between floral morphs, in accordance with nectary morphology. The estimated secretion rates for L. <i>salicaria</i> ranged from 61 83 µg of nectar sugar per flower per hour.<p>Hand-pollination experiments carried out over the summers of 2006 and 2007 at three field sites in and around Saskatoon have verified the strong self-incompatibility in the breeding system of this tristylous species. Intramorph pollination, using illegitimate pollen, did not result in fertilisation, whereas legitimate hand-pollination experiments yielded multiple pollen tubes at the style base, without exception.<p><i>Lythrum salicaria</i> in central Saskatchewan was visited by several bee taxa including honeybees (<i>Apis mellifera</i> L.), bumblebees (Bombus spp.), leafcutter bees (Megachile spp.), and sweat bees (Lasioglossum spp.). A single visit by <i>Anthophora furcata</i> (Panzer) was also recorded in 2007. Generally, bee visits led to high levels of pollination success as determined by fluorescence microscopy of pollen tubes following single insect visits to previously-unvisited flowers. However, most visits by hoverflies (Syrphidae) were non-pollinating. Visits by Pieris rapae (L.), yellowjacket wasps (Vespidae) and some non-syrphid flies (Diptera) also yielded no pollen tubes at the style base.<p>A study of the ultrastructure and development of the floral nectary of the purple loosestrife cultivar Morden Gleam (<i>Lythrum virgatum</i> L. x L. alatum Pursh.) showed that starch build up in pre-secretory nectary tissues declined throughout secretion, and is virtually absent in post-secretory nectary tissues. The lack of a direct vascular supply to the floral nectary suggests that the starch breakdown products likely make up most of the floral nectar carbohydrates. Surface features of the floral nectary in Morden Gleam closely resembled those of L. salicaria, located in the valley formed between the hypanthium and gynoecium. Nectary stomata, occasionally in pairs, likely serve as outlets for nectar in this cultivar.

Lignocellulosic bioethanol is currently being explored as a substitution to fossil fuels. Many lignocellulosic materials are being examined but the importance is to find those with attractive agro-energy features. Producing lignocellulosic ethanol is challenging because lignocellulosic biomass is resistant to chemical and biological degradation. To reduce biomass recalcitrance, a pretreatment stage is required. Pretreatment is considered to be the most intensive operating/operating cost component of cellulosic ethanol production. Therefore, research is heavily focused on understanding the effect of pretreatment technologies on the fundamental characteristics of lignocellulosic biomass. The first study in the thesis investigates Buddleja davidii as a potential biomass source for bioethanol production. The work focuses on the determination of ash, extractives, lignin, hemicellulose, and cellulose content in this plant, as well as detailed elucidation of the chemical structures of both lignin and cellulose by NMR spectroscopy. The study showed that B. davidii has several unique agro-energy features as well as some undesired characteristics. The second study presents research on the ethanol organosolv pretreatment (EOP) of B. davidii and its ability to produce enzymatically hydrolysable substrates. It was concluded that the removal of hemicellulose, delignification, reduction in the degree of polymerization (DP) of cellulose, and the conversion of crystalline cellulose dimorphs (Iα/Iβ) to the easily degradable para-crystalline and amorphous celluloses were the characteristics accounted for efficient enzymatic deconstruction of B. davidii after EOP. The third study provides a detailed elucidation of the chemical structure of ethanol organosolv lignin (EOL) of B. davidii by NMR spectroscopy. Such research was needed to understand the pretreatment mechanism in the context of delignification and alteration of the lignin structure. Future applications of the resulted EOL will be valuable for industrially viable bioethanol production process. EOP mainly cleaved β-O-4' interlinkages via homolysis, decreased the DP of lignin, and increased the degree of condensation of lignin. EOL had low oxygen content, molecular weight, and aliphatic OH as well as high phenolic OH, which are qualities that make it suitable for different co-product applications. The last study provides information on the anatomical characteristics of pretreated B. davidii biomass after EOP. The importance of this research was to further understand the alterations that occur to the cellular structure of the biomass which can then be correlated with its enzymatic digestibility. The results concluded that the physical distribution of lignin within the biomass matrix and the partial removal of middle lamella lignin were key factors influencing enzymatic hydrolysis.

Introdução: A hemodiluição normovolêmica aguda (HNA) é estratégia utilizada para diminuir a necessidade de transfusão em procedimentos cirúrgicos. O hematócrito alvo, a solução empregada e os riscos da HNA são controversos e, embora as ações sobre os sistemas nervoso e cardiocirculatório tenham sido extensamente pesquisados, há poucos estudos sobre implicações pulmonares da HNA. O objetivo deste estudo é avaliar os efeitos pulmonares da HNA comparando amido hidroxietílico e Ringer lactato. Métodos: Vinte e cinco porcos foram monitorizados e cateterizados. Utlizou-se anestesia intravenosa contínua com propofol, cetamina, fentanil e pancurônio. Após estabilização da anestesia, os animais foram randomizados: Grupo Controle (n=8), Grupo HES (amido hidroxietílico, n=7) e Grupo RL (Ringer lactato, n=10). Os animais dos grupos II e III foram submetidos a HNA até hematócrito pré-estabelecido de 15%. A retirada do sangue foi acompanhada da expansão plasmática com amido hidroxietílico ou Ringer lactato na proporção de 1:1 ou 3:1 respectivamente. Medidas de mecânica respiratória e oxigenação sangüínea foram realizadas antes da retirada de sangue (T0), ao término da hemodiluição (T1) e após uma e duas horas (T2 e T3). Ao final foram realizadas biópsias pulmonares. Os dados foram submetidos à análise de variância para medidas repetidas (ANOVA) seguidas pelo teste de Tukey. Resultados: Em relação aos parâmetros do grupo controle, não houve diferença significante durante todo o procedimento. O grupo Ringer lactato demonstrou diminuição da complacência estática e aumento da diferença alvéolo-arterial de O2 (DAa-O2) em relação ao controle (*P<0,05). A análise histopatológica evidenciou atelectasias e alargamento da membrana basal na barreira alvéolo-capilar no grupo Ringer lactato. O grupo amido hidroxietílico apresentou comportamento semelhante ao grupo controle. Conclusões: O estudo sugere que na HNA acentuada, o amido hidroxietílico é superior na preservação estrutural do pulmão, evidenciado pormanter a complacência pulmonar e a oxigenação, quando comparada à realizada com solução de Ringer lactato / Introduction: Acute normovolemic hemodilution (ANH) is a common procedure to decrease the needs of blood transfusion during a variety of surgical procedures. There are controversies regarding ANH such as the target hematocrit, the solution to be used and its risks. Although its effects in heart and brain have been extensively studied; investigations concerning its pulmonary effects are lacking. The aim of this experiment was to compare the pulmonary effects of ANH with hydroxyethyl starch and lactated Ringer\'s. Methods: Twenty five pigs were instrumented for standard hemodynamic monitoring. Intravenous anesthesia consisted continuous infusion of propofol, ketamine, fentanyl and pancuronium. After anesthesia stabilization animals were randomized: Control group (n=8), HES group (hydroxyethyl starch, n=7) and LR group (lactated Ringer\'s, n=10). Animals of Control Group were anesthetized and observed. Group Animals of group HES and LR were submitted to ANH to reach a pre-established hematocrit around 15%. Blood withdrawal was accomplished simultaneously with plasma expansion performed with hydroxyethyl starch (1:1) or with lactated Ringer\'s (3:1). Pulmonary mechanics and oxygen transport and were measured before blood withdrawal (T0), at the end of hemodilution (T1), and one and two hours after the end of the hemodilution (T2, T3). Lungs biopsies were performed at the end. Data were submitted to analysis of Variance for repeated measures followed by the Tukey test. Results: In regard to all parameters of Control group, there were no significant differences during the whole procedure. The LR group demonstrated a decrease in compliance and an increased in (a-A)DO2 (*P<0,05). Hystopathological lung analysis revealed moderate to serious collapses as well as basement membrane enlargement in group LR. The HES data were similar to that found in control group. Conclusion: These results suggest that in severe ANH, HES preserves pulmonary structure and seems to less interfere with pulmonary mechanics and oxygenation indexes when compared to lactated Ringer\'s

INTRODUÇÃO: O conhecimento sobre os tipos celulares envolvidos na patogenia da urticária constitui um elemento essencial para a compreensão da fisiopatologia desta doença. Poucos autores têm dado atenção às interações entre mastócitos e dendrócitos da derme na urticária. Os objetivos deste estudo são orientados no sentido de descreverem-se os tipos de degranulação mastocitária na urticária aguda associada a medicamentos, e o de analisarem-se as interações entre dendrócitos da derme e mastócitos. MÉTODOS: Sete doentes com urticária aguda associada com medicamentos foram incluídos neste estudo. Foram obtidas biopsias cutâneas das lesões urticadas e da pele aparentemente normal destes doentes. Os quatorze fragmentos coletados foram divididos em duas partes (28 fragmentos): uma das partes foi enviada para processamento pela coloração de hematoxilina-eosina, para a coloração de Azul de Toluidina e reações de imunoistoquímica com anticorpos anti-CD34, antifator XIIIA (anti- FXIIIa) e antitriptase e o outro fragmento foi processado para uso na microscopia imunoeletrônica, utilizando-se anticorpos para triptase e FXIIIa, além de dupla imunomarcação com ouro com o uso de anticorpos antitriptase e anti-FXIIIa. RESULTADOS: células imunomarcadas com anticorpos anti-CD34 foram observadas de forma esparsa na derme superficial e de forma mais proeminente na derme reticular. Havia múltiplos dendrócitos dérmicos FXIIIa+ na derme superficial e média, dispersos nas regiões subepidérmicas e em torno doa vasos da derme, tanto na pele urticada com na pele aparentemente normal. O número destas células foi similar nos dois grupos de amostras. Não houve diferença estatística entre o número de células triptase-positivas na pele aparentemente normal e na pele urticada, em todos os doentes. Nós observamos mastócitos íntegros na maioria das amostras da pele aparentemente normal. Tanto as amostras de pele aparentemente normal quanto as amostras de pele urticada apresentavam mastócitos em processo de degranulação do tipo anafilático, com inúmeros grânulos extruídos. Após a dupla imunomarcação com ouro, na imuno-microscopia eletrônica de transmissão foram observadas partículas de ouro de 10 nm (FXIIIa) e 15 nm (Triptase) marcando concomitante os grânulos dos mastócitos indicando que tanto a triptase como o FXIIIa encontraram-se presentes nos grânulos destas células. De forma interessante, nós encontramos uma forte evidência de que grânulos contendo tanto FXIIIa, como triptase, extruídos dos mastócitos são fagocitados pelos dendrócitos da derme. CONCLUSÕES: na urticária aguda associada a medicamentos o padrão de degranulação observado foi do tipo anafilático. Este estudo constitui a primeira demonstração da expressão do FXIIIa nos grânulos intracitoplasmáticos e nos grânulos extruídos dos mastócitos, dispersos na matriz extracelular, nos doentes com urticária aguda associada a medicamentos. Outro fato inédito foi a demonstração da fagocitose dos grânulos extruídos dos mastócitos pelos dendrócitos da derme FXIIIa+ / BACKGROUND: The knowledge about the cell types involved in urticaria is an essential element for understanding the pathophysiology of this disease. Few authors have been attempting on interactions among mast cells and dermal dendrocytes in urticaria. The aims of this study are to describe the types of mast cell degranulation in drug-induced acute, besides to analyze the interactions between mast cell and dermal dendrocyte in urticaria. METHODS: Seven patients with drug-induced acute urticaria were enrolled in the study. We token skin biopsies of urticarial lesion and apparently normal skin. The fourteen fragments collected were divided into two parts (28 sections): one to haematoxylin-eosin stain, Toluidine blue stain and immunohistochemisty reactions with anti-CD34, anti-FXIIIa and anti-tryptase antibodies and other part to immunogold electron microscopy using single antibodies to tryptase and FXIIIa, besides double immunogold labelling with anti-tryptase and anti-FXIIIa. RESULTS: immunolabelled CD34+ cells were observed scattered in the superficial dermis and more prominent in the reticular dermis. There were multiple FXIIIa+ dermal dendrocytes in upper and mid dermis, dispersed in subepidermal areas and around blood vessels, both in apparently normal skin and urticarial lesion of drug-induced acute urticaria. The number of these cells was similar in both groups. There was no difference in tryptase positive cells number between apparently normal skin and urticarial lesions, in all patients. We observed intact mast cells in the majority of the sections of the apparently normal skin. Some sections demonstrated a few mast cells in degranulation process, in anaphylactic degranulation type. In urticarial lesions, several mast cells showed degranulation process, in anaphylactic degranulation type. After double immunogold staining, 10 nm (FXIIIa) and 15 nm (Tryptase) gold particles were seen together over the granules in mast cells indicating that tryptase and FXIIIa are each localized into granules of these cells. Interestingly, we found a strong evidence of than the exocytosed mast cell granules contents both FXIIIa and Tryptase immunolabelled are phagocytised by dermal dendrocytes. CONCLUSIONS: In drug-induced acute urticaria the degranulation pattern of mast cells found was composed by anaphylatic. This is the first report, in acute urtuicaria, concern of the expression of FXIIIa in the cytoplasmic mast cell and in extruded granules into extracellular matrix. Phagocytosis of the extruded mast cell granules by FXIIIa+ dermal dendrocytes in urticaria was observed

Desde o início de 1990, a enxertia de gordura na prega vocal é descrita como um método para reparar a insuficiência glótica. O objetivo deste estudo é avaliar os efeitos da aplicação tópica de mitomicina-C no processo cicatricial de enxertos autólogos de gorduras inseridos em pregas vocais de coelhos através da medida da deposição de colágeno. Vinte e oito coelhos foram submetidos a enxertia de gordura em ambas pregas vocais. As pregas vocais direitas recebeu previamente a aplicação tópica de mitomicina-C (0,4mg/ml) durante cinco minutos enquanto que as pregas vocais esquerdas formavam o grupo controle (sem mitomicina-C). Quatro grupos com 6 coelhos cada foram sacrificados com 7, 14, 30 e 90 dias após a cirurgia de enxertia. As pregas vocais foram removidas para estudo histológico com a intenção de quantificar a deposição de colágeno através da coloração por Picrossírius Red sob microscopia polarizada. A deposição de colágeno foi menor em todos os grupos de pregas vocais que receberam aplicação tópica de mitomicina-C quando comparada com as pregas vocais do grupo controle. No presente estudo, a aplicação tópica de mitomicina-C antes da enxertia de gordura reduziu significativamente a deposição de colágeno (p = 0,05). / Since the early 1990s, fat implantation in the vocal fold is described as a method of repairing glottal insufficiency. The aim of this study was to evaluate the effect of topical application of mitomycin in the healing process with collagen deposition measurement around of autologous fat implants inserted in rabbits vocal folds. Twenty-eight rabbits were submitted to a fat implant in the both vocal folds. The right vocal folds received previously topical application of mitomycin (0,4mg/ml) for five minutes and the left vocal folds were the control group (without mitomycin). Four groups of 6 rabbits each were sacrificed 7, 14, 30 and 90 days after the implantation. The samples of the vocal folds were collected for histological analysis with the purpose of quantifying the collagen deposition by Picrosirius Red stain under polarization microscopy. The collagen deposition was lower in all groups of vocal folds with topical application of mitomycin than in control groups. In the present study, the topical application of mitomycin before the fat grafts reduced significantly the collagen deposition (p = 0,05).

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-07-01T13:22:09Z No. of bitstreams: 1 thiagopereiradasilva.pdf: 8248708 bytes, checksum: 846bfbc45d8748f458221b2646af213c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-13T16:00:55Z (GMT) No. of bitstreams: 1 thiagopereiradasilva.pdf: 8248708 bytes, checksum: 846bfbc45d8748f458221b2646af213c (MD5) / Made available in DSpace on 2016-07-13T16:00:55Z (GMT). No. of bitstreams: 1 thiagopereiradasilva.pdf: 8248708 bytes, checksum: 846bfbc45d8748f458221b2646af213c (MD5) Previous issue date: 2012-03-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O bacterioplâncton é um dos componentes da cadeia trófica em ecossistemas aquáticos, sendo regulado por fatores como disponibilidade de nutrientes, temperatura, predação e infecção viral. A morte de bactérias é caracterizada como o processo de perda funcional e morfológica da célula e tem função de controlar a abundância e produção bacteriana, com significado importante na ciclagem de carbono e nutrientes nos ecossistemas aquáticos. No entanto, o processo de morte celular em bactérias aquáticas é desconhecido. Ainda não se sabe se a morte celular programada (MPC), processo regulado e bem conhecido em organismos eucariotos, ocorre em bactérias aquáticas. Este trabalho teve por objetivo investigar a ocorrência de MCP em bactérias aquáticas utilizando dois modelos de ecossistemas: lago Batata e lago dos Manacás. O lago Batata é um ecossistema amazônico de inundação e encontra-se divido em duas áreas: impactada e natural. O lago dos Manacás é um ecossistema artificial localizado em Minas Gerais. Três grupos de bactérias, provenientes do lago dos Manacás, lago Batata – área natural e lago Batata – área impactada foram estudados. As amostras de água foram coletadas na sub-superfície dos lagos e processadas para análises da viabilidade celular por microscopia de fluorescência e da fragmentação de DNA por citometria de fluxo. Em paralelo, amostras do lago Batata – área impactada foram processadas para microscopia eletrônica de transmissão (MET) para caracterizar a diversidade ultraestrutural da comunidade bacteriana e investigar a ocorrência de alterações celulares bacterianas. Os resultados da viabilidade celular utilizando marcadores específicos para integridade de membrana (LIVE/DEAD BacLight) mostraram diretamente a presença de bactérias vivas/viáveis e mortas/inviáveis . O lago Batata apresentou maior proporção de morte celular bacteriana (36,20%) em comparação com o lago dos Manacás (19,66%). As análises de fragmentação de DNA (ensaio TUNEL) mostraram que a MCP constitui um fenômeno presente nos ecossistemas aquáticos estudados, com maior ocorrência na área impactada do lago Batata. A MET revelou a presença de bactérias aquáticas com grande diversidade ultraestrutural, representada por diferenças morfológicas quanto ao envoltório celular, cápsula, mesossomos, vesículas membranosas, partículas aderidas e tilacóides. Além disso, 34,28% das bactérias apresentavam vírus no citoplasma. A diversidade ultraestrutural pode representar a ampla diversidade metabólica e adaptativa bacteriana, enquanto a presença de vírus parece relacionada com a morte bacteriana. As análises de integridade ultraestrutural mostraram que (i) a maior proporção de bactérias encontrava-se com alterações ultraestruturais indicativas de processo de morte, (ii) a frequência de ocorrência de cápsula foi menor nas bactérias vazias, (iii) bactérias com alterações apresentaram maior frequência de partículas aderidas, e (v) o número médio de fagos por secção foi significativamente maior nas bactérias alteradas em comparação com as bactérias intactas. A MET mostrou a ocorrência de alterações ultraestruturais típicas de MCP, como retração e condensação citoplasmáticas. Em conjunto, nossos dados ressaltam morte bacteriana como um evento importante atuando na regulação da comunidade bacteriana e demonstra, pela primeira vez, a ocorrência de MCP em bactérias aquáticas. A MCP pode ter significado funcional como um dos mecanismos desenvolvidos para sobrevivência da comunidade bacteriana. / The bacterioplankton is an important component of the food web structure in aquatic ecosystems and it is regulated by many factors such as nutrient supply, temperature, predation and virus infection. Bacterial death is characterized by functional and morphological loss of the bacterial cell with roles in the control process of abundance and bacterial production of aquatic ecosystems and functional meaning in the carbon and nutrient cycles. However, the cell death process in aquatic bacteria remains to be defined. Programmed cell death (PCD) is a regulated process largely known in eukaryotic organisms. There are not studies dealing with PCD occurrence in aquatic bacteria. This study aimed to investigate the occurrence of PCD in free-living aquatic bacteria in two models of aquatic ecosystems: Batata lake and Manacás lake. Batata Lake is located on floodplain Trombetas river in northern of Brazilian Amazon. This ecosystem has been impacted for bauxite tailings over teen years and it is presently divided in impacted and natural stations. Manacás Lake is an artificial system located in Minas Gerais. Three groups of bacteria from Manacás Lake, Batata lake – impacted area - and Batata lake – natural area -were studied. Water samples were collected from the subsurface of these lakes and processed for analyses of bacterial viability by fluorescence microscopy and DNA fragmentation by flow citometry. In parallel, samples from Batata lake – impacted area - were processed for transmission electron microscopy (TEM) for characterization of the bacterial community ultrastructural diversity and investigation of bacteria alterations. Our cell viability results using specific markers for membrane integrity (LIVE/DEAD BacLight) showed directly the presence of live/viable and dead/ not viable bacteria. Batata lake presented a higher proportion of bacterial death (36,20%) compared to Manacás lake (19,66%). DNA fragmentation analyses (TUNEL assay) showed that PCD is a phenomenon occurring in all aquatic ecosystems investigated, with higher frequency in the Batata lakeimpacted area compared to the other ecosystems. Our TEM analyses revealed a great bacterial ultrastructural diversity represented by morphological differences in the cellular envelope, capsule, mesosomes, membrane vesicles, attached particles and thylacoids. Moreover, around 34,28% of the bacteria showed virus in their cytoplasm. The ultrastructural diversity may represent the large metabolic and adaptative diversity of aquatic bacteria while the presence of virus may be related to bacterial death. Our ultrastructural integrity analyses showed that (i) the higher proportion of bacteria was in death process or dead (damaged and empty), (ii) the capsule frequency is lower in empty bacteria, (iii) the higher bacteria-particle association was found in altered bacteria, (iv) the mean number of viruses per cell section was higher in altered compared to intact bacteria. TEM also showed the presence occurrence of typical ultrastructural changes indicative of MPC, such as cell retraction and condensation. Altogether, our data demonstrate that PCD occur in aquatic bacteria, and that this event may be a survival mechanism for bacterial communities in these ecosystems.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-26T20:24:00Z No. of bitstreams: 1 katiabatistadoamaral.pdf: 5840188 bytes, checksum: bc96f4a6457a961cca6e272ba1aaaa5d (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:29:35Z (GMT) No. of bitstreams: 1 katiabatistadoamaral.pdf: 5840188 bytes, checksum: bc96f4a6457a961cca6e272ba1aaaa5d (MD5) / Made available in DSpace on 2016-09-26T20:29:35Z (GMT). No. of bitstreams: 1 katiabatistadoamaral.pdf: 5840188 bytes, checksum: bc96f4a6457a961cca6e272ba1aaaa5d (MD5) Previous issue date: 2015-12-11 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O roedor Nectomys squamipes, também conhecido como rato d’água, é considerado o mais importante reservatório silvestre do parasito Schistosoma mansoni no Brasil, contribuindo para a epidemiologia da esquistossomose mansônica humana. Além disso, quando infectado, não apresenta sinais clínicos da doença, apresentando lesões teciduais extremamente brandas decorrentes da infecção. Parâmetros relacionados à infecção esquistossomótica em modelos murinos de infecção experimental já foram bem documentados; porém, em modelos de infecção natural, ainda são pouco conhecidos. A relação parasito-hospedeiro neste modelo de infecção natural é de grande relevância e motivou o desenvolvimento deste trabalho. Foi realizado estudo comparativo da esquistossomose mansônica em N. squamipes naturalmente infectado pelo parasito S. mansoni e no camundongo Swiss experimentalmente infectado, nas fases aguda e crônica da infecção, através de análises histopatológicas, bioquímicas e ultraestruturais, objetivando obter uma melhor compreensão de como o rato d’água lida com o parasitismo pelo S. mansoni. Foram realizadas análises de frequências e tipos de granulomas, suas áreas médias e do comprometimento tecidual dos órgãos alvos da infecção (fígado e intestinos), nos dois modelos experimentais. A participação dos eosinófilos durante a resposta inflamatória granulomatosa também foi avaliada, visto que estas células estão presentes em grandes números nos granulomas. Dosagens bioquímicas das transaminases hepáticas auxiliaram na avaliação do dano hepatocelular. Além disso, a influência da esteatose hepática neste modelo de infecção natural foi estudada, visto que seus efeitos não são conhecidos em animais silvestres. Assim, dosagens séricas de glicose, colesterol total e triglicerídeos foram importantes para avaliar o perfil glicêmico e lipídico dos animais estudados. Os tecidos hepáticos dos animais foram submetidos à espectroscopia Raman, para avaliar o grau de insaturação presente. Finalmente, análises ultraestruturais ajudaram a aprofundar o conhecimento sobre os papéis dos eosinófilos e dos corpúsculos lipídicos presentes nos hepatócitos nestes dois modelos de esquistossomose mansônica. Os resultados revelaram que N. squamipes apresentou excelente modulação das lesões teciduais no fígado, com baixo comprometimento tecidual neste órgão e uma reação granulomatosa mais exacerbada no intestino delgado, favorável à eliminação dos ovos do parasito nas fezes. Além disso, os níveis séricos das transaminases não se alteraram em decorrência da infecção neste roedor silvestre, ao contrário do que ocorreu no camundongo Swiss. N. squamipes infectados e não infectados apresentaram os maiores graus de insaturação presentes nos tecidos hepáticos. Características ultraestruturais intrigantes dos eosinófilos de N. squamipes foram observadas por MET. Através dos dados obtidos, concluímos que o rato d’água apresenta uma relação ecológica bem estabelecida com o parasito S. mansoni, sendo que esta adaptação ao parasitismo pode estar relacionada ao metabolismo lipídico deste reservatório silvestre. / The rodent Nectomys squamipes, also known as water rat, is considered the most important wild reservoir of the parasite Schistosoma mansoni in Brazil, contributing to the epidemiology of human schistosomiasis. Furthermore, when infected, it shows no clinical signs of the disease with extremely soft tissue injuries resulting from infection. Parameters related to the infection in murine models of experimental infection have been well documented; however, in natural infection models, they are still largely unknown. The host-parasite relationship in this model of natural infection is highly relevant and motivated the development of this work. So, It was conducted a comparative study of schistosomiasis in N. squamipes naturally infected by the parasite Schistosoma mansoni and in Swiss experimentally infected mice, at the acute and chronic phases of the infection through histological, biochemical and ultrastructural analysis, aiming to gain a better understanding of how the water rat handles with parasitism by S. mansoni. Analysis of frequencies and types of granulomas were held, their average areas and tissue impairment of the target organs of infection (liver and intestines) in both experimental models. The involvement of eosinophils during granulomatous inflammatory response was also evaluated, as these cells are present in large numbers in the granulomas. Biochemical testing of liver transaminases contributed to the evaluation of hepatocellular damage. Furthermore, the influence of hepatic steatosis in the natural infection model was studied, since their effects are not known in wild animals. Thus, serum levels of glucose, total cholesterol and triglycerides were important to assess the glycemic and lipid profile of the animals studied. The liver tissues of animals were subjected to Raman spectroscopy, to assess the degree of unsaturation. Finally, ultrastructural analysis helped to deepen understanding of the roles of eosinophils and lipid bodies present in hepatocytes in these two models of schistosomiasis. The results revealed that N. squamipes showed excellent modulation of tissue lesions in the liver, with low tissue impairment and a more exacerbated granulomatous reaction in the small intestine, favoring the elimination of the parasite eggs in feces. Furthermore, serum transaminases levels did not change as a result of the infection in wild rodents, unlike what occurred in Swiss mice. N. squamipes infected and uninfected showed the highest degree of unsaturation present in liver tissue. Intriguing ultrastructural characteristics of N. squamipes eosinophils were observed by TEM. Through the data obtained, we concluded that the water rat has a wellestablished ecological relationship with the parasite Schistosoma mansoni, and this adaptation to parasitism may be related to lipid metabolism of this wild reservoir.

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