Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2017-01-26T12:29:37Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_VariabilidadeMorfometricaMolecular.pdf: 4841557 bytes, checksum: f98bac4b966e74cac366c8276fdf7ac3 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-01-26T12:29:55Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_VariabilidadeMorfometricaMolecular.pdf: 4841557 bytes, checksum: f98bac4b966e74cac366c8276fdf7ac3 (MD5) / Made available in DSpace on 2017-01-26T12:29:55Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_VariabilidadeMorfometricaMolecular.pdf: 4841557 bytes, checksum: f98bac4b966e74cac366c8276fdf7ac3 (MD5) Previous issue date: 2011 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O presente estudo objetivou testar a hipótese da heterogeneidade populacional morfológica e molecular em diversos grupos de Desmodus rotundus na Amazônia oriental, bem como descrever a relação desta heterogeneidade com processos e padrões de produção da Raiva em humanos e bovinos. Para tanto, um total de 776 exemplares de vampiro comum, de 72 localidades do Pará foram cedidos pelo Instituto Evandro Chagas (IEC - Ministério da Saúde/Belém), Laboratório Nacional Agropecuário (Lanagro/Belém) e Fundação de Vigilância em Saúde do estado do Amazonas (FVG/Manaus). Quanto a descrição espacial e temporal da Raiva em humanos e bovinos no Pará, ao longo de uma década (1999-2008), tais registros foram obtidos junto a Secretaria Executiva de Saúde Pública do Pará (SESPA). Do total de espécimes de D. rotundus, apenas os indivíduos adultos (329 machos e 315 fêmeas) foram submetidos a 39 medidas fenotípicas (16 externas e 23 cranianas). Na abordagem genética, 236 indivíduos (53% fêmeas e 47% machos) foram caracterizados por meio de 10 marcadores do tipo microssatélites. Já para a descrição de áreas de risco foram utilizadas feições ecológicas, biológicas, socioeconômicas e de cobertura e uso do solo, georreferenciadas geograficamente. Como um dos principais resultados das inferências fenotípicas foi observado que apenas as fêmeas de D. rotundus no Pará, mostraram tendências a formação de grupos que reúnem espécimes da porção mais ao norte do estado (Baixo Amazonas, Marajó e Nordeste), como sendo menos similares as do sudeste e sudoeste. No geral, fenotipicamente D. rotundus não mostrou elevada estruturação entre os grupos no Pará. A maior ocorrência de variabilidade observada para D. rotundus não foi entre os grupos geográficos. Segundo os dados de análise molecular de variância (AMOVA) ocorreram variações em 96% dos acontecimentos dentro de cada grupo. No geral, muitos grupos estudados do Pará ainda encontram-se sobre equilíbrio de Hardy-Weinberg, levando a crer na existência de uma única população caracteristicamente panmítica, contudo, com tendências à formação de três grandes grupos (Baixo Amazonas, Marajó e Nordeste). Para tal população, os padrões reprodutivos e adaptativos da espécie, garantiriam a alta equidade da riqueza alélica e os bons índices de diversidade genética de D. rotundus na Amazônia oriental, mesmo sob os efeitos da fragmentação de áreas, que se processa principalmente no lado leste no estado do Pará. / At the present study the hypothesis of population morphological and molecular heterogeneity was tested in several groups of the common vampire bat Desmodus rotundus from oriental Amazonia as well the relationship among the environmental heterogeneity and the process and patterns of rabies outbreaks in human and bovine population was described. For this 776 individuals of common vampire bats from 72 different places of state of Pará were disposed by Instituto Evandro Chagas (IEC - Ministério da Saúde/Belém), Laboratório Nacional Agropecuário (Lanagro/Belém) and Fundação de Vigilância em Saúde do Amazonas (FVG/Manaus). Data from 1999 to 2008 of rabies upon humans and bovines in Pará were disposed by the Secretaria Executiva de Saúde Pública do Pará (SESPA). For morphometric analysis, 39 phenotypic measures (16 bodies and 23 cranials) all adult individuals (329 males and 315 females) were obtained. For genetic analysis, 258 bats (47% males and 53% females) were characterized by 10 microsatelite markers. To describe the areas of rabies risk we used the ecological, biological and socioeconomic shapes of environment as well the soil using, all these geographically georeferenced. One of our results of phenotypic inferences suggest that vampires from Pará showed the tendency to group formation with individuals of the north region of state (Amazonas basin, Marajóand Northeastern regions), only as well they were less similar to bats from southern and southwestern regions. In general, D. rotundus did not show high phenotypically structure among the groups in Pará. The highest variability occurrence in D. rotundus, was not observed among geographic groups. According to molecular analysis of variance (AMOVA), 96% of them were found within each studied group. Our data showed that several of the groups studied in Pará are still under Hardy-Weinberg equilibrium, suggesting the existence of one single panmitic population of the common vampire bat in this North state, but with tendencies to the formation of three great groups (Amazon basin, Marajóand Northeast). To this panmitic population, the reproduction and adaptation patterns garanteed the high equity of allelic richness and good rates of genetic diversity in D. rotundus from oriental Amazon, even under the fragmentation effects in many areas of Pará state, mainly in the east side.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-07-06T18:35:03Z No. of bitstreams: 1 texto completo.pdf: 2607465 bytes, checksum: 3b40f68f4d4225bf615db6cabfb8d333 (MD5) / Made available in DSpace on 2016-07-06T18:35:03Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2607465 bytes, checksum: 3b40f68f4d4225bf615db6cabfb8d333 (MD5) Previous issue date: 2016-04-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Existem poucos estudos sobre a caracterização do sistema reprodutor masculino, especialmente o epidídimo, em morcegos neotropicais. Estudos prévios em morcegos, Molossus molossus e Eumops glaucinus, mostraram que o epitélio epididimário apresenta características similares a de outros mamíferos, quando analisados sob microscopia de luz. Porém, não foi encontrado nenhum trabalho caracterizando o epidídimo do morcego vampiro comum, Desmodus rotundus. O epidídimo exerce diversas funções como concentração, maturação, transporte e estocagem do espermatozoide. Esse órgão é composto por epitélio pseudoestratificado, podendo ser dividido em quatro regiões, segmento inicial, cabeça, corpo e cauda. Assim, o objetivo desse trabalho foi caracterizar o epidídimo do morcego vampiro comum com base nos aspectos morfológicos, histomorfométricos e ultraestruturais, através do uso de microscopia de luz, epifluorescência e eletrônica. Para a imunohistoquímica, foram usados marcadores de diferentes tipos celulares, como aquaporina 9, citoqueratina 5 e H + V-ATPase, e proteínas de tight junction, como claudina 1, claudina 3, claudina 4 e zônula de oclusão 1. No presente estudo foi possível identificar a região proximal do epidídimo, segmento inicial e cabeça, e a região distal, corpo e cauda. Todas as regiões apresentaram epitélio pseudoestratificado composto por células principais, basais e claras. A altura do epitélio epididimário diminuiu a partir da região proximal para a distal, enquanto o diâmetro do ducto e o de interducto aumentaram. O compartimento do ducto foi o principal componente do epidídimo do morcego vampiro comum. Células principais apresentaram formato colunar com estereocílio na membrana apical da célula em contato com o lúmen e foram as mais frequentes.Ao longo de todo o epidídimo, foi observado marcação PAS- positiva, formando o glicocálice, e expressão da proteína aquaporina 9 no estereocílio. Em todas as regiões observamos grânulos PAS-positivo no citpolasma, além de características ultraestruturais relacionadas às funções de absorção e secreção. O segundo tipo celular mais observado foi a célula basal. Essas foram identificadas pela posição do núcleo, localizado na porção basal do epitélio, adjacente à membrana basal. Em todas as regiões do epidídimo as células basais expressaram citoqueratina 5 e formaram uma rede de comunicação, através de prolongamentos citoplasmáticos laterais, na base do epitélio. Células claras foram as células menos observadas no epitélio epididimário do morcego vampiro comum. Essas apresentaram formato caliciforme, com núcleo na região apical da célula, baixa frequência na região da cauda e expressaram H + V-ATPase. Ultraestruturalmente, os tipos celulares mostraram características similares ao longo do epidídimo. No morcego vampiro comum, observamos a expressão de proteínas de tight junction, claudinas e zônula de oclusão 1, na porção apical das membranas laterais das células adjacentes que compõem o epitélio epididimário. Entretanto, enquanto zônula de oclusão 1 foi observada somente em tight junction, as claudinas foram observadas na região de tight junction, e na membrana basolateral das células adjacentes, incluindo células basais. Houve diferença na distribuição das claudinas quando comparado o epitélio das regiões proximais com as distais do epidídimo e entre as estações de chuva e seca. Conclui-se que o conhecimento das características do epitélio epididimário do morcego comum aumenta as informações sobre o aparelho reprodutor masculino de espécies de morcegos neotropicais, uma vez que o epidídimo de animais dessa espécie apresenta algumas peculiaridades, como o formato caliciforme das células claras em todas as regiões e baixa quantidade dessas na região da cauda, e a ausência de prolongamento citoplasmático em direção ao lúmen das células basais. Entretanto, mais estudos são necessários para o entendimento de como ocorre a maturação espermática nessa espécie, e se variações hormonais e sazonais podem interferir de alguma forma nesse órgão. / There are few studies about the characterization of the male reproductive system, especially the epididymis, in neotropical bats. Previous studies in bats, Molossus molossus and Eumops glaucinus, showed that the epididymal epithelium presents similar characteristics to the other mammals, when analyzed by light microscopy. However, there is not any study focused on the morphological features of the epididymis in common vampire bat Desmodus rotundus. The epididymis is responsible for several functions such as concentration, maturation, transport and storage of sperm. This organ is composed of pseudostratified epithelium and may be divided into four regions, initial segment, caput, corpus and cauda. Therefore, the aim of this study was to characterize the common vampire bat’s epididymis based in morphological, histomorphometric and ultrastructural aspects using light, epifluorescence and electron microscopy. For the immunohistochemistry, were used different cell types markers, such as aquaporin 9, cytokeratin 5 and H + V-ATPase, and tight junction proteins, as claudin 1, claudin 3, claudin 4 and zonula occludens 1. In this current study, we observed the proximal region of the epididymis, initial segment and caput, and the distal region, corpus and cauda. All the regions showed a pseudostratified epithelium composed of principal, basal and clear cells. The epithelium height decreased from the proximal to the distal region, while the duct and interduct diameter increased. The duct compartment was the main component of the common vampire bat epididymis. Principal cells showed a columnar shape with stereocilia in the cell apical membrane in contact with the lumen, and were the most frequent cell type. Along the epididymis, was observed PAS-positive staining, forming the glycocalix, and expression of aquaporin 9 in the stereocilia. In all regions, we observed PAS-positive granules in the cytoplasm, besides of ultrastructural characteristics related to absorption and secretion functions. The second most frequent cell type was basal cells. These cells were identified by the nuclei position, located in the basal portion of the epithelium, adjacent to the basement membrane. In all epididymal regions, the basal cells expressed cytokeratin 5, and formed a dense network, through cytoplasmatic lateral extensions, at the basal portion of the epithelium. Clear cells were the less frequent cell type observed in the epididymal epithelium of common vampire bat. These cells showed a goblet-shaped body, with nuclei in the apical region, low frequency in the cauda region, and expressed H + V-ATPase. Ultrastructurally, the cell types showed the same cellular characteristics along the epididymis. In the common vampire bat, we observed expression of tight junction proteins, claudins and zonula occludens 1, at the apical portion of lateral membranes of the adjacent epididymis epithelial cells. However, only zonula occludens 1 was observed in the tight junction, while the claudins were observed in the tight junction and in the basolateral membrane of adjacent epithelial cells, including basal cell. There was difference in the claudins distribution when compared the epithelium of the proximal with the distal regions, and between rainy and dry seasons. In conclusion, the knowledge of the epididymal epithelium of the common vampire bat increases the information about the male reproductive system in neotropical bat species, since the epididymis of this specie shows peculiarities, as the shape of clear cells in all regions and low frequency in the cauda, and absence of the luminal-reaching body into the lumen in basal cells. Moreover, more studies are necessary to understand the process of sperm maturation in this species, and if hormonal and seasonality variations could interfere in this organ.

No Brasil a raiva dissemina-se de maneira insidiosa nos herbívoros domésticos, produzindo perdas à indústria pecuária. No estado de São Paulo, a última epizootia registrada ocorreu entre os anos 1997-2002, acometendo bovinos e equinos. Este estudo examinou a possível relação de alguns casos detectados no sul de Minas Gerais no período 2000 a 2009 com o foco paulista citado, mediante análise filogenética de segmentos do gene da glicoproteína (540 nucleotídeos) e nucleoproteína (416 nt) viral, usando o algoritmo de Neighbor-joining, modelo evolutivo Kimura 2- parâmetros com 1000 replicações, considerando sequências de isolados procedentes de diferentes regiões do interior paulista e do Brasil, tomadas do GenBank. Foi proposta uma análise geográfica mediante o programa ArcGis, localizando as coordenadas geográficas dos municípios de origem dos casos sobre mapas de relevo, bacias hidrográficas e distribuição de biomas. A análise filogenética dos dois genes estudados sugeriu que os focos mineiros podem ter a mesma origem genética da última epizootia paulista de raiva em herbívoros ocorrida entre 1997 e 2002. A análise filogenética baseada na nucleoproteína mostrou um maior nível de detalhamento sugerindo a ocorrência de diferentes eventos e/ou centros de dispersão de raiva em herbívoros na área de estudo. A análise geográfica insinuou que os casos aconteceram nas porções menos elevadas da Serra da Mantiqueira, na área de Mata Atlântica e em proximidades das bacias dos rios Piracicaba/Jaguarí, Paranaíba do Sul, Grande, Pardo e Mogi-Guaçu. / Bovine rabies is still spreading insidiously in Brazil, producing economic losses to livestock industry. A remarkable epizootics took place in São Paulo state between 1997 and 2002, affecting bovine and equines. This research was intended to examine genetic relations among some rabies outbreaks in Minas Gerais (MG), during 2000 and 2009 with São Paulo epizootics by phylogenetic analysis based on glycoprotein (540 nucleotides) and nucleoprotein (416 nt) genes partial sequences using Neighbor- joining algorithm, Kimura 2-parameters model, with 1000 bootstrap replications, considering sequences from different regions of São Paulo State (SP) and Brazil from GenBank. A geographic analysis was proposed, plotting geographic coordinates of municipalities with rabies cases on topographic, hydrographic and biome maps using ArcGis software. Phylogenetic analysis for both genes suggested that cases from MG can have the same genetic origin of SP epizootics. Phylogenetic analysis based on nucleoprotein gene showed a richer level of detailing than glycoprotein gene, suggesting different events and/or dispersion centers of livestock rabies in the studied area. Geographic analysis proposed that MG cases occurred at less elevated portions of Serra da Mantiqueira mountains in the area of Atlantic forest, near Piracicaba/Jaguarí, Paranaíba do Sul, Grande, Pardo and Mogi-Guaçu rivers.

Recent research has shown that climate change had and will likely continue to have impacts on biological processes, including the propagation of infectious and zoonotic diseases. Assessments of local level impacts at the human-wildlife interface are imperative for stakeholders and policy makers, and empirical review of such research is undoubtedly necessary to understand the current state of the field, gaps of knowledge, and to identify future lines of research. In that vein, this thesis focuses on the impacts of climate change on disease at the human-wildlife interface. Specifically, my thesis works to quantify the recent temporal and spatial distribution of empirical research linking climate change with changes in the burden of infectious diseases (Chapter 2). This retrospective scoping of the last five years of empirical research identified if, and to what extent, there are biases in the diseases, species, or geographic areas studied within this scientific field. My study revealed both geographic and topical biases within the scope of recent literature, with an overwhelming emphasis on vector-borne diseases in temperate areas. There was also unequal representation in publication demographics of authors and institutions with most research originating from well developed countries. As a proof-of-concept case study, my thesis provides an empirical assessment of the plausible climatic drivers of a wildlife-disease transmission risk in an understudied region (Chapter 3), which could function to fill some of the identified research gaps in Chapter 2. Therein, my work assessed the impacts of climate variation from the last century on the environmental suitability of the rabies host Desmodus rotundus (common vampire bat) in Latin America. Findings revealed that average and standard deviation of temperature were the most important drivers of D. rotundus geographic distribution according to species' records between 1901 and 2019. Nevertheless, high uncertainty was detected regarding the predictability of D. rotundus environmental suitability across the United States-Mexico border and in the Andes Mountains of Chile. The overall modeling efforts did, however, reveal a northward distributional shift of the rabies reservoir as a likely response to climate change. Together, studies contained in this thesis provide empirical, retrospective evidence that demonstrates the effects of climate change on the increased risk of disease transmission at the human-wildlife interface. / Master of Science / Climate change is understood as the change in global or regional climate patterns, including variations of temperature and humidity factors beyond normal ranges, mostly attributed to increased levels of atmospheric carbon dioxide. Climate change is expected to influence many biological systems and presents an imminent threat to almost all organisms and geographic areas across the globe. Previous studies suggest that climate change will increase the burden of infectious diseases, including those originating from wildlife. This thesis aims to assess the availability of empirical evidence supporting the idea of a link between climate change and infectious diseases of wildlife origin. Chapter 2 examines recent scientific literature assessing climate change and infectious disease, and identifies biases in the diseases, species, and geographic areas commonly studied. This study found that literature generally focused on diseases transmitted by arthropods (e.g., insects, arachnids, or crustaceans) in temperate areas. There was little focus on diseases transmitted directly (e.g., via bites) or in non-temperate areas (e.g., tropics). Chapter 3 attempts to address issues detected in Chapter 2 by studying a directly-transmitted infectious disease in the tropics. More specifically, Chapter 3 assessed the impacts of climate variation from the last century on the distribution of the common vampire bat (Desmodus rotundus), which is a known rabies host in Latin America. Chapter 3 revealed that temperature variables were the largest drivers of common vampire bat distribution. Nevertheless, high uncertainty was detected regarding the vampire bat's ability to invade new areas such as the continental United States-Mexico border or the lowlands to the Andes Mountains in Chile. Together, studies contained in this thesis provide support for current and future research on the study of climate change as an amplifier for the risk of infectious diseases.

Iron storage disease is a significant cause of liver disease and mortality in captive Egyptian fruit bats (Rousettus aegyptiacus). The nature of the susceptibility in this and other captive exotic species to iron storage disease is not clear. Hepcidin, a key iron regulatory hormone, is involved in the regulation of iron absorption in humans and other mammalian species and a deficiency in hepcidin has been associated with a number of genetic mutations resulting in hemochromatosis in humans. The objectives of this thesis were to identify whether there is a functional mutation in the hepcidin gene in the Egyptian fruit bat that may increase the susceptibility of this species to iron storage disease, and whether there is a functional deficiency in hepcidin gene expression in the Egyptian fruit bat in response to iron challenge. We compared the coding region of the hepcidin gene amongst several species of bats and investigated hepcidin response to intramuscular injection of iron dextran amongst three species of bats with variable susceptibility to iron storage disease; the Egyptian fruit bat, the straw-colored fruit bat (Eidolon helvum), and the common vampire bat (Desmodus rotundus). While a number of genetic differences were identified amongst species, a functional mutation that could result in decreased hepcidin activity was not identified in the Egyptian fruit bat. Bats exhibited marked variation in hepcidin gene expression, with the highest level of hepcidin response to iron challenge in the common vampire bat. While the Egyptian fruit bat exhibited significant hepcidin response to iron challenge, the magnitude of response was lower than that in the common vampire bat and lower than expected based on findings in healthy humans. The straw-colored fruit bat did not exhibit any hepcidin response despite a significant increase in iron stores, which suggests this species may have evolved an alternate mechanism for coping with excessive iron or may be more susceptible to iron overload than previously recognized. / Toronto Zoo Scholarship Fund