Made available in DSpace on 2015-03-03T15:02:46Z (GMT). No. of bitstreams: 1 AndreaFA_TESE.pdf: 1420174 bytes, checksum: c5656fd03fcd0a38acd22f419f0567c7 (MD5) Previous issue date: 2010-03-12 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Societal concerns about environmental sustainability has lead to the development of ecologically-friendly alternatives to chemical insecticides for crop protection. One such alternative is biological pest control. In particular, baculoviruses are well suited as insect biopesticides due to their narrow host specificity and relative ease of propagation. In Brazil, the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is the main biological control agent employed for the soybean pest, Anticarsia gemmatalis. This baculovirus biopesticide is currently produced using caterpillars, but increasing market demand for the product has encouraged the development of an in vitro manufacturing process, which can be scaled up to much higher virus productivities. In this study, three wild-type AgMNPV isolates (AgMNPV-2D, AgMNPV-MP2 and AgMNPV-MP5) and a recombinant form (vAgEGT-LacZ) were characterised in terms of occlusion body (OB) production and infection kinetics, to enable future optimisation of the in vitro production process. These viruses were propagated using a Spodoptera frugiperda (IPLB-SF21) insect cell line grown in shaker-flask batch cultures. Among the virus isolates tested, AgMNPV-MP5 was found to be the best producer, yielding (5.3?0.85)x108 OB/mL after 8 days post infection. The characterisation of vAgEGT-LacZ propagation in suspension cell cultures has not been previously reported in the literature; hence it became the main focus for this thesis. In particular, it was carried out a study on the effect of the multiplicity of infection (MOI) on OB production. Five successive batches were performed getting a final production (8.9?1.42)x1014 occlusion bodies, considering that production is related for a bioreactor with final volume of 10m3. A low MOI associated with a fed-batch process for vAgEGT-LacZ production was found to support a 3-fold higher OB yield when compared to the default batch process (1.8x107 and 5.3x107 OB/mL, respectively). This yield is competitive with regards to the production process. / A preocupa??o da sociedade com o meio ambiente tem levado a buscar alternativas de substitui??o dos inseticidas qu?micos por outros produtos menos agressivos ao homem e ao ambiente. Assim, a utiliza??o de controle biol?gico contra pragas ? altamente desej?vel, pois reduz os riscos ambientais e p?blicos da utiliza??o de produtos qu?micos convencionais. Em particular, os v?rus do tipo baculov?rus s?o uma grande alternativa devido ? especificidade oferecida aos seus hospedeiros e a sua forma de propaga??o. No Brasil, o baculov?rus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) ? o principal agente de controle biol?gico da praga da soja Anticarsia gemmatalis. A crescente demanda deste bioinseticida tem estimulado o interesse no desenvolvimento de processos com base na produ??o in vitro de baculov?rus. Deste modo, poder? aumentar a oferta de v?rus, suprindo a necessidade do mercado deste bioinseticida para o controle de A. gemmatalis. Neste trabalho, estrat?gias de produ??o in vitro do baculov?rus selvagem AgMNPV e do seu recombinante vAgEGT?-LacZ, como influ?ncia do isolado, da cin?tica e do modo de opera??o, foram analisadas como alternativa para futura amplia??o de escala na produ??o deste bioinseticida viral. A produ??o em batelada de tr?s isolados selvagens do baculov?rus AgMNPV (AgMNPV-2D, AgMNPV-MP2 e AgMNPV-MP5) utilizando o cultivo em shaker, foi realizada com a finalidade de selecionar o melhor produtor de corpos de oclus?o a partir das infec??es em c?lulas de inseto Spodoptera frugiperda, linhagem IPLB-SF21 para avalia??o comparativa com recombinante vAgEGT?-LacZ. A sele??o identificou o isolado AgMNPVMP5 como melhor produtor de corpos de oclus?o de (5,30?0,85) x108 OB/mL em 8 dias de infec??o. A produ??o in vitro do vAgEGT?-LacZ foi foco principal deste trabalho, pois n?o h?, na literatura, a produ??o deste recombinante em sistemas de cultivo em suspens?o. Foi realizado estudo da multiplicidade de infec??o para identificar a quantidade de in?culo viral para o cultivo. A partir da?, foram realizadas cinco bateladas sucessivas obtendo-se (8,9?1,42)x1014 corpos de oclus?o para um volume final de 10m? de suspens?o de c?lulas infectadas. O aumento da produ??o de corpos de oclus?o obtidos a partir do vAgEGT?-LacZ foi analisado utilizando a estrat?gia de cultivo em batelada alimentada utilizando baixa multiplicidade de infec??o. Esta estrat?gia permitiu aumento de tr?s vezes na produ??o de corpos de oclus?o quando comparada ? produ??o em cultivos em batelada (5,3x107 e 1,8x107 OB/mL, respectivamente). E ainda, o baculov?rus recombinante vAgEGT?-LacZ mostrou-se competitivo em rela??o ao baculov?rus selvagem AgMNPV-MP5

Made available in DSpace on 2015-04-14T13:53:50Z (GMT). No. of bitstreams: 1 443857.pdf: 104654 bytes, checksum: d14d1ff631f377fd5683daa19fc320f5 (MD5) Previous issue date: 2012-08-30 / Introduction: The immune risk phenotype (IRP), as shown by increased CD8+ and low CD4+ T cell counts, and cognitive impairment have been associated with increased morbidity and mortality in very old subjects. Recent evidence suggests that persistent viral infections of the herpesviridae family such as cytomegalovirus (CMV) are likely to be involved with these specific T cell changes. This study aims to identify the IRP and investigate possible associations with viral infections, cognitive and functional states of elderly in Brazil. Material and Methods: 360 elders aged between 60-103 years were recruited from the public health care system in Porto Alegre, Brazil. Peripheral blood was collected and lymphocyte subsets (CD4+, CD8+, NK, NK T, B and CD8+CD28-) assessed by multi-color flow cytometry. CMV and Epstein-Barr virus (EBV) serologies were determined by ELISAs. Cognitive function was evaluated by the word list memory and constructional praxis from the standard neuropsychological battery of Consortium to Establish a Registry for Alzheimer s Disease and Mini Mental Status Examination (MMSE) inventory. PFEFFER Functional Activities Questionnaire was used to determine activities of daily living. Results: 59 individuals were defined with IRP (CD4/CD8 ratio < 1). Increased IgG titers to CMV (p<0.01) but not to EBV were found in the IRP+ group as compared to non-IRP. The IRP+ elders had a greater late-memory deficit (p<0.05) and more functional disability and dependency (p=0.01) when compared with the non-IRP group. Subjects with both IRP and cognitive impairment did not show synergist effects upon lymphocytes or viral serologies. Conclusions: The IRP was identified in 16% of the Brazilian community dwellers. Our data further indicate the association of CMV with IRP and both cognitive and functional disability/dependency during aging. / Introdu??o: O fen?tipo risco imunol?gico (IRP) tem sido demonstrado pelo aumento de c?lulas T CD8+ e baixa contagem de T CD4+, e o comprometimento cognitivo t?m sido associado com aumento da morbidade e mortalidade em idiv?duos muito velhos. Evid?ncias recentes sugerem que infec??es virais persistentes da fam?lia Herpesviridae como o citomegalov?rus (CMV) s?o mais propensos a desenvolverem estas altera??es nas c?lulas T espec?ficas. Este estudo visa identificar o IRP e investigar poss?veis associa??es com infec??es virais, e com o estado cognitivo e funcional em idosos do Brasil. Material e M?todos: 360 idosos com idades entre 60-103 anos foram recrutados a partir do sistema p?blico de sa?de em Porto Alegre, Brasil. O sangue perif?rico foi recolhido e os subconjuntos de linf?citos (c?lulas T CD4 +, CD8 +, c?lulas NK, NK T, B e CD8+CD28-) foram avaliados por citometria de fluxo. A sorologias para CMV e para o v?rus de Epstein-Barr (EBV) foram determinadas por ELISA. A fun??o cognitiva foi avaliada atrav?s da lista de palavras e praxia construtiva do Consortium to Establish a Registry for Alzheimer s Disease (CERAD), do teste de mem?ria verbal e l?gica de Wechesler, e do Mini Exame de Estado Mental (MEEM). O question?rio de Atividade Funcionais PFEFFER foi utilizado para determinar as atividades da vida di?ria. Resultados: 59 indiv?duos foram definidos com IRP. T?tulos aumentados de IgG para CMV (p <0,01), mas n?o para o EBV foram encontrados no grupo IRP+ grupo quando comparados ao grupo Non-IRP. Idosos com IRP+ apresentaram um maior d?ficit de mem?ria tardia (p <0,05) e maior depend?ncia e incapacidade funcional (p = 0,01) em rela??o ao grupo Non-IRP. Indiv?duos com IRP e com d?ficit cognitivo n?o demonstraram efeitos sin?rgicos sobre as popula??es de linf?citos ou sorologias virais. Conclus?es: O IRP foi identificado em 16% dos moradores de comunidades brasileira. Nossos dados ainda indicam uma importante associa??o do CMV com IRP e ambos com o estado cognitivo e funcional defici?ncia/depend?ncia durante o envelhecimento.

The Novgorod icon of Divine Wisdom is a great innovation of fifteenth-century Russian art. It represents the winged female Sophia flanked by the Theotokos and John the Baptist. Although the icon has a contemporaneous commentary and it exercised a profound influence on Russian cultural history (inspiring, among others, the sophiological theory of the turn of the twentieth century), its meaning, together with the dating and localisation of the first appearance of the iconography, has remained a great art-historical conundrum. This thesis sheds new light on this icon and explores the message, roots, function and historical context of the first, most emblematic and most enigmatic Russian allegorical iconography. In contrast to its recent interpretations as a Trinitarian image with Christ-Angel, it argues that the winged Sophia is the personification of the Orthodox Church. The Novgorod Wisdom icon represents the Church of Hagia Sophia, that is Orthodoxy, as it was perceived in fifteenth-century Rus’: the icon together with its commentary was a visual-textual response to the Florentine Union between the Catholic and Orthodox Churches, signed in 1439 but rejected by the Russians in 1441. The thesis is based on detailed interdisciplinary research, utilising simultaneously the methodologies of philology, art history, theology and history. The combined analysis reveals that the great innovation of the Novgorod Sophia icon is that it amalgamates ecclesiological and sophiological iconographies in new ways. Hence the dissertation is also an innovative attempt to survey how Orthodoxy was perceived and visualized in medieval Rus’. It identifies the theological questions that constituted the basis of Russian Orthodox identity in the Middle Ages and reveals the significance of the polemics between the Catholic and Orthodox Churches for the history of Medieval Russian art.

This dissertation examines the ceremonies and rituals involving the princes of early Rus’ and their entourage, how these ceremonies and rituals are represented in the literature and artefacts of early Rus’, the possible cultural influences on ceremony and ritual in this emergent society, and the role of ceremony and ritual as representative of political structures and in shaping the political culture of the principalities of early Rus’. The process begins by introducing key concepts and historiographic considerations for the study of ceremony and ritual and their application to the medieval world. The textological survey that follows focusses on the chronicles of Rus’, due to their compilatory nature, and discusses the philological, linguistic, and contextual factors governing the use of chronicles in this study. This examination of the ceremonies and rituals of early Rus’, the first comprehensive study of its kind for this region in the early period, engages with other studies of ceremony and ritual for the medieval period to inform our understanding of the political culture of early Rus’ and its influences. The structure of this dissertation is dictated by the chronology of ceremonies and rituals that structure the reigns of Rus’ princes in literary sources. The first chapter investigates—both comparatively and locally—the development of enthronement rituals depicted in textual sources and on coins. The second chapter focusses on rituals of association that are represented as mediating relations between princes in a non-central functioning dynastic culture. Oath-taking (and breaking) and association through commensality—dining and gift-giving—are examined in terms of historical context and the internal categorisation of associative acts in textual sources from Rus’. The final chapter builds on recent studies of ritualised warfare in early Rus’ and examines the ritualisation of princely movement—the most common action associated with the princes of Rus’ in textual sources—in times of war. The celebration of triumph and princely entry along with ritualised invocations for intercession in war are acts examined—both in textual sources and iconographic artefacts—as rituals of triumphal rulership reflecting both Byzantine and wider medieval culture. This study concludes with a discussion of the themes explored in its three chapters and offers further considerations about the influence of the Church and monastic culture inherited from Byzantium (and developed in Rus’) on the preservation, creation, and promulgation of ritualised political power.

Made available in DSpace on 2015-04-14T14:50:57Z (GMT). No. of bitstreams: 1 413481.pdf: 1430442 bytes, checksum: e220e9605982f141faabbfa549358e7e (MD5) Previous issue date: 2009-04-23 / O c?ncer bucal atinge cerca de 7% da popula??o mundial, sendo um importante problema de sa?de p?blica, tanto pelas seq?elas apresentadas pelos pacientes, como pelos custos envolvidos com o seu tratamento. Entre os c?nceres da cavidade bucal, 95% s?o representados pelo carcinoma espinocelular bucal (CECB). Estudos t?m mostrado que esse tumor ocorre preferencialmente em homens com mais de 40 anos, de pele branca, que apresentam os fatores de risco tradicionais, principalmente o uso de tabaco e/ou de ?lcool e a exposi??o aos raios solares. Todavia, a literatura demonstra o desenvolvimento do CECB em pacientes que n?o apresentavam exposi??o aos fatores de risco acima citados, tendo sido sugerida a associa??o com o papilomav?rus humano (HPV). Apesar de ser um importante v?rus oncog?nico, o papel do HPV na carcinog?nese bucal ainda ? controverso. O objetivo deste estudo foi detectar e tipar o HPV e verificar sua associa??o com a exposi??o ao tabaco e ao ?lcool em uma popula??o do Rio Grande do Sul/Brasil. Dezessete amostras de tecido fresco congelado foram obtidas de bi?psias de CECB. O ?cido desoxirribonucl?ico (DNA) do HPV foi extra?do por meio de m?todo padr?o que utiliza proteinase K. Para testar a viabilidade do DNA extra?do, foi realizada uma rea??o em cadeia da polimerase (PCR) para o gene da R-globina humana. A pesquisa molecular de HPV foi realizada utilizando os testes comerciais da Roche AMPLICOR (AMP) e LINEAR ARRAY (LA) HPV. Os resultados mostraram que 76,5% da popula??o estudada eram do g?nero masculino, com idade m?dia de 61,71 (?12.96), 29,4% dos indiv?duos n?o fumavam e 29,4% pertenciam ao grupo dos que n?o bebiam. O DNA do HPV foi detectado em 47,1% dos casos de CECB. Em todas as 8 amostras positivas, foi identificado o HPV 16, 75% (6/8) dos tumores foram diagnosticados histologicamente como moderadamente diferenciados e 37,5% (3/8) estavam localizados no assoalho da boca. Nenhuma associa??o foi observada entre a infec??o por HPV e a exposi??o ao tabaco ou ao ?lcool. O estudo encontrou uma preval?ncia consideravelmente alta de infec??o por HPV 16 nas les?es de CECB (aproximadamente a metade), suportando a possibilidade de um poss?vel papel do HPV na carcinog?nese bucal.

Made available in DSpace on 2015-04-14T14:50:58Z (GMT). No. of bitstreams: 1 416220.pdf: 5780942 bytes, checksum: 72898bdecabb1e0a005cf610ebdcc238 (MD5) Previous issue date: 2009-06-29 / O c?ncer cervical ? um problema que vem afetando cada vez mais mulheres de todo o mundo e, se n?o detectado em tempo, pode resultar em alta taxa de letalidade. Virtualmente todos os c?nceres cervicais t?m como agente causal essencial o Papilomav?rus Humano (HPV). A Organiza??o Mundial da Sa?de (OMS) estima que cerca de 660 milh?es de pessoas s?o infectadas todos os anos, al?m de ser o v?rus que causa Doen?as Sexualmentes Transmiss?veis (DST) mais comum do trato genital. Os HPVs s?o subdivididos em v?rias categorias, sendo classificados por tipo e subtipo, grau de risco (alto e baixo risco) e pelo s?tio preferencial de infec??o (cut?neo, genital, etc). Os HPV genitais de alto risco (HR-HPV) dos tipos 16 e 18 s?o respons?veis por aproximadamente 70% dos casos de c?ncer cervical no mundo, com alguma diferen?a de preval?ncia dependendo da regi?o. Estudos t?m aumentado muito o conhecimento acerca destes v?rus nos ?ltimos anos, mas algumas quest?es ainda ficam em aberto devido a dificuldades t?cnicas e experimentais. Um foco recente importante dos estudos sobre estes v?rus tem sido buscar o entendimento sobre a estrutura e fun??es das oncoprote?nas E6 e E7 e seus alvos moleculares, com intuito de buscar novos alvos ou metodologias para o desenvolvimento de vacinas ou f?rmacos contra o HPV. Neste trabalho, buscamos complementar os trabalhos j? desenvolvidos na ?rea de modelagem estrutural da prote?na E6 de HPV 16, que tem como principal fun??o a condu??o de p53, prote?na celular supressora de tumores, ? degrada??o. O trabalho foi desenvolvido atrav?s de modelagem por homologia utilizando como molde o modelo experimental parcial mutado publicado por Nomin? et al (2006). O modelo obtido apresenta dois dom?nios parcialmente independentes (N- e C-terminal) ligadas por um conector central (linker) sem estrutura secund?ria definida. Apesar de a prote?na apresentar grande tend?ncia ? desordem intr?nseca, os dom?nios mantiveram a maior parte de suas topologias ao longo da simula??o em meio aquoso, indicando boa qualidade do modelo sugerido.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-07-10T11:53:31Z No. of bitstreams: 1 471993 - Texto Completo.pdf: 2126269 bytes, checksum: f86f75c4cb3e97d7b63c12ed82e07361 (MD5) / Made available in DSpace on 2015-07-10T11:53:31Z (GMT). No. of bitstreams: 1 471993 - Texto Completo.pdf: 2126269 bytes, checksum: f86f75c4cb3e97d7b63c12ed82e07361 (MD5) Previous issue date: 2015-04-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Head and neck cancer represents a group of diseases affecting the face and upper aerodigestive tract. Early diagnosis of these neoplasms and their etiologic factors is crucial for patient?s survival and treatment. In the last decades, cause-and-effect relationship between some viruses and malignancies has been widely accepted. Recently, the role of Human papillomavirus (HPV) and Epstein-Barr virus (EBV) has been shown in the pathogenesis of oropharyngeal carcinoma, undifferentiated nasopharyngeal carcinoma and lymphomas. Nevertheless, the role of such viral agents in oral squamous cell carcinoma (OSCC) is still controversial. This study aimed to investigate HPV, EBV, p53 and Ki-67 in OSCC and oral lymphoma, considering clinical and histological features. The sample was composed of archived material (medical records and paraffin blocks of biopsied specimens) from patients with OSCC, oral lymphoma, and oral traumatic fibrous hyperplasia. The biopsied specimens and medical records were allocated into five groups: (1) 16 samples from patients with OSCC grade I (well-differentiated); (2) 16 samples from patients with OSCC grade II (moderately-differentiated); (3) 19 samples from patients with OSCC grade III (poorly-differentiated); (4) 14 samples from patients with oral non-Hodgkin lymphoma; and (5) 19 samples from patients with oral traumatic fibrous hyperplasia. Biopsied specimens were analyzed for HPV, EBV, p53 and Ki-67 expression by immunohistochemistry, and clinical data concerning age and gender of the patients, size and duration of the lesion as well as habits and comorbidities were collected. Marker expression was evaluated according to clinical features and histological grade of the tumors. There was no association of HPV and EBV with the tumors analyzed. Ki-67 expression was significantly lower in the fibrous hyperplasia group with no other significant differences. p53 expression was significantly lower in the lymphoma and fibrous hyperplasia groups than the OSCC groups, with no other significant differences. OSCC grade II and III were associated with male gender and tobacco smoking, while grade III was also associated with alcohol consumption. There was no association of the tumors with either chimarr?o or comorbidities. Duration of the lesion was inversely correlated to lesion size, which in turn was inversely correlated to HPV. EBV and HPV were positively correlated to each other, as was Ki-67 to lesion size and p53, which was correlated to HPV. / Os tumores malignos da regi?o de cabe?a e pesco?o constituem um grupo de doen?as que afeta a face e o trato aerodigestivo superior. O diagn?stico precoce dessas neoplasias e de seus fatores etiol?gicos ? de extrema import?ncia para a sobrevida e para o tratamento do paciente. Nas ?ltimas d?cadas, a rela??o de causa e efeito entre certos v?rus e tumores malignos tem sido amplamente aceita. Recentemente, foi relatado o papel do Human papillomavirus (HPV) e do Epstein-Barr virus (EBV) na patogenia do carcinoma espinocelular de orofaringe, no carcinoma indiferenciado de nasofaringe e em linfomas, sendo ainda controverso o papel desses agentes virais no carcinoma de c?lulas escamosas oral (CCEO). O presente estudo teve por objetivo investigar, por meio de exame imunoistoqu?mico, os v?rus HPV e EBV, bem como os marcadores Ki-67 e p53 em carcinomas e linfomas orais considerando fatores cl?nicos e histol?gicos. Esp?cimes de bi?psia arquivados em blocos de parafina e prontu?rios de pacientes portadores de les?es orais de CCEO, linfoma e hiperplasia fibroepitelial foram distribu?dos em cinco grupos: (1) 16 amostras de CCEO grau I (bem diferenciado); (2) 16 amostras de CCEO grau II (moderadamente diferenciado); (3) 19 amostras de CCEO grau III (pobremente diferenciado); (4) 14 amostras de linfoma n?o-Hodgkin e (5) 19 amostras de hiperplasia fibroepitelial. Os esp?cimes de bi?psia foram submetidos a processamento imunoistoqu?mico para HPV, EBV, p53 e Ki-67. Dados cl?nicos referentes a idade e sexo dos pacientes, tamanho e dura??o da les?o, comorbidades e h?bitos foram coletados dos prontu?rios. A express?o dos marcadores foi analisada de acordo com os aspectos cl?nicos e grau histol?gico dos tumores. N?o houve associa??o entre HPV e EBV com os tumores analisados. A express?o do Ki-67 foi significativamente menor no grupo hiperplasia fibroepitelial, n?o havendo outras diferen?as significativas entre os demais grupos. Os linfomas e as hiperplasias fibroepiteliais exibiram express?o de p53 significativamente menor que os carcinomas, sem outras signific?ncias. CCEO grau II e III foram associados ao sexo masculino e ao tabagismo, enquanto o CCEO grau III foi tamb?m associado ao consumo de ?lcool. N?o houve associa??o dos tumores avaliados com chimarr?o ou comorbidades. Houve correla??o negativa entre dura??o e tamanho da les?o, bem como entre tamanho da les?o e HPV. EBV e HPV exibiram correla??o positiva, assim como o Ki-67 foi correlacionado ao tamanho da les?o e ? p53, que por sua vez exibiu correla??o positiva com o HPV.

Made available in DSpace on 2016-04-28T20:16:25Z (GMT). No. of bitstreams: 1 2008- Daniel de Barros Macieira.pdf: 1044606 bytes, checksum: 797036e36833a30609c06a68880f37c8 (MD5) Previous issue date: 2008-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Infectious diseases present high morbidity among domestic cats and a delayed or misdiagnosis may result in patient death. The association between feline immunodeficiency (FIV) and/or feline leukemia (FeLV) viruses infections with hemoplasmas has great importance in veterinary practice, since agents may act synergically increasing risks for infected animals. To date, there are no studies in Brazil critically examining whether or not hemoplasma infections are associated with retroviral infection in cat. Thus, the aim of this study was to determine the prevalence and risk factors for Mycoplasma haemofelis (Mhf) and Candidatus Mycoplasma haemominutum (Mhm) infections in domestic cats tested for FIV and FeLV with a commercial ELISA kit. Based on serologic testing, cats were grouped as i) FIV-positive (n=25); ii) FeLV-positive (n=39); iii) FIV/FeLV-positive (n=8); and iv) FIV/FeLV-negative (n=77), or base-comparison group. CBCs were followed by DNA extraction, species-specific PCR (16S rRNA gene) for Mhf and Mhm and Southern blotting for all animals. Among all animals tested for retroviral infection (n=149), 16,8%, 26,2% and 5,4% were seropositive for FIV, FeLV and both retroviruses respectively. Associated factors to retroviral infections included an increased mean age (FIV, p<0,001), neutered status and history of fights/bites (FIV, p=0,033 and <0,001 respectively); presence of fleas (FeLV, p=0,018); history of blood transfusions and roaming outdoors (FIV/FeLV, p=0,028 and 0,033 respectively). Among evaluated clinical signs, anorexia and pale mucous (FIV, p=0,004 and 0,034 respectively); lethargy/depression, jaundice and tachycardia (FeLV, p=0,033, 0,036 and 0,003 respectively) were identified. Among CBC findings, anemia was related to FIV (p=0,012) and FeLV (p<0,001) infections, whereas thrombocytopenia was only related to FIV infection (p=0,015). Animals with FIV or double FIV and FeLV infections had a reduced WBC count (p=0,021 and 0,012 respectively). Mhf DNA was found in 4.0%, 2.6%, 12.5% and 7.8% of the cats from groups i, ii, iii and iv respectively, while 32.0%, 5.1%, 50.0% and 5.2% of these animals had a Mhm infection. Host factors that were associates to the presence of hemoplasma DNA included the retroviral status (p=0,001), history of fights/bites (p=0,003), roaming outdoors (p=0,001), living with other cats (p=0,008) and pale mucous (p=0,033). The logistic regression model used herein showed that cats with FIV (OR=4,25; p=0,009) and both FIV and FeLV (OR=7,56; p=0,014) were at greater risk of being hemoplasma infected than retroviral negative cats, mainly due to Mhm infection (OR=8,59, p=0,001 and OR=18,25, p=0,001 respectively). Among pure-breed cats, FIV-positive status was associated with hemoplasma infection (OR 45,0; p=0,001). / As doen?as infecto-contagiosas possuem uma alta morbidade entre os gatos dom?sticos e um diagn?stico impreciso ou retardado pode resultar no ?bito do paciente. A associa??o entre infec??es pelos v?rus da imunodefici?ncia (FIV) e/ou leucemia (FeLV) felina com hemoplasmas possui grande import?ncia na cl?nica m?dica, uma vez que os agentes podem agir de modo sin?rgico elevando os riscos para os animais acometidos. Devido ao fato de inexistirem no Brasil estudos que analisem criticamente a associa??o entre estes retrov?rus e os hemoplasmas, o presente trabalho teve como objetivo determinar a preval?ncia e poss?veis fatores associados para as infec??es por Mycoplasma haemofelis (Mhf) e Candidatus M. haemominutum (Mhm) em gatos dom?sticos, testados para a presen?a de FIV e FeLV, atrav?s de um kit comercial baseado na metodologia de ELISA. Com base no teste sorol?gico, os gatos foram agrupados como i) FIV-positivos (n=25); ii) FeLV-positivos (n=39); iii) FIV/FeLV-positivos (n=8); e iv) FIV/FeLV-negativos (n=77), ou grupo de refer?ncia para as compara??es. Hemogramas foram seguidos da extra??o/purifica??o de DNA, rea??o em cadeia da polimerase esp?cie-espec?fica para a detec??o de Mhf e Mhm e Southern Blot/hibridiza??o (SB) para todos os animais. Entre os 149 animais testados, 16,8%, 26,2% e 5,4% foram soropositivos para FIV, FeLV e ambos os retrov?rus respectivamente. Fatores associados ? infec??o por retrov?rus inclu?ram idade m?dia mais elevada (FIV, p<0,001), condi??o reprodutiva castrado e hist?rico de brigas/mordidas (FIV, p=0,033 e <0,001 respectivamente); presen?a de pulgas (FeLV, p=0,018); transfus?o de sangue e acesso ao ambiente externo (FIV/FeLV, p=0,028 e 0,033 respectivamente). Dentre as altera??es cl?nicas avaliadas, anorexia e mucosas p?lidas (FIV, p=0,004 e 0,034 respectivamente); letargia/depress?o, icter?cia e taquicardia (FeLV, p=0,033, 0,036 e 0,003 respectivamente) foram identificadas. Dentre os par?metros hematol?gicos a anemia esteve associada a presen?a de FIV (p=0,012) e FeLV (p<0,001) enquanto a trombocitopenia esteve apenas associada a infec??es por FIV (p=0,015). Animais com FIV ou com dupla infec??o (FIV e FeLV) tiveram uma contagem de leuc?citos reduzida (p=0,021 e 0,012 respectivamente). O DNA de Mhf foi achado em 4,0%, 2,6%, 12,5% e 7,8% dos gatos dos grupos i, ii, iii e iv, respectivamente, enquanto 32,0% 5,1%, 50,0% e 5,2% desses animais tinham infec??es por Mhm. Fatores inerentes ao hospedeiro que apresentaram associa??o com a presen?a do DNA de hemoplasmas inclu?ram o estado retroviral (p=0,001), hist?rico de brigas/mordidas (p=0,003), acesso ao ambiente externo (p=0,001), coabitar com outros gatos (p=0,008) e mucosas p?lidas (p=0,033). O modelo de regress?o log?stica usado mostrou que gatos com FIV (OR=4,25, p=0,009) e animais duplamente infectados para FIV e FeLV (OR=7,56, p=0,014) estavam em maior risco de serem infectados por hemoplasmas do que animais negativos para ambos os retrov?rus, principalmente em virtude de infec??es por Mhm (OR=8,59, p=0,001 e OR=18,25, p=0,001 respectivamente). Entre os animais de ra?a definida, a positividade para FIV estava associada com infec??es para hemoplasmas (OR=45, p=0,001).

Made available in DSpace on 2015-04-14T14:51:32Z (GMT). No. of bitstreams: 1 458474.pdf: 1757493 bytes, checksum: c5379d3dddf546f5888748177b083560 (MD5) Previous issue date: 2014-03-07 / Acute viral bronchiolitis is the most common respiratory illness in children in the first years of life and at least half of patients are diagnosed with respiratory tract infection by viruses such as Respiratory Syncytial Virus (RSV). Because RSV is extremely common disease it generates large number of hospitalizations and large costs to health systems. The RSV Fusion (F) Protein is essential for the infective cycle of the virus. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease due to viral infection. Neutrophil Extracellular Traps (NETs) are formed by the release of the inner content of neutrophils (proteins from granules and DNA) in the extracellular space in response to different stimuli. They are essential to prevent the spread of microorganisms, which are killed by antimicrobial proteins that are anchored in networks of DNA, such as elastase and myeloperoxidase. The objective of this work is to characterize the effect of the RSV F protein on the generation of NETs. The F protein was capable of inducing the formation of NETs in vitro in a dose-dependent way with co-expression of neutrophil elastase and myeloperoxidase. The production of NETs in response to F protein was mediated by the TLR-4-receptor and was dependent on ROS production, p38 and ERK MAPK. Together, these data provide evidence that support the activation of specific signaling pathways by the F protein to induce the production of NETs. Excessive production of NETs can aggravate the inflammatory symptoms induced by infection with RSV. / A bronquiolite viral aguda ? a doen?a respirat?ria mais frequente em crian?as nos primeiros anos de vida, sendo que ao menos a metade dos pacientes ? diagnosticado com infec??o respirat?ria por v?rus como o V?rus Sincicial Respirat?rio (RSV). Por ser uma doen?a extremamente comum gera grande n?mero de interna??es e grandes custos aos sistemas de sa?de. A prote?na de fus?o (F) do RSV ? essencial para o ciclo infectivo do v?rus. Neutr?filos e seus produtos est?o presentes nas vias a?reas de pacientes infectados por RSV que desenvolveram doen?a pulmonar aumentada devido a infec??o viral. As Redes Extracelulares de Neutr?filos (NETs) s?o formadas pela libera??o do conte?do nuclear dos neutr?filos no espa?o extracelular em resposta a diferentes est?mulos, sejam patog?nicos ou n?o. Elas s?o fundamentais para impedir a dissemina??o de microrganismos, que s?o mortos pelas prote?nas antimicrobianas que ficam ancoradas nas redes de DNA, como elastase e mieloperoxidase. O objetivo desta disserta??o ? caracterizar o efeito da prote?na F do RSV sobre a gera??o de NETs. A prote?na F foi capaz de induzir a forma??o de NETs in vitro de forma dose-dependente e com a co-express?o de elastase neutrof?lica e mieloperoxidase. A produ??o de NETs pela prote?na F foi mediada pelo TLR-4, e dependente da produ??o de ROS e de ERK-p38 MAPK. Juntos, estes dados fornecem evid?ncias que suportam a ativa??o de vias de sinaliza??o espec?ficas pela prote?na F para induzir a produ??o de NETs. A produ??o excessiva de NETs pode agravar os sintomas inflamat?rios induzidos pela infec??o com RSV.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-08-24T19:19:55Z No. of bitstreams: 1 2016 - Marli Sidoni.pdf: 1735106 bytes, checksum: 03907fc3437a44bf96b94d6040184407 (MD5) / Made available in DSpace on 2018-08-24T19:19:55Z (GMT). No. of bitstreams: 1 2016 - Marli Sidoni.pdf: 1735106 bytes, checksum: 03907fc3437a44bf96b94d6040184407 (MD5) Previous issue date: 2016-12-01 / This work focused on deploying IgY technology into developing ELISA immunoenzymatic test to FIV diagnose, contributing to the establishment of a production model for a kit in the field of veterinary medicine. The first stage of this work consisted in analyzing the literature of the veterinary documents aimed to diagnose animal diseases, as stated in our current legislation. The access to both national and international optimized the enlightenment of developing parameters and method validation criteria for the development of this new product, named ELISA r-p24 IgY. The second stage consisted in establishing purification production and physicochemical characterization of the recombinant protein p24 of FIV. The biological activity maintenance was proved by way of Western Blot test with the banda presence of approximately 25 kDa, referring to the p24 protein. The third stage was to obtain IgY cat anti-IgG, derived from hens inoculation. The kinetics were monitored by ELISA and the outcome demonstrated that as of the second week, there was a gradual increase in antibody in the yolk, and remained high throughout the period of five months. Reference to the chicken 1, the average concentration was 40,1 mg/mL e for the chicken 2 was 32,2 mg/mL, throughout the period of 5 months. The fourth stage was the use of IgY technology to develop, standardize e validate the r-p24 IgY ELISA related to its use to diagnose the infection caused by FIV. The results were: 99% accuracy, 97.7% sensitivity, 99.5% specificity and 99.1% kappa index. In the fifth stage was carried out a comparative study between ELISA r-p24 IgY and ELISA r-p24 IgG, and it was demonstrated superior performance of the ELISA r-p24 IgY. The ELISA r-p24 IgY to have favorable characteristics from a commercial perspective, such as high precision and maintenance of reactivity for a minimum period of 12 months. Therefore, the above described procedure was efficient and enabled the development of a FIV test. The predominance of IgY technology may contribute to research and development of new tests, following both international regulation related to animal welfare and validation, thus boosting national development of diagnostic kits, for the benefit of human health or animal. / O objetivo deste trabalho foi aplicar a tecnologia IgY no desenvolvimento de um teste imunoenzim?tico, ELISA, para o diagn?stico do FIV, contribuindo no estabelecimento de um modelo de produ??o para um kit nacional na ?rea de medicina veterin?ria. A primeira etapa do desenvolvimento deste trabalho consistiu na revis?o da literatura dos documentos pr?prios para produtos de uso veterin?rio, destinados a diagnosticar doen?as dos animais, disponibilizados na legisla??o vigente. A consulta aos documentos nacionais e internacionais potencializou o esclarecimento de par?metros de desempenho ou crit?rios de valida??o de m?todos, para o desenvolvimento deste novo produto, denominado ELISA r-p24 IgY. A segunda etapa consistiu no estabelecimento da produ??o, purifica??o e caracteriza??o f?sico-qu?mica da prote?na recombinante p24 do FIV. A preserva??o da atividade biol?gica foi demonstrada por Western Blot com a presen?a de uma banda pept?dica de aproximadamente 25 kDa, referente ? prote?na r-p24. A terceira etapa deste trabalho, consistiu na obten??o de anticorpos IgY anti-IgG de gato, a partir da inocula??o em galinhas poedeiras. A cin?tica foi acompanhada por ELISA demonstrando um aumento gradativo do t?tulo de anticorpos na gema a partir da segunda semana, com um aumento significativo no 2? m?s, e mantendo-se elevado durante todo o per?odo de cinco meses. As concentra??es m?dias de prote?nas na galinha 1 foi de 40,1 mg/mL a partir de uma gema e na galinha 2 foi de 32,2 mg/mL por gema, no per?odo de 5 meses. A quarta etapa deste trabalho consistiu no emprego da tecnologia IgY para o desenvolvimento, padroniza??o e a valida??o do teste de Elisa r-p24 IgY para o diagn?stico da infec??o causada pelo FIV. Os resultados obtidos foram: a acur?cia de 99%, a sensibilidade de 97,7%, a especificidade de 99,5%, e o ?ndice kappa de 99,1%. Na quinta etapa deste trabalho realizou-se o estudo comparativo do ELISA r-p24 IgY frente ao ELISA r-p24 IgG, e foi demonstrado o desempenho superior no ELISA r-p24 IgY. A valida??o do ELISA r-p24 IgY mostrou caracter?sticas desej?veis para o uso comercial, tais como alta precis?o e manuten??o da reatividade por um per?odo m?nimo de 12 meses. Conclui-se que o procedimento elaborado foi eficiente e possibilitou o desenvolvimento de um teste para o diagn?stico do FIV. O dom?nio da Tecnologia IgY poder? contribuir com a pesquisa e o desenvolvimento de novos ensaios atendendo ?s normas e diretrizes nacionais e internacionais, tanto de bem-estar animal como de valida??o, impulsionando o desenvolvimento nacional de kits diagn?sticos de interesse em sa?de humana ou animal.