Made available in DSpace on 2014-06-11T19:32:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-16Bitstream added on 2014-06-13T20:44:07Z : No. of bitstreams: 1 alexandrino_b_dr_jabo.pdf: 403623 bytes, checksum: a596d9d91434dd37dd44458b1b70eb74 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A presente pesquisa teve como objetivo avaliar a utilização de cobaias e caprinos para teste de vacinas contra o herpesvírus bovino tipo 1 (BoHV-1) e os vírus da diarreia viral bovina tipos 1 (BVDV-1) e 2 (BVDV-2). Inicialmente foi realizada a infecção experimental em cobaias e bovinos, com esses três vírus, para verificar a máxima resposta imunogênica em ambas as espécies. As médias geométricas dos títulos de anticorpos (GMT) dos bovinos foram altas para todos os agentes virais; em cobaias foi possível observar que o BoHV-1 induziu elevadas GMT, para o BVDV-1 a indução foi moderada, enquanto para o BVDV-2 foi baixa, podendo comprometer a viabilidade dos testes antigênicos. Após essa etapa, foram vacinados bovinos, cobaias e caprinos, com seis vacinas comerciais de antígenos inativados, para verificar a indução de anticorpos por estímulo vacinal. Nos bovinos, as GMT obtidas para o BoHV-1 mostraram que as vacinas utilizadas promoveram a indução moderada na resposta imunológica, e apenas três delas foram consideradas satisfatórias. Para o BVDV-1, apenas uma vacina pode ser considerada eficiente, e em relação ao BVDV-2 nenhuma delas poderia ser assim classificada. Os resultados da vacinação com os caprinos mostraram que, apesar de os animais serem reagentes ao BoHV-1 no início do experimento, as formulações foram capazes de induzir a produção de anticorpos com títulos elevados contra os três vírus estudados. As cobaias, por sua vez, receberam doses fracionadas de uma vacina comercial nacional e, das frações testadas, 3,2mL foi a que apresentou melhores resultados, sendo esta estabelecida para testar as demais vacinas nesta espécie. Depois da imunização, os resultados mostram que para o BoHV-1 as GMT foram altas; em relação ao BVDV-1, apenas duas vacinas foram capazes de induzir... / The present research had as objective to evaluate the use of guinea pigs and goats in test of vaccines against the bovine herpesvirus type 1 (BoHV-1) and the bovine viral diarrhea viruses types 1 (BVDV-1) and 2 (BVDV-2). First, an experimental infection was realized in guinea pigs and bovines, using these three viruses, to verify the maximum immune response in both species. The geometric means of antibodies titres (GMT) for bovines were high for the viral agents; in guinea pigs was possible to observe that the GMT induced by BoHV-1 was high, by BVDV-1 was moderate and by BVDV-2 was weak and could compromise the viability of antigenic tests. After this stage, the bovines, guinea pigs and goats were vaccinated using six commercial vaccines with inactivated antigens to verify the induction of antibodies production by vaccinal stimulus. In bovines, the GMT obtained for BoHV-1 showed the vaccines induced a moderate immune response, being only three of them considered satisfactory. For BVDV-1 only one and for BVDV-2 none of them can have the same classification. The results obtained with the goats, despite they were positive to BoHV-1 in the sorting, showed the formulations were able to induce high antibody production against the three virus studied. The guinea pigs, on the other hand, received fractional doses of a commercial vaccine well-established in the domestic trade and, from the fractions tested, 3.2 mL showed the best results, being established to test the other vaccines. After immunization, the results of GMT for BoHV-1 was high; relating to BVDV-1, only two vaccines were capable to induce antibodies production, but their GMT were weak; and there was no immune response against BVDV-2. Thus, goats can be ... (Complete abstract click electronic access below)

abstract: Background: This study examines how pro-vaccine flu messages, guided by the Extended Parallel Process Model (EPPM), affect parents’ intentions to vaccinate their children. Methods: Parents of children six months to five years old (N = 975) were randomly exposed to one of four high-threat/high-efficacy messages (narrative, statistical, combined, control) and completed a follow-up survey. Differences between message conditions were assessed with one-way ANOVAs, and binary logistic regressions were used to show how constructs predicted intentions. Results: There were no significant differences in the ANOVA results at p = .05 for EPPM variables or risk EPPM variables. There was a significant difference between message conditions for perceived manipulation (p = 0.026), authority, (p = 0.024), character (p = 0.037), attention (p < .000), and emotion (p < .000). The EPPM model and perceptions of message model (positively), and the risk EPPM model and fear control model (negatively), predicted intentions to vaccinate. Significant predictor variables in each model at p < .05 were severity (aOR = 1.83), response efficacy (aOR = 4.33), risk susceptibility (aOR = 0.53), risk fear (aOR = 0.74), issue derogation (aOR = 0.63), perceived manipulation (aOR = 0.64), character (aOR = 2.00), and personal relevance (aOR = 1.88). In a multivariate model of the significant predictors, only response efficacy significantly predicted intentions to vaccinate (aOR = 3.43). Compared to the control, none of the experimental messages significantly predicted intentions to vaccinate. The narrative and combined conditions significantly predicted intentions to search online (aOR = 2.37), and the combined condition significantly predicted intentions to talk to family/friends (aOR = 2.66). Conclusions: The EPPM may not be effective in context of a two-way threat. Additional constructs that may be useful in the EPPM model are perceptions of the message and fear control variables. One-shot flu vaccine messages will be unlikely to directly influence vaccination rates; however they may increase information-seeking behavior. The impact of seeking more information on vaccination uptake requires further research. Flu vaccine messages should be presented in combined form. Future studies should focus on strategies to increase perceptions of the effectiveness of the flu vaccine. / Dissertation/Thesis / Doctoral Dissertation Public Health 2015

abstract: The ability to manipulate the interaction between small molecules and biological macromolecules towards the study of disease pathogenesis has become a very important part of research towards treatment options for various diseases. The work described here shows both the use of DNA oligonucleotides as carriers for a nicotine hapten small molecule, and the use of microsomes to study the stability of compounds derived to treat mitochondrial diseases. Nicotine addiction is a worldwide epidemic because nicotine is one of the most widely used addictive substances. It is linked to early death, typically in the form of heart or lung disease. A new vaccine conjugate against nicotine held within a DNA tetrahedron delivery system has been studied. For this purpose, several strands of DNA, conjugated with a modified dTpT having three or six carbon atom alkynyl linkers, have been synthesized. These strands have later been conjugated to three separate hapten small molecules to analyze which conjugates formed would be optimal for further testing in vivo. Mitochondrial diseases are hard to treat, given that there are so many different variations to treat. There is no one compound that can treat all mitochondrial and neurodegenerative diseases; however, improvements can be made to compounds currently under study to improve the conditions of those afflicted. A significant issue leading to compounds failing in clinical trials is insufficient metabolic stability. Many compounds have good biological activity, but once introduced to an animal, are not stable enough to have any effect. Here, several synthesized compounds have been evaluated for metabolic stability, and several showed improved stability, while maintaining biological activity. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2016

Made available in DSpace on 2014-06-11T19:27:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-21Bitstream added on 2014-06-13T20:36:07Z : No. of bitstreams: 1 milbradt_el_me_botfmvz.pdf: 618674 bytes, checksum: f9989c313b2f2cc46addf64bb9019c37 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Com o objetivo de avaliar a eficiência de diferentes métodos preventivos de coccidiose para perus de corte, foram utilizados 420 perus de corte, fêmeas da linhagem BUT 9 distribuídas em um delineamento experimental inteiramente casualizado dividido em quatro tratamentos: T1- dieta controle sem vacinação contra coccidiose e droga anticoccidiana, T2- dieta acrescida de droga anticoccidiana do 1º até os 60 dias de vida das aves (maduramicina 1%, 5ppm), T3- vacinação contra coccidiose (vacina comercial), T4- imunização pela exposição à cepas de campo (pool de oocistos). A administração da vacina e do pool de oocistos foi realizada via ração, no sétimo dia de vida das aves. As aves foram alojadas com densidade de 21 aves/m² até o sétimo dia, 9,8 aves/m² entre o oitavo dia e a sexta semana e, 4,2 aves/m² até a idade do abate, 70 dias. Aos 21 dias de idade, as aves foram submetidas ao desafio de coccidiose, representado por um pool de oocistos, sem a identificação das espécies, na dosagem de 20.000 oocistos por ave, a qual foi aplicada diretamente no esôfago. Os resultados foram submetidos à análise de variância (ANOVA) com auxílio do programa estatístico SAS, as médias comparadas pelo teste “t” a 5% de significância. A análise do experimento foi dividida em duas partes, sendo, fase inicial compreendida entre o dia do alojamento e o 28º dia de vida e fase final, compreendida entre o 29º dia até o abate. Na fase inicial, os tratamentos influenciaram (P≤0,05) o peso médio semanal, o ganho de peso médio, a conversão alimentar e o consumo médio de ração semanal, sendo que as aves do tratamento controle apresentaram desempenho inferior quando comparado aos demais. Na fase final, as aves apresentaram excelente recuperação do desempenho, sendo que somente o peso médio, aos 70 dias, foi afetado pelos tratamentos (P≤0,05)... / In order to evaluate the effectiveness of various preventive methods of coccidiosis control for turkeys, four hundred and twenty females BUT 9 strain were used distributed in a completely randomized design divided into four treatments: T1- control diet without coccidiosis vaccination and anticoccidial drug, T2- control diet increased by anticoccidial drug at the 1st until 60 days of age (maduramicin 1%, 5ppm), T3- control diet and vaccination (commercial vaccine), T4- control diet and oocysts mixed pool administration. The vaccine and pool administration was done into diet, on the seventh day of age. The birds were housed with a 21 birds / m² density by seventh day, 9.8 birds / m² of at the eighth day until sixth week, and 4.2 birds / m² up to the age of slaughter, 70 days. At 21 days of age, birds were submitted to the challenge of coccidiosis, represented by an oocysts pool without identifying the species, the dose of 20,000 oocysts/bird, which was applied directly in the birds’ esophagus. The results were submitted to variance analysis (ANOVA) using the SAS statistical program and compared by means of Test “t” to 5%. The trial analysis was divided into two parts, the initial stage understood at housing day until 28th day of age, and the final stage, at 29th day until killing. In the initial stage, the treatments affected (P ≤ 0.05) the weekly weight average, weight gain average, feed conversion, weekly consumption average and total feed intake, and the birds in the control treatment showed worse performance compared with others. In the final stage, the birds showed excellent performance recovery, with only the weight average, at 70 days, was affected by treatments (P ≤ 0.05). Therefore, all prevention and control methods of coccidiosis applied were efficient, as well as no adverse effect on the parameters evaluated, were still able to protect the birds... (Complete abstract click electronic access below)

Orientador: Samir Issa Samara / Banca: Moacir Marchiori Filho / Banca: Fabio Carvalho Dias / Banca: Adolorata Aparecida Bianco Carvalho / Banca: Luís Antonio Mathias / Resumo: A presente pesquisa teve como objetivo avaliar a utilização de cobaias e caprinos para teste de vacinas contra o herpesvírus bovino tipo 1 (BoHV-1) e os vírus da diarreia viral bovina tipos 1 (BVDV-1) e 2 (BVDV-2). Inicialmente foi realizada a infecção experimental em cobaias e bovinos, com esses três vírus, para verificar a máxima resposta imunogênica em ambas as espécies. As médias geométricas dos títulos de anticorpos (GMT) dos bovinos foram altas para todos os agentes virais; em cobaias foi possível observar que o BoHV-1 induziu elevadas GMT, para o BVDV-1 a indução foi moderada, enquanto para o BVDV-2 foi baixa, podendo comprometer a viabilidade dos testes antigênicos. Após essa etapa, foram vacinados bovinos, cobaias e caprinos, com seis vacinas comerciais de antígenos inativados, para verificar a indução de anticorpos por estímulo vacinal. Nos bovinos, as GMT obtidas para o BoHV-1 mostraram que as vacinas utilizadas promoveram a indução moderada na resposta imunológica, e apenas três delas foram consideradas satisfatórias. Para o BVDV-1, apenas uma vacina pode ser considerada eficiente, e em relação ao BVDV-2 nenhuma delas poderia ser assim classificada. Os resultados da vacinação com os caprinos mostraram que, apesar de os animais serem reagentes ao BoHV-1 no início do experimento, as formulações foram capazes de induzir a produção de anticorpos com títulos elevados contra os três vírus estudados. As cobaias, por sua vez, receberam doses fracionadas de uma vacina comercial nacional e, das frações testadas, 3,2mL foi a que apresentou melhores resultados, sendo esta estabelecida para testar as demais vacinas nesta espécie. Depois da imunização, os resultados mostram que para o BoHV-1 as GMT foram altas; em relação ao BVDV-1, apenas duas vacinas foram capazes de induzir... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present research had as objective to evaluate the use of guinea pigs and goats in test of vaccines against the bovine herpesvirus type 1 (BoHV-1) and the bovine viral diarrhea viruses types 1 (BVDV-1) and 2 (BVDV-2). First, an experimental infection was realized in guinea pigs and bovines, using these three viruses, to verify the maximum immune response in both species. The geometric means of antibodies titres (GMT) for bovines were high for the viral agents; in guinea pigs was possible to observe that the GMT induced by BoHV-1 was high, by BVDV-1 was moderate and by BVDV-2 was weak and could compromise the viability of antigenic tests. After this stage, the bovines, guinea pigs and goats were vaccinated using six commercial vaccines with inactivated antigens to verify the induction of antibodies production by vaccinal stimulus. In bovines, the GMT obtained for BoHV-1 showed the vaccines induced a moderate immune response, being only three of them considered satisfactory. For BVDV-1 only one and for BVDV-2 none of them can have the same classification. The results obtained with the goats, despite they were positive to BoHV-1 in the sorting, showed the formulations were able to induce high antibody production against the three virus studied. The guinea pigs, on the other hand, received fractional doses of a commercial vaccine well-established in the domestic trade and, from the fractions tested, 3.2 mL showed the best results, being established to test the other vaccines. After immunization, the results of GMT for BoHV-1 was high; relating to BVDV-1, only two vaccines were capable to induce antibodies production, but their GMT were weak; and there was no immune response against BVDV-2. Thus, goats can be ... (Complete abstract click electronic access below) / Doutor

La maladie de Newcastle est une de deux pestes aviaires qui, comme l’influenza, impactent fortement les élevages d’oiseaux domestiques par leur incidence clinique et leurs conséquences économiques sur la filière (contrôle des mouvements d’animaux, abattages sanitaires et préventifs). Des vaccins contre cette maladie ont été développés il y a plusieurs décennies à base de souches virales isolées dans les années 60. Ils assurent normalement une excellente protection clinique. Toutefois, depuis une dizaine d’années, des observations de terrain, principalement en Afrique et en Asie, font état d’échecs partiels de vaccination avec occurrence de foyers réduits dans des élevages a priori correctement vaccinés. En parallèle, des essais in vivo en conditions contrôlées ont établi que les vaccins actuels protégeaient bien cliniquement contre une épreuve avec des souches virulentes récentes mais n’empêchaient pas leur excrétion par les animaux vaccinés. Pour résoudre cette problématique, l’objectif de ce travail a été de générer une souche vaccinale plus efficace contre les souches virulentes circulant actuellement à l’échelle du globe. Pour générer des virus atténués modifiés, nous avons dû dans un premier temps améliorer le système conventionnel de génétique inverse. Nous montrons que la réduction du nombre de plasmides à 2 dans le système, permet de générer plus de virus atténués que le système conventionnel basé sur 4 plasmides. Dans un second temps, nous nous sommes intéressés à étudier le comportement in vitro de virus atténués et virulents équipés de marqueurs fluorescents. Nous montrons que seuls les virus virulents induisent un effet cytopathique in vitro. En revanche, les deux types de virus induisent une infection persistante à long terme sans effet cytopathique. Les cellules infectées de façon persistante résistent à une surinfection par un autre virus. En revanche, lors de co-infections simultanées, nous établissons qu’une cellule infectée par un premier virus peut s’infecter par un second virus lors d’un transfert direct de matériel viral d’une cellule à une autre par des extensions membranaires. Cette observation est remise en perspective par rapport à la capacité de ces virus à se recombiner chez l’animal telle qu’identifiée par des analyses bioinformatiques comparatives de différents isolats. En effet, nous montrons que des cellules peuvent s’infecter avec plusieurs virus par contact direct. Dans un dernier travail, une nouvelle souche vaccinale a été générée consistant à insérer des antigènes immunoprotecteurs d’un virus original isolé à Madagascar en 2008, dans un génome d’une souche vaccinale conventionnelle utilisée depuis plus de 50 ans. Nous montrons que cette nouvelle souche protège efficacement contre trois génotypes viraux dont deux circulant actuellement en Afrique et en Asie. / In addition to influenza, Newcastle disease is one of the two major diseases of poultry that strongly impact the animal health and farming owing to animal bans and depopulations. Vaccines against Newcastle disease are available. They have been developed some decades ago from isolates collected in the 60’s. They usually provide an excellent clinical protection. However, field reports of the last decade, mainly from Africa and Asia, suggest partial vaccination failures in some farms despite proper vaccination. In parallel, in vivo trials have shown that current vaccines provide a good clinical protection against a challenge with recent field strains, but do not prevent shedding of the challenge virus from vaccinated birds. To address this issue, one of the objectives of this study was to generate a new vaccine prototype with improved efficacy against virulent strains circulating worldwide. To generate new engineered attenuated viruses, we first developed an improved reverse genetics system. We demonstrate that the reduction of the number of plasmids to 2 compared to the conventional system based on 4 plasmids does not affect the performances of reverse genetics for virulent strains but significantly increases the yield of attenuated viruses. In a second step, we focused on the behavior of the attenuated and virulent viruses generated by reverse genetics. The viruses were tagged with fluorescent reporter genes to make easier they follow up in cell culture. We show that only virulent strains produce cytopathic effects in vitro. However, both attenuated and virulent strains are able to establish persistent infection in cells without cytopathic effects. Persistently infected cells resist to a super-infection by another virus. In contrast, after co-infection by two different viruses, we show that a cell infected by one virus can be infected by a second one by direct virus trafficking between the cells through cell membrane extensions. This observation supports the possibility of recombination events in the field which are frequently claim in the literature from comparative bioinformatics of field isolates and vaccine strains. Indeed, we show that cells can be infected by multiple viruses through direct contacts between cells. In a last step, a new vaccine prototype has been produced consisting in the substitution of immune-protective antigens in the conventional LaSota vaccine by their homologues derived from an original isolate of Madagascar (2008). We show that this prototype is protective against challenges with three different viruses, including two recent isolates from Africa and Asia.

Objetivo: Estimar o impacto da vacina conjugada contra o meningococo C (VCMC), na incidência e mortalidade, nas coortes de nascidos com e sem indicação de vacinação, no município de São Paulo (MSP); e estimar a efetividade direta da VCMC segundo esquema do Programa Nacional de Imunização. Métodos: O impacto foi avaliado por estudo descritivo, abrangendo casos de doença meningocócica (DM) notificados ao MSP, de 1998 a 2012. A definição de caso é a adotada pelo Ministério da Saúde. Descreveu-se o comportamento da DM no MSP para todo período e analisou-se a tendência da incidência e mortalidade da DM global e por faixa etária de 2008 a 2012, utilizando o modelo de Poisson. O impacto da VCMC foi analisado por meio das razões de taxas de incidência e mortalidade nos períodos anterior e posterior a introdução da VCMC. Estimou-se a fração prevenida na população (FPP) para mensurar o impacto, comparando-se taxas de incidência e mortalidade globais da DM, por faixa etária e sorogrupo C, de 2012 com as de 2009. Para estimativa da efetividade da VCMC utilizou-se estudo de caso-controle de base populacional, com quatro controles para cada caso, pareado pela área de residência dos casos. Casos e controles foram selecionados entre nascidos a partir de janeiro/2009. Casos eram aqueles com DM pelo sorogrupo C confirmado por cultura e/ou reação em cadeia de polimerase em tempo real, de 2011 a 2013, internados em hospitais do MSP, notificados à vigilância do município. Controles foram selecionados entre crianças residentes na vizinhança dos casos, sem história de DM. A efetividade da vacina foi estimada pela fórmula (1-odds ratio para vacinação). As odds ratios (OR) não ajustadas e ajustadas e respectivos intervalos de confiança (IC95 por cento ) foram estimados por regressão 11 logística condicional múltipla. A associação entre ser vacinado com VCMC e a variável dependente, DM pelo sorogrupo C, foi mensurada pela estimativa da OR após ajuste para potenciais confundidores. Resultados: O impacto da VCMC na incidência da DM por todos os sorogrupos, mensurado pela FPP foi de 62,7 por cento , 69,6 por cento e 61,4 por cento para, respectivamente menores um, um e dois anos; na DM pelo sorogrupo C de 81,6 por cento e 67,9 por cento para menores de dois anos e de dois a três anos. Houve impacto na taxa de mortalidade global da DM medido pela FPP de 86,2 por cento e 77,8 por cento respectivamente para menores de dois anos e de dois a três anos e na mortalidade da DM pelo sorogrupo C a FPP foi de 84,2 por cento para menores de quatro anos. A efetividade da VCMC foi de 97,7 por cento (IC95 por cento :99,6 por cento -89,6 por cento ) ajustada para idade, número de pessoas no quarto da criança e renda familiar. Conclusões: A estratégia brasileira com a VCMC resultou em elevado impacto nas coortes de nascidos com indicação de vacinação, mais acentuado nas taxas de mortalidade, sugerindo que a vacina confere não só proteção para a doença, mas também para formas mais graves. A VCMC foi altamente efetiva na faixa etária alvo. / Objective: To assess the impact of meningococcal C conjugate vaccine (MCCV), to estimate incidence and mortality rates of meningococcal disease (MD) in birth cohorts recommended and not recommended for vaccination and to measure direct vaccine effectiveness of the National Vaccination Program immunization schedule. Methods: We assessed the impact of MCCV in a descriptive study including cases of MD reported in the city of Sao Paulo, Brazil, from 1998 to 2012. We used the standard case definition recommended by the Brazilian Ministry of Health for MD reporting. We assessed changes in the disease epidemiology in the city for the entire study period and estimated incidence and mortality rates of MD (overall and by age group) from 2008 to 2012 using Poisson regression models. We conducted an impact analysis of MCCV by comparing incidence and mortality rates of MD before and after vaccine introduction. We also estimated the population prevented fraction (PPF) by comparing incidence and mortality rates of MD between 2009 and 2012 in the entire population and by age group and serogroup C. To measure vaccine effectiveness, we carried out a population-based case-control study matched for area of residence with a 4-to-1 ratio of controls to cases. Cases and controls were selected among children born from January 2009. Cases were those children admitted to the citys hospitals who were diagnosed with MD serogroup C (MDC) confirmed by culture and/or real-time polymerase chain reaction and reported to the surveillance system from 2011 to 2013. Controls were selected among children with no history of MD from neighboring areas of cases. We calculated vaccine effectiveness using the formula (1 odds ratio [OR] for 13 vaccination) and estimated crude and adjusted ORs and related 95 per cent confidence intervals (95 per cent CI) by conditional multiple logistic regression. We assessed the association between MCCV vaccination and MDC the dependent variable by estimating OR after adjustment for the potential confounders. Results: There was an impact of MCCV on the incidence of MD in all serogroups, the PPF among children under age one, age one, and age two were 62.7 per cent , 69.6 per cent , and 61.4 per cent , respectively; and in serogroup C, the PPF in children under age two and age two to three were 81.6 per cent and 67.9 per cent . There was also an impact on the overall mortality rate of MD, the PPF in children under age two and age two to three were 86.2 per cent and 77.8 per cent ; and on mortality of MDC, the PPF was 84.2 per cent in children under age four. MCCV effectiveness in children was 97.7 per cent (95 per cent CI 99.6 per cent 89.6 per cent ) after adjusting for age, number of persons per room, and household income. Conclusions: The MCCV strategy implemented in Brazil had a high impact on birth cohorts recommended for vaccination. This impact was more pronounced on mortality rates, which suggests that, in addition to preventing disease, MCCV can prevent more severe forms of MD. MCCV proved highly effective in the age groups targeted.

Introdução: A infecção pelo Papiloma Virus Humano (HPV) vem sendo reportada como uma das doenças sexualmente transmissíveis com maior incidência na atualidade, porém a sua prevalência não é bem esclarecida em homens, principalmente devido a baixa presença de sintomas. Além disso, poucos estudos foram realizados nesta população até o momento para verificar a resposta imune pós-vacinação. As hipóteses testadas serão fundamentais para aprofundar o conhecimento da imunopatogênese, da resposta vacinal em pacientes infectados pelo HIV e colaborar no desenho e estratégias de vacinação anti-HPV na população infectada pelo HIV Objetivos: Analisar a resposta específica aos antígenos da vacina anti-HPV em homens infectados pelo HIV. Métodos: Um total de 24 pacientes infectados pelo HIV que preencheram os critérios de inclusão durante o período de coleta foram vacinados pela vacina anti-HPV bivalente em três doses nos períodos: zero, dois e seis meses. Os grupos foram divididos em: Grupo Controle (Cinco indivíduos sadios, com sorologia negativa para HIV); Grupo A (Nove pacientes com CD4 <500 celulas mm³); Grupo B (10 pacientes com CD4 >=500 celulas mm³). Foram realizados ELISA para a detecção de anticorpos Anti-HPV nos momentos pré e pós-vacinação nos grupos estudados; posteriormente realizamos nos mesmos o ensaio de cultura celular para detecção de citocinas (IFN?, IL17, TNF, IL6 e IL10) pela técnica de CBA . Resultados: Obtivemos soroconversão da primeira dose da vacina para o grupo A 55,6%, grupo B 30%, grupo controle 60%; na segunda dose obtivemos para o grupo A 88,8%, grupo B 80%, grupo controle 80%, e por final a terceira dose no grupo A 88,8%, grupo B 90%, grupo controle 100%. A citocina IL 6 (perfil TH2) demonstrou níveis mais elevados, comparados entre os grupos A, B e grupo controle (p<0.001). A partir da 3° dose da vacinação observamos baixos níveis de INF-? (perfil TH1) A e B (p<0.0006). O grupo controle apresentou produção de INF- ? quando comparado com grupos A e B (p<0.001). Conclusão: Os pacientes soropositivos e grupo controle foram respondedores a vacinação anti-HPV. Foi demonstrada uma elevada produção das citocinas entre os grupos sugerindo uma imunomodulação do grupo HIV+. Esse trabalho apresenta informações relevantes que estimulam a realização de novos estudos nessa população, avaliações de reações cruzada da vacina que pode resultar em proteção a outros tipos de HPV não presentes na vacina, além de analisar por mais tempo as titulações no soro desses pacientes. Os dados do nosso estudo podem corroborar para a vacinação nessa população, diminuindo assim o risco de uma infecção, mortalidade e morbidade das doenças causadas pelo HPV em homens. / Introduction: Infection with Human Papilloma Virus (HPV) has been reported as one of the sexually transmitted diseases with a higher incidence nowadys, but its prevalence must be clarified in men, mainly due to low presence of symptoms. Moreover, few studies have been performed in this population until now to verify the immune response post-vaccination. The hypothesis here suggested will be the key for better understanding of the immunopathogenesis, the vaccine´s response in HIV-infected patients and collaborate in the design and strategies of vaccination against HPV in HIV-infected population. Objectives: Analyze the specific response to antigens of HPV vaccine in HIV-infected men. Methods: A total of 24 HIV-infected patients who were in accordance with the inclusion criteria during the data collection period were vaccinated with anti-HPV bivalent vaccine in three period doses: zero, two and six months. The groups were distributed in: Control group (five healthy subjects with negative serology against HIV); Group A (nine subjects with CD4 <500 cells/mm³; Group B (10 subjects with CD4 >500 cells/mm³). ELISA was performed to detect the level of antibodies anti-HPV before and after vaccination in the studied cohort. Postenarly, cells of these groups were submitted in culture to verify citokynes production (IFN?, IL17, TNF, IL6 and IL10) using CBA methodology. Results: We obtained seroconversion after the first dose of anti-HPV vaccine: control group 60%, group A 55,6% and group B 30%. In the second dose: control group 80%, group A 88,8% and Group B 80%. And at last, the third dose: Control Group 100%, Group A 88,8% and group B 90%. IL 6 citokyne (TH2 response) was detected in higher level when compared Control, A and B groups (p<0.001). IFN? citokyne (TH1 response) was detect in low level only after the third dose of vaccination, showing relevance between A and B groups (p<0.0006). Additionally, higher IFN? production was detected when compared the control with A and B groups (p<0.001). Conclusion: HIV patients and controls (HIV-) were responders to anti-HPV vaccination. It was clear that an elevated cytokine production was detected between groups, suggesting immunomodulation of HIV + group. This work suggests relevant information that challenge: new studies in this population, verification of cross-reactions of the vaccine resulting in protection of other HPV types not present in this vaccine, and analyze for longer period the titers of anti-HPV antibodies in these patients. All together, our data can corroborate for vaccination in this population, thus decreasing the risk of infection, mortality and morbidity of the disease caused by HPV in men.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-04T18:49:12Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2011-Dissertação-ElydaLima.pdf: 2803583 bytes, checksum: 5b93b96556bc230adb5bb7ca5a4fb423 (MD5) / Made available in DSpace on 2017-04-04T18:49:12Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2011-Dissertação-ElydaLima.pdf: 2803583 bytes, checksum: 5b93b96556bc230adb5bb7ca5a4fb423 (MD5) Previous issue date: 2011-03-16 / A bovinocultura é um dos principais destaques do agronegócio brasileiro no cenário mundial. No entanto, algumas doenças vêm causando prejuízos consideráveis entre elas está a papilomatose bovina, uma doença infectocontagiosa, de caráter tumoral, com etiologia relacionada a infecção pelo papilomavírus bovino (BPV) que se caracteriza pela formação de tumores em tecidos da pele e mucosa. Hoje se conhecem 11 tipos diferentes de Papilomavírus bovino, sendo os BPvs tipos 1, 2 e 4 oncogênicos. Até o momento não existe vacinas para o controle ou tratamento das papilomaviroses. Diferentes estudos vêm demonstrando que a proteína L2 pode ser uma candidata ao desenvolvimento de estratégias vacinais profiláticas contra o BPV. Neste trabalho, tivemos como objetivo construir um vetor vacinal a partir do plasmídeo pCINeo (Promega®) e do gene L2 de BPV1, avaliando in vitro a expressão do antígeno L2 em células de mamíferos. O gene L2 foi amplificado pela técnica de PCR a partir do genoma completo de BPV1, com uso de oligonucleotídeos específicos contendo um epítopo AU1 no primer forwarde posteriormente clonado em vetor de passagem pGEM-T Easy (Promega®) e subclonado no vetor de expressão pCIneo, gerando a construção pCIL2B1. O plasmídeo foi transfectado in vitroem células 293 para análise funcional da expressão de L2. Os resultados obtidos confirmaram a construção pCIL2B1 por PCR e sequenciamento. A capacidade desta construção expressar o gene L2 e produzir a respectiva proteína em células de mamífero foi confirmada por RT-PCR e western blot (usando anticorpo contra o epítopo AU1). / The cattle industry is one of the main highlights of the Brazilian agribusiness on the international stage. However, some diseases have caused considerable damage including bovine papillomatosis which is an infectious tumorous disease related to bovine papillomavirus (BPV) etiology and characterized by the formation of tumors in tissues of the skin and mucosa. Currently we know 11 different types of bovine papillomavirus, among which the BPv types 1, 2 and 4 are oncogenic. So far there is no vaccine or treatment for the papilomaviroses control. Different studies have reported that the L2 protein may be a candidate for the development of prophylactic vaccine strategies against BPV. The L2 protein has a potential cross-reaction with different BPVs and HPV (HumanPapillomavirus). On this paper, our objective was to construct a vector vaccine from pCINeo plasmid (Promega ®) and the gene of BPV1 L2, evaluating in vitro L2 antigen expression in mammalian cells.The L2 gene was amplifiedby PCR from thecomplete genome ofBPV1, using specifico ligo nucleotidescontainingan AU1epitopein the for wardprimerand subsequently cloned intop GEM-T Easyvector(Promega ®)and subclonedin expression vector pCIneo, pCIL2B1generatingbuilding. The plasmid was transfectedinto 293 cellsin vitrofunctional analysisforthe expression ofL2. ObtainingconstructionpCIL2B1was confirmed by PCRand sequencing.The capacityof this construction to express the geneand produce their L2 protein inmammalian cellswas confirmed by RT-PCR and western blot (usinganti body against the epitopeAU1). The results confirmed the L2 gene expression in mammalian cellsand the consequent production of the protein L2 BPV-1.

Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T01:23:26Z (GMT). No. of bitstreams: 1 Pivetta_LucianeBeneditaDuarte_M.pdf: 4595229 bytes, checksum: 95f48747caf7eb8ddce4f6ccacc9d5d4 (MD5) Previous issue date: 2011 / Resumo: Sorovariedades patogênicas de Salmonella enterica constituem um problema de saúde global, que abrange desde gastroenterites até doenças sistêmicas, sendo que ambas podem ser letais. Linhagens atenuadas de S. enterica são promissoras como vacinas vivas, pois são facilmente administradas via oral e capazes de induzir o sistema imune sistêmico e de mucosas do hospedeiro. Além disso, apresentam o potencial de atuar como vacinas multifatoriais, expressando antígenos de outros agentes infecciosos. Neste trabalho, linhagens recombinantes de Salmonella enterica foram construídas e a atenuação da virulência e a estimulação do sistema imune foram avaliadas no modelo murino da salmonelose. As linhagens recombinantes foram desenvolvidas a partir do sistema de recombinação homóloga ?Red, com a deleção de um ou mais genes com efeitos pleiotrópicos na virulência de Salmonella. Após a confirmação da deleção do gene alvo por PCR, as linhagens foram transduzidas com o bacteriófago P22, visando eliminar possíveis problemas com a integridade de lipopolissacarídeos. Posteriormente, caracterizações fenotípicas foram realizadas, por meio de curvas de crescimento in vitro, verificação da capacidade de invasão e sobrevivência no interior de macrófagos e resistência a espécies reativas de oxigênio e nitrogênio. A atenuação da virulência das linhagens foi avaliada in vivo por meio de inoculações orais e intraperitoneais em camundongos BALB/c/AnUnib e a dose letal média também foi estabelecida. Variados níveis de atenuação foram atingidos e dentre as linhagens desenvolvidas, a duplo mutante para os genes hupA e hupB foi a que apresentou os resultados mais promissores. Nos ensaios de proteção e desafio, 100% dos animais vacinados com duas doses da linhagem 'delta'hupA'delta'hupB sobreviveram ao desafio com doses letais da linhagem selvagem. Para a linhagem duplo mutante também foi verificada a capacidade de colonização bacteriana no sangue, placas de Peyer e baço, assim como a produção de anticorpos IgG e IgA. A obtenção de uma linhagem atenuada, porém capaz de induzir com eficácia o sistema imune do hospedeiro é idealmente almejada no desenvolvimento de uma vacina viva. No entanto, atingir esse balanço é um desafio, já que a deleção de genes de virulência de Salmonella acarreta em diferentes níveis de atenuação, mesmo quando os genes são intimamente relacionados. Uma perspectiva futura é o refinamento das linhagens atenuadas por meio da introdução de antígenos heterólogos e de um sistema de expressão regulado, com o estabelecimento de vacinas multifatoriais / Abstract: Pathogenic Salmonella enterica serovars are the cause of a global health problem that ranges gastroenteritis to lethal systemic disease. Attenuated strains of Salmonella enterica are promising as live vaccines because they are orally administrated and capable of inducing a mucosal and systemic immune response in the host. Besides, they can act as multifactorial vaccines delivering antigens from other diseases. In this work, attenuated strains of Salmonella enterica were produced and the attenuation of the virulence and the induction of an immunologic response were evaluated in the murine model of salmonellosis. The recombinant strains were constructed using the ?Red system of homologous recombination deleting one or more genes with pleiotropic effects on Salmonella virulence. After that, a PCR to confirm the deletion of the target gene was made and the strains were transduced with bacteriophage P22 to avoid problems with lipopolysaccharide's integrity. Phenotypic characterization as the in vitro growth curve of the strains, the resistance to ROI and RNI species and the ability of survival inside J774 macrophages cultures were performed. The attenuation of the strains was evaluated in vivo trough oral and intraperitoneal inoculations of the strains into BALB/c/AnUnib mice and the median lethal dose was also established. Many levels of attenuation were reached and among the developed strains, the double mutant strain for the genes hupA and hupB was the one with the best results. In the protection and challenge assays, 100% of the animal vaccinated with two doses of the 'delta'hupA'delta'hupB strain survived to the challenge with lethal doses of the wild type strain. For this strain it was also verified the capacity of colonization into blood, peyer's patches and spleen as well as the production of IgG and IgA antibodies. In the developmente of a live vaccine it is ideal to have a strain that is attenuated but still capable of inducing an immune response in the host. Reaching this balance is a challenge and the deletion of virulence genes in Salmonella entails different levels of attenuation although they are closely related. A future prospect is the improvement of these attenuated strains with the insertion of controlled expression systems and heterologous antigens, creating a multifactorial vaccine / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular