MicroRNAs (miRNAs) have been identified as a key regulator in various biological processes and different diseases including cancer, heart disease, and viral infections. In the context of virus-host interactions, previous genome wide functional screen involving overexpression and inhibition of murine miRNAs in vitro identified several miRNAs that suppressed viral replication in diverse herpesviruses including herpes simplex virus 1 (HSV-1), murine cytomegalovirus (MCMV) and murine gamma herpesvirus 68 (MHV-68) (Santhakumar, D. et al, 2010). One of the top broad-spectrum anti-viral miRNAs, miR-542-5p, also suppressed human cytomegalovirus (HCMV) as well as a Semliki Forest virus (SFV) and two subtypes of influenza A virus (H1N1 & H3N2) in vitro. Following the previous study, this thesis focuses on generation of in vivo anti-viral efficacy data using miR-542-5p against two diverse viruses: MCMV and influenza (H1N1) in a pre-clinical model (mouse). One of the key challenges for generating in vivo efficacy data with miRNAs is the choice of delivery vehicle. To address this issue the first part of the project focused on optimising delivery conditions (dose, route of administration) for miR-542-5p mimic to target the lungs of mice (as both MCMV and H1N1 replicate in lungs). Initially, delivery was optimised using two cationic polymers: linear polyethylenimine (in vivo-JetPEI) and branched polyethylenimine (25KDa bPEI) that have been widely used previously to deliver nucleic acids in mouse. In parallel, two novel delivery systems were tested as an alternative delivery vehicle for miRNA mimics: biodegradable cationic lipids (Lifectin) and exosomes, natural vesicles produced by cells that can transport RNA. Results from in vivo delivery studies indicate that nebulisation of miR-542-5p mimic complexed with bPEI (25KDa) showed a more significant increase in the level of miRNA in the lung compared to other delivery systems and did not result in an immune response. Using bPEI as the delivery vehicle, the miR-542-5p mimic was administered to mouse lungs to test its anti-viral function against H1N1 and MCMV. Delivery of the miR-542-5p mimic resulted in 4.6 fold reduction of H1N1 virus titre in lungs (averaged across multiple experiments). The miR-542-5p mimic also had a 2 fold reduction in MCMV titre in the lungs. These data confirm the broad-spectrum anti-viral effect of miR-542-5p in mouse as observed in previous in vitro studies. Preliminary microarray analysis of genes regulated by miR-542-5p in vitro suggest this miRNA may target multiple genes required by diverse viruses during their life cycles and may modulate the PI3K-Akt signalling pathway.

Many behavioral interventions have attempted to increase sports performance. These interventions include: goal setting and feedback, behavioral coaching, and acoustical guidance. The use of technological devices in all areas of life continues to steadily increase. Therefore, behavioral interventions should also adapt to meet these changes in technology. One such intervention is in-vivo audio feedback in which participants receive live feedback about their current performance while practicing the skill. In-vivo audio feedback has not yet been rigorously evaluated as a means of improving sports performance. This study used a multiple baseline across participants design to evaluate the effects of in-vivo audio feedback on cycling performance. Results from this study suggest that in-vivo audio feedback was an effective method for improving cycling performance for individuals wishing to increase their average cycling speed.

The gastrointestinal microbiota of livestock is a complex and diverse ecosystem, which strongly depends and correlates with animals health. One of the many ways, how to improve the composition of this microbiota in the gastrointestinal tract, is application of probiotics, prebiotics or an appropriate combination called as synbiotics. Using subtle interventions can be achieved health benefits, increase of some indicators livestock performance and improving the economic situation of farming. The aim of the thesis was to find out the effect of prebiotics on the currently applied probiotics and the composition of the intestinal microbiota. Three groups of calves were studied. In the first one the mixture of five strains of rifampicin resistant variants of Bifidobacterium (RRBif): Bifidobacterium animalis ssp. animalis (2 strains), B. choerinum, B. thermophilum and B. longum ssp. suis was fed to 2 days old calves in a form of fermented milk. In the second group probiotics were adminestered together with prebiotics mixture contains fructooligosaccharides and galactooligosaccharides everyday. The third group without synbiotic treatment was used as a control. The numerous of main bacterial groups in gastrointestinal tract were determined by cultivation. Our results showed that applied bacteria were able to survive the passage through the gastrointestinal tract of calves. Administered bacteria persist in counts more than 107 CFU/g in the group of calves, that were fed only by probiotics and in numbers 108 CFU/g in the group of calves, that were treated by synbiotics. Furthermore a slow decrease of bifidobacterial numbers to 104 CFU / g was observed in both groups. Significant difference (p = 0.05) between the count of RRBif in probiotic and synbiotic group were determined only two days after administration of that additive. Administration of probiotics and synbiotics to calves increased the count of naturally occurring bifidobacteria, but these differences were not significant.

Probiotics are living microorganisms, which contribute to health improvement. The bacteria of the genus Bifidobacterium are one of the most commonly used probiotics. These bacteria are naturally occurring especially in the digestive tract of mammals. They affect the digestive process by helping to optimize the composition of the intestinal microbiota. The probiotic bacteria are expected to survive in the digestive tract and to be able to colonize the intestinal mucosa. The aim of this diploma thesis was to test bifidobacteria of different origin for certain functional properties. Three strains of bifidobacteria were selected: Bifidobacterium animalis ssp. lactis DAN (isolated from Danone yoghurt), Bifidobacterium bifidum JKM (isolated from infant feces) and Bifidobacterium longum ssp. suis 5/9 (isolated from feces of a calf); and tested in vitro a in vivo. The ability to survive in simulated conditions of digestive tract was tested. The bifidobacteria were exposed to low pH and bile salts. All the strains survived the conditions in unaltered amount till the end of the test. Autoaggregation was observed as well. The strain Bifidobacterium animalis ssp. lactis DAN showed average autoaggregation only 7,69 %. The other two strains had average autoaggregation 16,10 % and 79,05 %. The autoaggregation correlated with adherence. Adherence was tested on intestinal cell lines HT-29, Caco-2 and HT29-MTX, which differed in their ability to produce mucin. The strain Bifidobacterium bifidum JKM showed the best ability to adhere to intestinal cells among all the tested bifidobacteria. They best adhered to Caco-2 cells. Further, it was necessary to determine if the in vitro study is in correlation with in vivo testing and the bifidobacteria strain with the best ability to adhere will survive in the digestive tract of adult volunteers for the longest time. Rifampicin resistant variants of tested bacteria were given to adult volunteers to distinguish the introduced bifidobacteria and the bifidobacteria naturally occurring in intestine. All the tested bacteria survived the passage through the digestive tract in large numbers (106 a 107 KTJ/g), the bacteria were not detected 12th day after the introduction. None of the strains is able to permanently colonize the colon of the volunteers. The hypothesis was not confirmed that the most adherent strain in the digestive tract will survive the longest.

LOPES NETO, José Joaquim. Bioacessibilidade e biodisponibilidade de compostos fenólicos de sementes de Triplaris Gardneriana Wedd (Polygonaceae). 2017. 95 f. Dissertação (Mestrado em Bioquímica)- Universidade Federal do Ceará, 2017. / Submitted by Weslayne Nunes de Sales (weslaynesales@ufc.br) on 2017-07-10T12:20:53Z No. of bitstreams: 1 2017_dis_jjlopesneto.pdf: 1657114 bytes, checksum: 3ec381c68cd046900e25b007ed35226a (MD5) / Approved for entry into archive by Weslayne Nunes de Sales (weslaynesales@ufc.br) on 2017-07-11T11:56:07Z (GMT) No. of bitstreams: 1 2017_dis_jjlopesneto.pdf: 1657114 bytes, checksum: 3ec381c68cd046900e25b007ed35226a (MD5) / Made available in DSpace on 2017-07-11T11:56:07Z (GMT). No. of bitstreams: 1 2017_dis_jjlopesneto.pdf: 1657114 bytes, checksum: 3ec381c68cd046900e25b007ed35226a (MD5) Previous issue date: 2017 / Triplaris gardneriana Wedd is a plant species from Brazilian semiarid region with seeds rich in phenolic compounds and high antioxidant capacity.The consumption of fruits and vegetables has been associated to low incidence of chronic diseases caused by oxidative stress. Among the food components capable of combating excess free radicals arethe phenolic compounds, with antioxidant capacity, which have been extensively studied. However, the total quantity of these substances present in foods does not necessarily reflect the bioactive amount absorbed and metabolized by the body. In this context, the present work aimed the development of an ethanolic extract obtained from the seeds of T. gardneriana (EETg), and from this, to evaluate its antioxidant activity through different methodologies, to investigate the bioaccessibility of EETg by determination of phenolic composition before and after in vitro digestion as well as to estimate its indirect bioavailability by chemical analysis of plasma and urine in rodents after oral administration. In general, EETg presented levels of antioxidant activity by DPPH, ABTS.+, FRAP and TBARS tests comparable to those described in specialized literature. The bioaccessibility indexes of phenolic compounds in EETg were 48.65 and 69.28% in the presence and absence of enzymes, respectively. Among the identified phenolics classes, flavonoids, represented by galloylated procyanidins, proved to be more bioaccessible (81.48 and 96.29% in the post-intestinal phase with and without enzymes, respectively). The oral administration in Wistar rats resulted in a significant decrease in plasma total antioxidant capacity (TAC) by FRAP assay 4 h after beginning the experiment. As to urine analysis, an increase in TAC by DPPH and FRAP was observed from 1 and 4 h after administration, respectively. UPLC-QTOF analysis of urine detected 2 metabolites originated from the degradation of phenolic compounds: hippuric acid and phenylacetil glycine. These results suggest that phenolic compounds in T. gardneriana, despite their antioxidant potential, are unstable under gastrointestinal conditions, being flavonoids the components with higher bioaccessibility; besides that, they showed limited bioavailability due to their rapid biotransformation and urinary elimination. / Triplaris gardneriana Wedd é uma espécie vegetal do semiárido brasileiro com sementes ricas em compostos fenólicos e alta capacidade antioxidante. O consumo de frutas e vegetais tem sido associado à baixa incidência de doenças crônicas causadas por estresse oxidativo. Dentre os componentes alimentares capazes de combater o excesso de radicais livres, os compostos fenólicos, de caráter antioxidante, têm sido bastante estudados.Contudo, a quantidade total destas substâncias presente nos alimentos não reflete necessariamente a porção bioativa absorvida e metabolizada pelo organismo. Neste contexto, o presente trabalho objetivou o desenvolvimento de um extrato etanólico obtido a partir das sementes de T. gardneriana(EETg),e a partir deste,caracterizar sua composição fenólica, avaliar sua atividade antioxidante através de diferentes metodologias, além deinvestigar a bioacessibilidade in vitro dos polifenóis presentes em EETg após digestão gastrointestinal simulada e estimar a biodisponibilidade indireta dos mesmos a partir da análise química do plasma e urina em roedores após sua administração oral. Em linhas gerais, EETg apresentou níveis de atividade antioxidante pelos ensaios DPPH, ABTS.+, FRAP e TBARS comparáveis àqueles descritos naliteratura especializada. Os índices de bioacessibilidade de compostos fenólicos em EETg foram de 48,65 e 69,28% na presença e ausência de enzimas, respectivamente. Dentre as classes fenólicas identificadas, os flavonoides, representados porprocianidinas acrescidas de grupos galoil, mostraram-se mais bioacessíveis (81,48 e 96,29% na fase pós-intestinal com e sem enzimas, respectivamente). A administração oral de EETg em ratos Wistar resultou em uma significativa diminuição da capacidade antioxidante total (CAT) do plasma pelo ensaio FRAP 4 h após o início do experimento. Para as amostras de urina, um aumento naCAT pelos testes DPPH e FRAP foi observado a partir de 1 e 4 h após a administração, respectivamente. Análises por UPLC-QTOF daurina detectou 2 metabólitos oriundos da degradação de compostos fenólicos: ácido hipúrico e fenilacetil glicina. Estes resultados sugerem que os compostos fenólicos deT. gardneriana, apesar do seu potencial antioxidante, são instáveis em condições gastrointestinais, sendo flavonoides os componentes com maior bioacessibilidade. Além disto, mostraram biodisponibilidade limitada devido à sua rápida biotransformação e eliminação urinária.

Made available in DSpace on 2014-07-28T18:10:46Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 88.pdf: 1790108 bytes, checksum: ed390bad661dddf012595ee892a4b51c (MD5) Previous issue date: 2008 / Made available in DSpace on 2014-12-22T16:56:36Z (GMT). No. of bitstreams: 2 88.pdf: 1790108 bytes, checksum: ed390bad661dddf012595ee892a4b51c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Todos produtos injetáveis devem estar livres de pirogênio. Existem dois métodos oficiais para a detecção da contaminação pirogênica: o teste em coelhos e o teste do Lisado do Amebócito de Limulus (LAL), também denominado como ensaio de endotoxina. Entretanto, o LAL tem como limitação detectar somente endotoxinas e sofrer interferências do ajuste do pH, por cátions que neutralizam as cargas negativas das endotoxinas, das altas concentrações de sais, por quelantes que capturam cátions bivalentes e etc. No caso das análises de produtos biológicos (soros hiperimunes, hemoderivados etc), não há reação, possibilitando a resposta de resultados falsos negativos. O Instituto Nacional de Controle de Qualidade em Saúde (INCQS) analisa diversos produtos injetáveis, entre eles, os soros hiperimunes (antivenenos, anti-rábico, anti-tetânico etc) que são distribuídos pelo Programa Nacional de Imunização(PNI/MS) aos postos de saúde. Pela Farmacopéia Brasileira, coelhos que tenham recebido estes produtos no teste de pirogênio não podem ser reutilizados com outros produtos biológicos. Este fato implica na utilização de um grande número de animais para estas análises. / Injectable products must to be free of any kind of pyrogenic contamination. Two methods are the official ones for testing this contamination: the Rabbit Test and the Limulus Amoebocyte Lysate Test (LAL) or so called, Endotoxin Assay. However, LAL is limited only for detecting endotoxin, besides suffering some kind of interferences, it is not possible to test biological products, such as hyperimmune sera, blood derivates etc), since these products produce false-negative results. The National Institute of Quality Control in Health (INCQS) perform analysis of a great variety of injectable products, among them, we find anti-venom, anti-rabies and anti-tetanus sera. The Brazilian Pharmacopoeia states that rabbit which have received previous injection of a biological products is not allowed to be re-used in another assay for the same kind of product. This fact implies on using a great number of animals. Due to the lack of information in the scientific literature about the re-use of rabbits which have received previous injection of hyperimmune sera, this study was elaborated using an experimental design where animals were divided into four groups that received from a single injection of 1ng/mL spiked sera up to a group that received 3 negative sera injections in a 48 hours interval and challenged on day 7 with spiked serum. Results showed that the response to the positive stimulus did not presented significant statistical difference, independently of the amount of negative sera previously injected. So, it was possible to conclude that for Anti-Bothrops, Anti-rabies and Anti-tetanus sera it is possible to reuse rabbits for 4 times within a 7-day period.

O carrapato Rhipicephalus microplus é um ectoparasito hematófago de grande importância para a pecuária por ser responsável por perdas massivas na produção animal, de forma que o seu controle é economicamente relevante. Este carrapato, além dos danos que causa por si só, é também um importante vetor para a transmissão de microorganismos patogênicos, entre eles o hemoprotozoário intraeritrocítico Babesia bovis. O presente trabalho descreve o desenvolvimento de uma linhagem de B. bovis capaz de expressar um antígeno protetor, uma glutationa S-transferase do carrapato Haemaphysalis longicornis (HlGST), e o teste desta linhagem como uma vacina de vetor vivo para o controle do carrapato R. microplus. B. bovis, em cultivo, da linhagem S74-T3B foram eletroporados em presença de plasmídeo contendo o promotor bidirecional de B. bovis Ef-1 aresponsável pela expressão independente de dois genes: o repórter fusionado ao agente para seleção (GFP-BSD) e HlGST fusionada à sequência codificadora do peptídeo sinal de MSA-1 (merozoite surface antigen-1). Após a eletroporação, foi feita a seleção com blasticidina para obtenção da linhagem nomeada HlGST. A linhagem HlGST é composta por parasitos contendo diferentes padrões de inserção dos genes exógenos, tanto dentro quanto fora do locus Ef-1. Uma linhagem clonal denominada HlGST-Cln expressando HlGST e GFP-BSD foi obtida a partir da linhagem HlGST. Dois ensaios, independentes, de imunização de bovinos com os parasitos clonais foram realizados, sendo usado como controle uma linhagem clonal previamente caracterizada denominada GFP-Cln. Todos os animais inoculados desenvolveram uma forma branda de babesiose, indicando que ambas as linhagens clonais são atenuadas, mas apenas os animais imunizados com a linhagem HlGST-Cln foram capazes de produzir anticorpos anti-HlGST. O segundo procedimento de imunização foi seguido por um desafio com larvas de R. microplus. O desenvolvimento dessas larvas no hospedeiro levou a fêmeas adultas de menor peso e fertilidade. Coletivamente, esses dados mostram a possibilidade de uso de linhagens transfectadas de B. bovis como vacinas de vetor vivo. / The tick Rhipicephalus microplus is a notorious blood-feeding ectoparasite of cattle, responsible for massive losses in animal production. It is the main vector of pathogenic microorganisms, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74- T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1 promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD, and HlGST fused to the MSA-1 (merozoite surface antigen-1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1 a locus. A B. bovis clonal line termed HlGST-Cln expressing HlGST and GFP-BSD was then derived from the mixed parasite line HlGST. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1 locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis indicating that both transfected cloned parasite lines are attenuated. All animals immunized with HlGST-Cln produced detectable anti-glutathione-S-transferase antibodies. After immunization with HlGST-Cln, calves were challenged with R. microplus larva. Development of these larva produce fully engorged female tick with reduced weight and fertility. Collectively, these data show that transfected B. bovis parasites can be used as vectors in live vectored vaccines.

Tese (doutorado)—Universidade de Brasília, Faculdade de Educação, Programa de Pós-Graduação em Educação, 2017. / Submitted by Raquel Almeida (raquel.df13@gmail.com) on 2017-05-25T19:50:21Z No. of bitstreams: 1 2017_LucianadeMayaRicardo.pdf: 24850627 bytes, checksum: f3318bf02c402dc0f3ea0d7b3c97f986 (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2017-06-13T18:39:56Z (GMT) No. of bitstreams: 1 2017_LucianadeMayaRicardo.pdf: 24850627 bytes, checksum: f3318bf02c402dc0f3ea0d7b3c97f986 (MD5) / Made available in DSpace on 2017-06-13T18:39:56Z (GMT). No. of bitstreams: 1 2017_LucianadeMayaRicardo.pdf: 24850627 bytes, checksum: f3318bf02c402dc0f3ea0d7b3c97f986 (MD5) Previous issue date: 2017-06-13 / Esta tese tem como campo de reflexão o trabalho de educação desenvolvido junto à comunidade, na década de 1990, no Museu Vivo da Memória Candanga, por sua prática de caráter pioneiro e inovador por meio do patrimônio cultural. Traz em seu arcabouço o histórico de ocupação do Distrito Federal no início da construção de Brasília, as diversas vilas que se formaram, a retirada da população de algumas áreas até o fechamento do Hospital Juscelino Kubistchek de Oliveira (HJKO) e a criação do Museu Vivo da Memória Candanga (MVMC). Expõe a estrutura educativa pensada, proposta e posta em prática no MVMC, apresentando, previamente, outras práticas de educação em museus que são de interesse desta pesquisa, como é o caso do Projeto Interação, na década de 1980. Discute a questão da educação formal, informal e não formal e sua atuação dentro de um espaço de memória, incluindo o museu às cidades educadoras, uma nova proposta de educação fora do espaço da sala de aula formal, que dialoga com toda a comunidade e, naturalmente com o museu, integrante desta cidade. A pesquisa termina com uma proposta de educação para o Museu da Educação do Distrito Federal, que interage com a comunidade que o recebe. / This thesis has a reflection field the work of education developed with the community, in the 1990s, in the Museu Vivo da Memória Candanga (MVMC), for its pioneering and innovative practice through cultural heritage. It brings in its history the occupation of the Federal District at the beginning of the Brasilia construction, the several towns that were built, and the withdrawal of the population from some areas until de closure of the Juscelino Kubistchek de Oliveira and the creation of the Museu Vivo da Memória Candanga. It exposes the educational structure thought out, proposed and put into practice in the MVMC, presenting, previously, other practices of education in museums that are the interest of this research, as it is the Projeto Interação casa, in the 1980’s decade. It discusses the issue of formal, informal and non-formal education and its performance within a memory space, including the museum to educate cities, a new education proposal outside the formal classroom space, which dialogues with the whole community and, naturally with the museum, a member of this city. The research ends with an education proposal for the Museum of Education of the Federal District, which interacts with the community that receives it.

Tese (doutorado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-graduação em Ciências Animais, 2016. / Submitted by Camila Duarte (camiladias@bce.unb.br) on 2017-01-23T14:45:02Z No. of bitstreams: 1 2016_OscarOliveiraBrasil.pdf: 1386481 bytes, checksum: 21c30909a66af7329e43406c6d853c0f (MD5) / Approved for entry into archive by Ruthléa Nascimento(ruthleanascimento@bce.unb.br) on 2017-03-13T16:41:43Z (GMT) No. of bitstreams: 1 2016_OscarOliveiraBrasil.pdf: 1386481 bytes, checksum: 21c30909a66af7329e43406c6d853c0f (MD5) / Made available in DSpace on 2017-03-13T16:41:43Z (GMT). No. of bitstreams: 1 2016_OscarOliveiraBrasil.pdf: 1386481 bytes, checksum: 21c30909a66af7329e43406c6d853c0f (MD5) / A tecnologia de múltipla ovulação e transferência de embriões (MOTE) é uma ferramenta importante para o melhoramento genético e para a conservação de embriões de animais de raças ameaçadas ou que apresentem características de interesse. Atualmente, ainda existe uma grande variabilidade na resposta superovulatória de ovelhas doadoras de embriões, limitando a difusão dessa biotécnica reprodutiva em condições de campo. Portanto, objetivou-se avaliar diferentes alternativas para aumentar a produção de embriões in vivo e reduzir a variabilidade nos resultados de superovulação. Entre as abordagens utilizadas estão: (1) a superestimulação na emergência da onda folicular; (2) metodologias para selecionar as doadoras mais responsivas ao FSH exógeno; e (3) aumento da dose de FSH em ovelhas com histórico de baixa ovulação. A melhoria da superovulação foi conseguida através seleção das doadoras pelo número de folículos ≥ 2 mm, realizada 4,5 dias após pré-tratamento de sincronização e pelo histórico de um prévio protocolo superovulatório. Contudo, a superestimulação iniciada na emergência da onda folicular, a seleção das doadoras pelas medidas ovarianas e o aumento da dose de FSH em ovelha com histórico de baixa ovulação não melhoram a resposta superovulatória e o rendimento embrionário, tornando inviável essas abordagens em programas MOTE. / Technology of multiple ovulation and embryo transfer (MOET) is an important tool for genetic improvement and embryo conservation of endangered breeds or those exhibit characteristics of interest. Currently, there is a great variability in the super ovulatory response of donor sheep embryos, limiting the spread of this reproductive biotech in field conditions. This study aimed to evaluate different alternatives to increase in vivo embryo production and reduce variability in superovulation results. Among the approaches used are (1) superestimulation in the emergence of the follicular wave; (2) methodologies to select the most responsive donors to exogenous FSH; and (3) increase in the dose of FSH in sheep with low ovulation historic. The improvement of superovulation was achieved by donors’ selection through of the number of follicles ≥ 2 mm and historic of a previous superovulatory protocol. However, the superestimulation initiated in follicular wave emergence, the selection of the donor by ovarian measures and increasing FSH dose in sheep with low ovulation history does not improve superovulatory response and embryonic yield, making it unnecessary these approaches to MOET programs.

O carrapato Rhipicephalus microplus é um ectoparasito hematófago de grande importância para a pecuária por ser responsável por perdas massivas na produção animal, de forma que o seu controle é economicamente relevante. Este carrapato, além dos danos que causa por si só, é também um importante vetor para a transmissão de microorganismos patogênicos, entre eles o hemoprotozoário intraeritrocítico Babesia bovis. O presente trabalho descreve o desenvolvimento de uma linhagem de B. bovis capaz de expressar um antígeno protetor, uma glutationa S-transferase do carrapato Haemaphysalis longicornis (HlGST), e o teste desta linhagem como uma vacina de vetor vivo para o controle do carrapato R. microplus. B. bovis, em cultivo, da linhagem S74-T3B foram eletroporados em presença de plasmídeo contendo o promotor bidirecional de B. bovis Ef-1 aresponsável pela expressão independente de dois genes: o repórter fusionado ao agente para seleção (GFP-BSD) e HlGST fusionada à sequência codificadora do peptídeo sinal de MSA-1 (merozoite surface antigen-1). Após a eletroporação, foi feita a seleção com blasticidina para obtenção da linhagem nomeada HlGST. A linhagem HlGST é composta por parasitos contendo diferentes padrões de inserção dos genes exógenos, tanto dentro quanto fora do locus Ef-1. Uma linhagem clonal denominada HlGST-Cln expressando HlGST e GFP-BSD foi obtida a partir da linhagem HlGST. Dois ensaios, independentes, de imunização de bovinos com os parasitos clonais foram realizados, sendo usado como controle uma linhagem clonal previamente caracterizada denominada GFP-Cln. Todos os animais inoculados desenvolveram uma forma branda de babesiose, indicando que ambas as linhagens clonais são atenuadas, mas apenas os animais imunizados com a linhagem HlGST-Cln foram capazes de produzir anticorpos anti-HlGST. O segundo procedimento de imunização foi seguido por um desafio com larvas de R. microplus. O desenvolvimento dessas larvas no hospedeiro levou a fêmeas adultas de menor peso e fertilidade. Coletivamente, esses dados mostram a possibilidade de uso de linhagens transfectadas de B. bovis como vacinas de vetor vivo. / The tick Rhipicephalus microplus is a notorious blood-feeding ectoparasite of cattle, responsible for massive losses in animal production. It is the main vector of pathogenic microorganisms, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74- T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1 promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD, and HlGST fused to the MSA-1 (merozoite surface antigen-1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1 a locus. A B. bovis clonal line termed HlGST-Cln expressing HlGST and GFP-BSD was then derived from the mixed parasite line HlGST. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1 locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis indicating that both transfected cloned parasite lines are attenuated. All animals immunized with HlGST-Cln produced detectable anti-glutathione-S-transferase antibodies. After immunization with HlGST-Cln, calves were challenged with R. microplus larva. Development of these larva produce fully engorged female tick with reduced weight and fertility. Collectively, these data show that transfected B. bovis parasites can be used as vectors in live vectored vaccines.