Neste trabalho foram estudados os perfis renal, hepático, energético, hormonal e de proteínas de fase aguda (proteinograma) para avaliar possíveis interações com o desempenho reprodutivo em bovinos. Para isso, foram delineados três experimentos. No experimento 1 o objetivo foi verificar se a inseminação artificial (IA) causa alterações nos perfis renal, hepático, energético, hormonal e de proteínas de fase aguda e estudar as relações entre esses perfis e a hemodinâmica uterina. Foram utilizadas amostras de sangue de vacas Nelore que foram inseminadas (GIA, n=9) ou não (GC, n=9). As amostras foram coletadas 30 horas antes da IA, 4, 24, 48 e 168 horas após a IA. No experimento 2 o objetivo foi estudar os efeitos da qualidade do sêmen sobre o perfil hepático e proteinograma, e estudar a relação dos perfis renal, hepático, energético, hormonal e proteinograma sobre a vascularização uterina. Foram utilizadas amostras sanguíneas de 362 vacas, que foram divididas em três grupos experimentais de acordo com a qualidade do sêmen: Boa (n=121), Média (n=121) e Regular (n=120). As amostras foram coletadas 30 horas antes da IA, 4 e 24 horas após a IA. Por fim, o experimento 3 é um estudo retrospectivo, realizado com o objetivo de comparar os perfis renal, hepático, energético, hormonal, e proteínas de fase aguda entre animais prenhes e vazios após a IA, e verificar se há relação entre a hemodinâmica uterina e a fertilidade. Neste experimento, os animais foram divididos em dois grupos experimentais de acordo com o resultado da IA (prenhe, n=76 X vazia, n=45). Em todos os experimentos, nos mesmos momentos da coleta de sangue, foram realizadas avaliações ultrassonográficas do útero no modo color Doppler e espectral. As amostras dos experimentos 1, 2 e 3 foram submetidas à quantificação das proteínas de fase aguda e dos componentes metabólicos utilizando analisador bioquímico automático (RX Daytona) e à dosagem hormonal, pela técnica de radioimunoensaio. Os dados foram analisados pelo PROC MIXED (SAS, versão 9.2, 2010). Foram consideradas diferenças estatísticas quando P<0,05. No experimento 1, os grupos não diferiram quanto aos perfis renal, hepático, energético, hormonal e proteinograma, no entanto, o RI apresentou correlações positivas com AST e BHB e correlação negativa com estradiol. O estradiol também foi correlacionado com EV, entretanto essa correlação foi positiva. No experimento 2, os animais inseminados com sêmen B, M ou R apresentaram concentrações semelhantes das variáveis do perfil hepático e proteínograma. O RI foi correlacionado positivamente com colesterol, HDL, LDL, e progesterona, e negativamente com glicose, estradiol, albumina e proteína total. Já o EV apresentou correlações negativas com ureia, GGT e cortisol. No experimento 3, os grupos Vazio e Prenhe foram semelhantes quanto aos perfis renal, hepático, energético, hormonal, proteiograma e hemodinâmica uterina. Sendo assim, conclui-se que o processo da IA e a qualidade do sêmen utilizado não causam alterações sistêmicas, bem como a fertilidade não pode ser explicada por estas alterações. Adicionalmente, a hemodinâmica uterina é correlacionada com diversos parâmetros, no entanto, o padrão vascular do útero não mostrou relação com a fertilidade / In this study, were evaluated kidney, liver, energy, hormonal and acute phase proteins profiles to evaluate the possible interactions with the reproductive performance in cattle. For this, three experiments were designed. In experiment 1 the objective was to verify if artificial insemination (AI) causes changes in renal, liver, energetic, hormonal and acute phase proteins profiles and to study the relationship between these profiles and uterine hemodynamics. Blood samples from inseminated (GIA, n = 9) or non inseminated Nellore cows (CG, n = 9) were used. Samples were collected 30 hours before AI, 4, 24, 48 and 168 hours after AI. In experiment 2 the objective was to study the effects of semen quality on liver and protein profiles and study the relationship of renal, liver, energetic, hormonal and protein profiles on uterine vascularization. Blood samples of 362 cows were used, which were divided into three groups according to semen quality: Good (n = 121), medium (n = 121) and Regular (n = 1200. Samples were collected 30 hours before AI, 4 and 24 hours after AI. Finally, experiment 3 is a retrospective study, carried out in order to compare the renal, liver, energetic, hormonal, and acute phase proteins profiles between pregnant and non pregnant animals after AI, and check for relationship between uterine hemodynamics and fertility. In this experiment, animals were divided into two groups according to the result of AI (pregnant, n = 76 and non pregnant, n = 45). In all experiments, at the same time of blood sampling were performed sonographic evaluations of the uterus in color Doppler and spectral mode. The samples of experiments 1, 2 and 3 were subjected to quantification of acute phase proteins and metabolic components using automatic biochemical analyzer (RX Daytona) and to hormone dosage, by radioimmunoassay. Data were analyzed using PROC MIXED (SAS, version 9.2, 2010). Statistics differences were considered when P<0,05. In experiment 1, the groups did not differ about kidney, liver, energetic, hormonal and protein profiles, however, the RI showed positive correlations with AST and BHB and negative correlation with estradiol. Estradiol was also correlated with EV, however this correlation was positive. In experiment 2, the animals inseminated with semen B, M or R showed similar concentrations of the variables of liver and proteinogram profiles. The RI was positively correlated with cholesterol, HDL, LDL, and progesterone, and negatively with glucose, estradiol, albumin and total protein. EV showed negative correlations with urea, GGT and cortisol. In Experiment 3, the non pregnant and pregnant groups were similar about kidney, liver, energetic, hormonal, proteiogram profiles and uterine hemodynamics. Thus, in this study were not observed systemic changes caused by AI process and by quality of semen, and systemic differences did not notice is between non pregnant and pregnant animals. Additionally, uterine hemodynamic is correlated with various parameters, however, the vascular pattern of the uterus was not correlated with fertility

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Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 139-155).

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-04-27T13:18:30Z No. of bitstreams: 1 IsabellaOB_TESE.pdf: 902178 bytes, checksum: d42880aee64b712a238f040f4408a579 (MD5) / Approved for entry into archive by Vanessa Christiane (referencia@ufersa.edu.br) on 2017-05-02T15:22:15Z (GMT) No. of bitstreams: 1 IsabellaOB_TESE.pdf: 902178 bytes, checksum: d42880aee64b712a238f040f4408a579 (MD5) / Approved for entry into archive by Vanessa Christiane (referencia@ufersa.edu.br) on 2017-05-02T15:23:03Z (GMT) No. of bitstreams: 1 IsabellaOB_TESE.pdf: 902178 bytes, checksum: d42880aee64b712a238f040f4408a579 (MD5) / Made available in DSpace on 2017-05-02T15:23:13Z (GMT). No. of bitstreams: 1 IsabellaOB_TESE.pdf: 902178 bytes, checksum: d42880aee64b712a238f040f4408a579 (MD5) Previous issue date: 2016-10-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Acute phase proteins are glycoproteins produced by hepatocytes and released into the bloodstream in response to tissue damage resulting from acute inflammatory processes. Currently, they are considered biomarkers of inflammation or infection, compared with other variables such as hyperthermia, leukocytosis and neutrophilia, but in donkeys have not yet been analyzed. This study aimed to evaluate acute inflammatory response in donkeys submitted to ovariectomy by two different approaches. We randomly selected 18 adult females animals weighing on average 100 kg, which were divided into two groups: Group I, 9 animals were ovariectomized by laparotomy and group II, 9 animals by laparoscopy. After the surgeries were evaluated the following variables in whole blood: blood count, white blood cell count differential cell count, fibrinogen, haptoglobin, albumin, antitrypsin, alpha-1-acid glycoprotein, ceruloplasmin, transferrin, immunoglobulins A and G (acute phase proteins) . In the same time, the peritoneal fluid were measured: number of erythrocytes and leukocytes, cytological analysis differential (leukocytes and mesothelial cells), total protein, pH and acute phase proteins. All variables were analyzed before surgery, 12, 24, 48, 72 hours, 8 and 16 days after surgery. There was an increase (p <0.05) in the number of leukocytes with neutrophilia after surgery, regardless of the surgical serum and peritoneal procedure. Acute phase proteins had different behaviors in blood and peritoneal fluid. For the first time, we identified a P23.000kD protein in serum and peritoneal samples in donkeys. The interest on the role of acute phase proteins in the veterinary medicine, particularly in horses is growing, mainly aiming to use them as early markers of inflammation / Proteínas de fase aguda são glicoproteínas produzidas pelos hepatócitos e liberadas na corrente sanguínea em resposta a dano tecidual, decorrente de processos inflamatórios agudos. Atualmente, são consideradas biomarcadores da resposta inflamatória e infecciosa, quando comparada com outras variáveis como hipertermia, leucocitose e neutrofilia, porém em asininos ainda não foram bem analisadas. Objetivou-se avaliar resposta inflamatória aguda em asininos submetidos à ovariectomia por duas diferentes abordagens. Foram utilizados 18 animais, fêmeas adultas, pesando em média 100 kg, as quais foram divididas em dois grupos, de 09 animais cada, onde grupo I, foram ovariectomizadas por laparotomia e o grupo II por laparoscopia. Após as cirurgias foram avaliadas as seguintes variáveis no sangue total: hemograma, fibrinogênio, haptoglobina, albumina, antitripsina, alfa-1 glicoproteína ácida, ceruloplasmina, transferrina, imunoglobulinas A e G. Nos mesmos momentos, no líquido peritoneal, foram mensuradas: número de hemácias e leucócitos, análise citológica diferencial (leucócitos e células mesoteliais), proteínas totais, pH e proteínas de fase aguda. Todas as variáveis foram analisadas antes das cirurgias, 12, 24, 48, 72 horas, 8 e 16 dias após os procedimentos cirúrgicos. Houve um aumento (p<0,05) do número de leucócitos com neutrofilia após as cirurgias, independente do procedimento cirúrgico, no soro e líquido peritoneal. As proteínas de fase aguda tiveram comportamentos diferentes no sangue e líquido peritoneal. Neste trabalho identificamos, pela primeira vez, a proteína P23.000kD no soro e amostras peritoneais de asininos. O interesse sobre o papel das proteínas de fase aguda na medicina veterinária, em especial em equídeos vem crescendo, principalmente, visando utilizá-las como marcadores precoces do processo inflamatório / 2017-04-26

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Trypanosomiasis is a disease of worldwide distribution which in Brazil is called "mal das cadeiras", owing the clinical signs shown by horses, which are the major species affected. The disease is caused by the protozoan Trypanosoma evansi, characterized by clinical signs such as weight loss, pale mucous membranes, swelling of the eyelid and vulva. Rabbits have been reported as a resistant species to T. evansi, however, is not yet known how immune aspects, such as innate immune and cholinergic system, behave in this species, not even as this inflammations pathways influence in rabbits's trypanotolerance. Thus, the aim of this study was to investigate the inflammatory response and cholinergic anti-inflammatory pathway and its relation to trypanotolerance in rabbits infected with T. evansi. For this, twelve female adult New Zealand rabbits, weighing 3.6 4.5 kg were used in this study. The animals were divided into two groups, a control group and an infected group, both with six animals. The rabbits belonging to the infected group received, intraperitoneally, 0.5 ml of rat blood containing 108 parasites per animal, while the control group received physiological solution by the same route. The experimental period reached 118 days. Blood was collected on days 0, 2, 7, 12, 27, 42, 57, 87, 102, and 118, for to determine the cholinesterases, and days 0, 5, 20, 35, 65, 95 and 118, for to evaluate blood count, total protein, serum proteinogram, immunoglobulins and acute phase proteins. There was an increase in the activity of butyrylcholinesterase (BChE), at 7th day PI, and in the activity of acetylcholinesterase (AChE), in 27th day PI. Furthermore, the infected group showed an increase in total protein and the fractions alpha, beta and gamma globulins along of experimental period. The reduction in albumin and hematocrit were observed in precise periods of experimental infection, as well as the increase in immunoglobulin G. Infection with T. evansi stimulated the production of acute phase proteins, such as C-reactive protein, haptoglobin, α-2 macroglobulin, being observed increase in immunoglobulin M (IgM) throughout the experimental period (118 days post-infection). From these results, it is concluded that the cholinergic pathway had influence the inflammatory response, through the action of AChE and BChE in the regulation of concentrations of acetylcholine, resulting in increased concentrations of cytokines and, consequently, in the production phase protein acute. The increase in IgM, associated with increased C-reactive protein and haptoglobin, suggests the involvement of these proteins in host defense against flagellated, with possible participation in trypanotolerance of rabbits infected with T. evansi. / A tripanossomose é uma enfermidade de distribuição mundial que, no Brasil, é denominada mal das cadeiras , em função dos sinais clínicos apresentados pelos equinos, principal espécie afetada. A doença é causada pelo protozoário Trypanosoma evansi, sendo caracterizada por sinais clínicos como perda de peso, mucosas pálidas, edema de pálpebra e vulva. Coelhos têm sido relatados como uma espécie resistente ao T. evansi, contudo, ainda não se sabe de que modo aspectos imunitários, como a imunidade inata e o sistema colinérgico, comportam-se nessa espécie, nem mesmo como essas vias da inflamação influenciam a tripanotolerância de coelhos. Assim, o objetivo deste trabalho foi investigar a resposta inflamatória e a via anti-inflamatória colinérgica e sua relação na tripanotolerância em coelhos infectados experimentalmente pelo T. evansi. Para tanto, foram utilizados 12 coelhos adultos, fêmeas, da raça Nova Zelândia, com peso corporal entre 3,6 a 4,5 Kg, divididos em dois grupos, um grupo controle e um grupo infectado, ambos com seis animais. Os coelhos pertencentes ao grupo infectado receberam, pela via intraperitoneal, 0,5 mL de sangue de rato contendo 108 tripanossomas por animal, enquanto que o grupo controle recebeu, pela mesma via, solução fisiológica. O período experimental foi de 118 dias, sendo o sangue coletado nos dias 0, 2, 7, 12, 27, 42, 57, 87, 102, e 118, para determinar as colinesterases, e nos dias 0, 5, 20, 35, 65, 95 e 118, para avaliar hemograma, proteínas totais, proteinograma sérico, imunoglobulinas e proteínas de fase aguda. Foi observado aumento na atividade da butirilcolinesterase (BChE) no 7° dia PI e da atividade da acetilcolinesterase (AChE) no 27° dia PI. Além disso, o grupo infectado apresentou aumento nas proteínas totais, bem como nas frações alfa, beta e gama globulinas ao longo do período experimental. A redução na albumina e no hematócrito foram observados em períodos pontuais da infecção experimental, bem como o aumento na imunoglobulina G. A infecção pelo T. evansi estimulou a produção de proteínas de fase aguda como a proteína C-reativa, haptoglobina, α-2 macroglobulina, sendo observado um aumento na imunoglobulina M (IgM) em todo o período experimental (118 dias pós-infecção). A partir desses resultados, conclui-se que a via colinérgica pode ter influência na resposta inflamatória, através da ação da BChE e AChE na regulação das concentrações de acetilcolina, acarretando em aumento nas concentrações de citocinas e, consequentemente, na produção de proteínas de fase aguda. A elevação na IgM, relacionada ao aumento da proteína C-reativa e haptoglobina, sugere o envolvimento dessas proteínas na defesa do hospedeiro contra o flagelado, e, possivelmente, na tripanotolerância de coelhos infectados com T. evansi.

Made available in DSpace on 2014-06-11T19:27:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-06-21Bitstream added on 2014-06-13T19:36:20Z : No. of bitstreams: 1 carvalho_wa_me_jabo.pdf: 654248 bytes, checksum: 619fb98a9565e57850118d52b19f5a0b (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / No hospedeiro bovino o nível de resistência ao carrapato Rhipicephalus (Boophilus) microplus varia de acordo com a raça sendo os fenótipos contrastantes herdáveis. O presente trabalho explora esses fenótipos a fim de estabelecer os perfis das respostas imunes, humoral e inflamatória, correlacionados com resistência em raça zebuína (Nelore) e susceptibilidade em raça taurina (HPB). Os animais foram expostos a infestação natural pelo R.(B.) microplus e amostras de soros foram coletadas em pontos estratégicos da cinética das infestações. Os níveis de imunoglobulinas totais (IgG1 e IgG2), bem como os de anticorpos IgG1, IgG2 e IgE anti-extrato de ovo, anti-extrato de larva não alimentada e anti-saliva foram determinados. Elementos da resposta inflamatória, como as principais proteínas de fase aguda e óxido nítrico, também foram dosados. Os resultados obtidos mostram que as infestações muito intensas, em hospedeiros suscetíveis, são capazes de modular os níveis séricos de IgG1 e IgG2 total, diminuindo-os significativamente em relação aos níveis observados durantes infestações menores. Também modulam negativamente a produção de todos os anticorpos específicos IgG1 e IgG2 avaliados a nível sistêmico. Bovinos susceptíveis ao carrapato produzem níveis mais altos de anticorpos IgE para todos os antígenos. O fenótipo susceptivel de infestação se diferencia pela maior freqüência do alótipo de IgG?2a, herdado por herança Mendeliana co-dominante. Animais suscetíveis, quando infestados, produzem níveis mais altos da proteína de fase aguda a1- glicoproteína ácida, de padrão anti-inflamatório, enquanto que animais resistentes produziram relativamente mais proteínas de fase aguda pró-inflamatórias, haptoglobina e amilóide sérica A. Em ambas as raças não houve diferença nos níveis de transferrina e óxido nítrico sistêmico, porém a produção de ambos é influenciada pelos níveis de infestação. / In bovine hosts resistance to the cattle tick, Rhipicephalus (Boophilus) microplus, varies according to the breed, being the phenotypes of infestations inherited. The present work exploits these contrasting phenotypes in order to determine the profile of the humoral, inflammatory and acute phase responses that are correlated with resistance seen in a zebuine breed (Nelore) and susceptibility seen in a taurine breed (Holstein). Bovines were exposed to natural infestations with R.(B.) microplus and they presented, as expected, different levels of infestation that also varied in intensity according to the season of the year. Samples of sera were collected at strategic points during the kinetics of different cycles of infestations. The levels of total serum IgG1 and IgG2 immunoglobulins, as well as those of IgG1, IgG2 and IgE anti-egg extracts, anti-unfed larvae extracts and anti-saliva antibodies were measured. Components of the inflammatory response, nitric oxide, as well as acute phase proteins, were also measured. The results show that very intense infestations in ticksusceptible bovines modulate the serum levels of IgG1 and IgG2, which are significantly diminished relative to those observed during less intense infestations. Intense infestations also modulate the production of all specific IgG1 and IgG2 antibodies. Susceptible animals produced more specific IgE, suggesting that this isotype does not participate in resistance against ticks. The susceptible cattle also have a higher frequency of IgG?2a , which are encoded by Mendelian co-dominant alleles. When infested susceptible animals produced higher levels of the anti-inflammatory acute phase protein, a1-acid glycoprotein, whereas resistant animals produced relatively higher levels of the pro-inflammatory proteins, haptoglobin and serum amyloide A.

Resumo: Este trabalho objetivou avaliar a anatomopatologia, o hemograma e o perfil bioquímico sérico de aves de postura inoculadas por Salmonella Gallinarum (SG) contendo os genes cobS e cbiA inoperantes (SGcobScbiA) que mostrou ser avirulenta em trabalhos anteriores, comparando-a com cepas virulentas SGNalr e SGcobS, para mostrar se SGcobScbiA pode ser componente de vacina contra cepas selvagens de SG e S.Enteritidis. 280 pintainhas foram distribuídas em 4 grupos (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) e G4 (controle). Com exceção do G4, os grupos receberam 0,2 mL de suas respectivas cepas contendo aproximadamente 108 UFC/mL de inóculo, aos 5 dias de idade. A eutanásia foi realizada 24h antes (1DAI) e após a inoculação (1DPI), e 3 (3DPI), 5 (5DPI), 7 (7DPI), 10 (10DPI) e 15 (15DPI) dias após a administração do inóculo, sacrificando-se, em cada momento, dez aves de cada grupo. As aves foram sacrificadas, obtendo-se amostras de sangue utilizadas para os exames hematológicos e bioquímicos. Fragmentos de fígado, baço, timo, bursa de Fabricius, rins e coração foram destinados aos exames histológicos. As aves inoculadas com a cepa SGcobS tiveram comportamento semelhante às aves inoculadas por SGNalr, porém com algumas respostas diferentes nos exames hematológicos e bioquímicos. As aves inoculadas com a cepa SGcobScbiA tiveram comportamento semelhante ao grupo controle, entretanto foi verificado alterações brandas em alguns parâmetros, mostrando que estudos futuros devem ser feitos, verificando se as alterações constatadas não irão interferir no desempenho de aves vacinadas com a cepa SGcobScbiA. / Abstract: The aim of the present study was to evaluate anatomopathology, hemogram and blood serum components of commercial layers experimentally inoculated with SGcobScbiA, which is a Salmonella Gallinarum (SG) strain it shows attenuation of the virulence in previous research and it was compared with high virulence SGNalr and SGcobS strains in order to show if SGcobScbiA has potential to be use as a vaccine against SG and S. Enteritidis wild strains. 280 commercial layers were divided into 4 groups (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) and G4 (control group). With exception of G4, all the other groups received 0,2 mL of their respective strain containing about 108 CFU/mL of the inoculum with five days of age. Birds were sacrificed 24 hours before (1DBI) and 24 hours after the inoculation (1DAI), and three (3DAI), five (5DAI), seven (7DAI) ten (10DAI), and fifteen (15DAI) days after the administration of the inoculum, slaughtering ten birds at a time in each group. Birds were submitted to euthanasia and blood samples were collected in order to make the hematological and blood serum components test. Samples of liver, spleen, thymus, bursa of Fabricius, kidneys and heart were collected for the histological test. The birds inoculated with SGcobS strain had similar behavior when compared with that ones who received SGNalr strain, however some different responses in the hematological and blood serum components were found. On the other hand, the birds inoculated with SGcobScbiA strain had similar behavior when compared with the control group, however, lower alterations in some parameters were found. Further studies must be done to verify if these alterations will not interfere in the performance of the vaccinate birds with SGcobScbiA strain. / Orientador: Ângelo Berchieri Júnior / Coorientador: José Jurandir Fagliari / Banca: Antonio Carlos Alessi / Banca: Raimundo Souza Lopes / Mestre

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-09-22T19:23:19Z No. of bitstreams: 1 thiagodealmeidaoliveira.pdf: 1376099 bytes, checksum: 1223858331fe263a8bdcce2a114266c5 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-09-27T13:42:30Z (GMT) No. of bitstreams: 1 thiagodealmeidaoliveira.pdf: 1376099 bytes, checksum: 1223858331fe263a8bdcce2a114266c5 (MD5) / Made available in DSpace on 2017-09-27T13:42:30Z (GMT). No. of bitstreams: 1 thiagodealmeidaoliveira.pdf: 1376099 bytes, checksum: 1223858331fe263a8bdcce2a114266c5 (MD5) Previous issue date: 2017-06-28 / A modulação da resposta imune inata seria uma ferramenta capaz de elhorar a capacidade do sistema imune de combater a infecção por patógenos e outros estímulos externos. A utilização de vias sinalizadoras com a participação de alguns receptores específicos, como o peroxisome proliferator-activated receptor alpha (PPARα) surgiria como importante hipótese para modular a produção de proteínas de fase aguda e do complemento em hepatócitos bovinos, órgão principal envolvido nessa síntese. Nesse sentido, o presente trabalho se utilizou de técnicas de cultivo primário de hepatócitos bovinos, provenientes de 5 animais diferentes, que foram isolados, cultivados e estimulados com um ligante desse receptor nuclear, o fenofibrato. Após 48 horas de cultivo as células tiveram seu ácido ribonucleico (RNA) extraído e quantificado, seguindo-se à síntese de ácido desoxirribonucleico complementar (cDNA) e execução das reações em cadeia da polimerase quantitativo (qPCR), a fim de se verificar a expressão e produção de proteínas de fase aguda e do sistema complemento por meio de de reações de qPCR. Os sobrenadantes de cultura também foram avaliados após esse período de tempo, onde foram dosadas as mesmas proteínas, por meio da técnica de Enzyme-linked immunosorbent assay (ELISA). Os resultados demonstraram que o cultivo se provou ineficiente no que diz respeito à viabilidade celular e à quantificação de RNA extraído, indicando que mais estudos devem ser feitos para padronização dessa técnica. Além disso, os resultados demonstraram que o PPARα, ativado frente ao seu ligante específico, o fenofibrato, não foi capaz de modular a expressão e tampouco a produção dos fatores de complemento e das proteínas de fase aguda analisadas, podendo indicar que em hepatócitos bovinos a modulação por esse receptor não ocorre da mesma maneira como em humanos e em murinos. No entanto, devido à grande variabilidade racial dos animais cujos fígados foram coletados e ao pequeno número amostral utilizado, análises mais refinadas são necessárias. Dessa maneira, estudos devem ser realizados visando novas moléculas capazes de modular proteínas relacionadas ao sistema imune em bovinos. / The innate immune modulation is a tool capable of improve the immune system capacity to fight infections by pathogens and other external stimuli. The use of signaling paths with participation of some specific receptors, such as peroxisome proliferator-activated receptor alpha (PPARα) arises as an important hypothesis to modulate the acute phase proteins (APP) and complement proteins in bovine liver cells, where most of these molecules are produced in a systemic level. In this way, this work aimed to establish a protocol of primary culture of bovine hepatocytes suitable to analyze intracellular signaling paths and genetic expression and synthesis of proteins related to innate immune system. For that, bovine hepatocytes from five different animals were isolated, cultivated and activated by a receptor ligand - fenofibrate, both alone and associated with proinflammatory stimuli, in different concentrations. After 48 hours of culture, ribonucleic acid (RNA) was extracted and quantified, complementary deoxyribonucleic acid was synthetized and real-time polymerase chain reactions were setup to verify the acute phase and complement proteins expression and synthesis. The culture supernatant was used to analyze the protein dosage by Enzyme-Linked Immunosorbent Assay (ELISA). The results showed that the hepatocytes isolation and culture were not very effective, since the extracted RNA showed low concentration and bad integrity, indicating that more studies must be performed to standardize this technique. Considering the low quantification and bad quality of RNA, it was not possible to access the role of the receptor PPARα in complement factors and acute phase proteins genetic expression and production, when activated by its specific ligand (fenofibrate). Thus, the development of isolation and culture techniques for bovine hepatocytes must continue to be developed in order to better evaluate the intracellular signaling pathways and the search for new molecules capable to modulate proteins related to the immune system in bovines.

The analysis of acute phase protein serum amyloid A (SAA) has recently been brought into clinical use in veterinary medicine. Some of the difficulties with incorporating the SAA method in clinical practice have been the expensive and rather large equipment required for the method. Due to these difficulties only larger clinics can afford to use the SAA analysis. The company Equinostic has recently developed a smaller instrument that costs one-tenth of a larger instrument. The instrument is named EVA1 and has so far only been used to analyze SAA in horses. The aim of this study was to investigate if the EVA1 instrument could be used to analyze SAA in cats. This study included 24 serum samples from cat, which were first analyzed twice on the EVA1 instrument and then sent to the Strömsholm Referral Animal Hospital in Sweden where they reanalyzed the samples using a validated reference method. Both instruments are based on an immunoturbidimetric assay. The correlation between the two instruments was good (r=0.97) but the EVA1 instrument showed constantly lower results than the reference method. The difference between the duplicates when analyzed on the EVA1 instrument was larger than expected. The conclusion is that EVA1 could be used to analyze SAA in cats. However, before it could be used clinically in veterinary practice an extended study is recommended.

Abstract Feline immunodeficiency virus (FIV) is an important infectious agent of domestic cats worldwide. It has been classified into the Lentivirus genus of the Retroviridae family, together with human immunodeficiency virus (HIV). Five FIV subtypes (A, B, C, D and E) have been described based on sequence variation of the V3-V5 region of the envelope (env) gene. There is considerable sequence diversity within and between subtypes, which has been a major obstacle in the development of a successful vaccine. However, an FIV vaccine that incorporates inactivated whole viruses from subtypes A and D is now commercially available. Although the vaccine has been shown to be efficacious in protecting against challenge with homologous and a heterologous (subtype B) subtypes, its effectiveness against other viral variants is unknown. Therefore, identifying the type and diversity of FIV strains in different regions is important to establish the potential efficacy of the vaccine in areas where vaccination is to be implemented. The proviral DNA sequence of the V3-V5 region of the env gene was determined for 102 FIV-infected cats from locations in Australia, New Zealand and South Africa. Subtype A was the predominant subtype in Australia and South Africa, although subtype B and C were also identified in each of these countries, respectively. Both subtypes A and C were also present in New Zealand. Of interest, there were some samples in New Zealand and South Africa that demonstrated subtype assignment discrepancies when different regions of the genome were analysed, suggesting co-infection and/or recombination. Cats infected with FIV exhibit varying degrees of immunological impairment. Currently, prognosis for an FIV-infected cat is based on clinical signs alone, which is a relatively subjective measure. In HIV-infected patients it is recognised that viral RNA load correlates with disease stage and prognosis. This PhD research tested whether viral RNA load may be a useful prognostic marker in FIV infection. A real-time PCR assay was developed to quantify plasma viral RNA load in 42 FIV-infected cats at three different clinical stages (1:healthy, 2:unwell without signs of immunodeficiency, 3:unwell with signs of immunodeficiency). In cats older than 5 years of age, log-transformed viral RNA loads were significantly higher in cats in category 3 compared to cats in category 1. There were no significant differences in the viral RNA load of older cats in category 2 compared to category 1. There were no cats younger than 5 years of age in category 3 and there was no significant difference in viral RNA load between young cats in categories 1 and 2. Of the 15 cats for which follow-up data was available, eight showed no change in clinical signs, and seven showed a worsening of clinical signs with six of these showing a progression of clinical category including death. One of the cats in category 2 that progressed clinically had one of the highest viral RNA loads of cats in that category. Three of four cats from category 3 that were followed had either died or been euthanised. Two of these cats had among the highest viral RNA loads in the whole study, while the remaining cat (for which the definitive cause of death was not confirmed) had a relatively low viral RNA load. In summary, measurement of viral RNA load was found to be a potentially useful clinical and prognostic marker but further work is required to better assess its usefulness to veterinarians. Serum acute phase proteins were investigated as possible candidate markers of FIV disease with the aim of developing a more simplified assay that could be used as a prognostic marker for FIV infection. Blood samples from 43 FIV-infected and 25 FIV-negative cats were assayed for the concentration of four acute phase proteins. Both healthy and sick cats were included in the study. Compared to healthy cats, sick cats had significantly higher concentrations of serum amyloid A (P<0.05). Alpha 1-acid glycoprotein and haptoglobin were also found to be in higher concentrations in sick cats (P<0.1). Other variables such as age and gender were also associated with acute phase protein concentrations. With respect to FIV infection, it was found that in sick cats, serum amyloid A, in combination with the age of the cat, was the best predictor of FIV viral RNA load. Alpha 1-acid glycoprotein and haptoglobin were not significantly associated with FIV viral RNA load. Although health status did not influence albumin levels, they were found to be significantly lower in FIV-positive cats in comparison to FIV-negative cats (P<0.05). The frequent monitoring of viral RNA loads and CD4+ lymphocyte counts that is performed on HIV-infected patients is cost prohibitive in veterinary patients. This study showed that there is potential for the use of acute phase protein concentrations (in particular serum amyloid A) as alternative prognostic tools in FIV-infected cats. Further work, particularly longitudinal studies, is required to more definitively define changes in viral RNA load and acute phase protein concentrations throughout the course of FIV infection.