Malignant brain tumors are diseases with poor prognosis and/or severe long-term side effects of treatment. This thesis aimed to discover novel regulators in brain tumor development, based on studying neural stem cell and progenitor cell (NSPC) differentiation and using animal models to introduce new insights to mechanisms of human brain tumors. The enzyme heparanase (HPSE) that degrades heparan sulfate (HS) is active in cell signaling and ECM remodeling. In paper I, we found an enhanced differentiation to oligodendrocytes in ES cell-derived NSPCs overexpressing HPSE. Further analysis suggested that this enhanced formation of oligodendrocytes was associated with alterations in receptor tyrosine kinase signaling, and that HPSE might also exert anti-apoptotic functions. Subsequently, in paper II we studied the involvement of HPSE in glioma development. We observed that high HPSE levels associated with poor survival in glioma patients. In experimental models, we found that HPSE promoted glioma growth, and that an inhibitor of HPSE reduced glioma progression both in vitro and in vivo. We hypothesize that regulators in NSPC differentiation could have a potential role in brain tumor development. In paper III, we explored the function of NRBP2, a pseudokinase that is up-regulated during NSPC differentiation. We found low expression of NRBP2 in brain tumors, in comparison to normal brain. In medulloblastoma, in particular, low NRBP2 expression is linked to poor prognosis. Overexpression of NRBP2 in medulloblastoma cells led to impaired cell growth and migration, concomitant with an increased cell death. In paper IV, we searched for novel glioma susceptibility genes by sequencing dog breeds from the same ancestor but with different glioma incidence. In this way we identified three new glioma-associated genes. Two of these are significantly regulated in human glioma and one of those might have a role in glioblastoma stem cell differentiation.

Master of Science / Department of Grain Science and Industry / C.G. Aldrich / Novel ingredients have been a source of innovation and growth in the pet food market. Further, with rising trends in the humanization of pet food, there has been increased competition between the human food systems and pet food industry for high quality ingredients. Next-generation distillers dried grains (NG-DDG) are a sustainable alternative protein source that show a strong potential for use in companion animal diets. The objectives of this work were to determine the effect of NG-DDG on the extrusion of dry kibbles, the utilization of diets by dogs, the palatability of diets by dogs and cats, and to evaluate the amino acid profile and protein quality through a chick growth assay. Corn gluten meal (CGM) and soybean meal (SBM) were used as standards for comparison. Diets were extruded over 3 days in a complete block design. During extrusion, the NG-DDG kibbles had less radial expansion (P<0.05) compared to the CGM and SBM kibbles (2.62 vs. average 3.10 mm²/mm², respectively). The NG-DDG kibble also required a smaller (P<0.05) mass restriction-valve opening to increase die back-pressure. No other differences in extrusion parameters or kibble texture were observed. Twelve beagle dogs were arranged in a 3x3 replicated Latin Square and were each fed the 3 experimental diets to evaluate digestibility by use of titanium dioxide. Diet produced with CGM was more digestible (P<0.05) in terms of dry matter, organic matter, crude protein, crude fat, and gross energy. Additionally, dogs fed NG-DDG diets had larger (P<0.05) fecal mass than both CGM and SBM (55.65 vs 35.91 and 43.25 g/d, respectively), and a higher (P<0.05) fecal score than dogs fed the CGM diet (3.63 vs. 3.27). Diets were fed to both dogs and cats to assess palatability via a two-bowl test. Dogs had a preference (P<0.05) for CGM over SBM and NG-DDG, but cats showed a preference (P<0.05) for SBM and NG-DDG over CGM. To assess protein quality, one-day old chicks (CobbxCobb; n=120) were fed semi-purified diets containing test ingredients at a 10% crude protein inclusion level, as well as spray dried granulated egg (SDG) and a nitrogen-free basal diet (NEG) to serve as positive and negative controls, respectively. Chicks were arranged in a randomized block design with 6 chicks per pen, 1 pen per battery, and 4 pens per treatment. The protein efficiency ratio (PER) of each treatment was calculated as weight gain (g) per protein intake (g). All experimental treatments had a lower (P<0.05) PER value than the positive control. The PER for NG-DDG and CGM did not differ from each other and had the lowest value of all treatments (P<0.05; average 1.17). In summary, next-generation distillers dried grains can be used to make a similar kibble to CGM and SBM, are similar to SBM in terms of digestibility, and would be an acceptable source of protein in companion animal diets when paired with a supplemental protein source.

Resumo: Sabendo-se da influência das mutações no gene TP53 no desenvolvimento das neoplasias e da discrepância entre os resultados obtidos pelas técnicas de sequenciamento e imunoistoquímica, esta pesquisa teve como objetivo avaliar a sequência do TP53 e a imunorreatividade da p53, relacionando-as com fatores clínicopatológicos em cães com linfoma. Foram avaliados 12 cães com linfoma e cinco cães sadios. Amostras de neoplasia/linfonodo e de bulbo piloso foram obtidas. O diagnóstico histopatológico foi determinado pela classificação de Kiel. O imunofenótipo e a imunomarcação da p53 foram determinados por imunoistoquímica. Para a imunomarcação com a p53, utilizou-se anticorpo policlonal anti-p53 (CM1) na diluição de 1:500. A região do gene TP53 compreendida entre os exons quatro e nove (incluindo íntrons) foi amplificada por PCR e submetida ao sequenciamento. A média da imunorreatividade da p53 nos cães com linfoma foi 27,33%±12,14%, diferente da média do grupo controle (0,75±0,5) (P<0,05). A ausência de marcação da p53 foi observada em apenas um caso de linfoma de baixo grau de malignidade, cujo cão apresentou um ano de sobrevida. O outro caso de linfoma de baixo grau de malignidade apresentou a menor marcação da p53 (14,12%). O linfoma com maior marcação da p53 (50,56%) consistiu em um linfoma alimentar cujo cão evoluiu para o óbito no dia da consulta. As maiores médias de marcação foram observadas nos linfomas cutâneos, estádio clínico V, subestádio "b", imunofenótipo B e tempo de sobrevida de até trinta dias, sem diferença estatística (P>0,05). Apesar dos resultados obtidos pela imunoistoquímica, nenhuma mutação foi encontrada nas sequências analisadas ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: TP53 mutations are usually involved in cancer, but sequencing and immunohistochemistry results are often controversial. Thus, the aim of this study was to analyse both TP53 sequence and p53 immunostaining and to associate their findings to clinical-pathological features in dogs with lymphoma. Twelve dogs with lymphoma and five healthy dogs were included in this study. Tumor and hair sample was collected. Histopathological diagnosis was performed according to Kiel classification. Immunohistochemistry was performed to identify immunophenotype as well as p53 expression. Polyclonal antibody anti-p53 (CM1) was used at a 1:500 dilution. The region that encompasses exons 4-9 was amplified by means of PCR reactions and sequencing was then performed. p53 immunolabeling mean was 27,33%±12,14 in canine lymphoma group. It was statistically different from control group mean (0,75±0,5) (P<0,05). Only one p53-negative case was observed in a dog with low grade lymphoma, which presented one-year survival. Other low grade lymphoma presented lowest p53 immunoreactivity (14,12%). Highest reactivity (50,56%) was observed in dog with alimentary lymphoma, which died in the day of diagnosis. The highest immunolabeling means were verified in cutaneous presentation, clinical stage V, substage "b", immunophenotype B and survival time up to 30 days, but there was no significant difference (P>0,05). Nevertheless, gene mutations were not observed in any sequence. In conclusion, p53 immunostaining is not essentially related to mutation in the gene TP53, particularly to the region between exons 4-9 / Orientador: Carlos Roberto Daleck / Coorientador: Janete Apparecida Desidério Sena / Banca: Andrigo Barboza De Nardi / Banca: Julio Lopes Sequeira / Banca: Renée Laufer Amorim / Banca: Manoel Victor Franco Lemos / Doutor

Orientador: Gilson Helio Toniollo / Banca: Maria Denise Lopes / Banca: Fabiana Ferreira de Souza / Banca: Paulo Henrique Franceschini / Banca: Aulus Cavalieri Carciofi / Resumo: O crescimento acelerado na produção de alimentos para cães e gatos nem sempre é acompanhado pelo aumento de sua qualidade. Os desequilíbrios alimentares são diretamente responsáveis ou predispõem a inúmeras doenças ósseas, metabólicas e podem ser potenciais causadores de alterações testiculares, com reflexo na fertilidade de cães. Mediante isso, objetivou-se: avaliar as possíveis alterações de testículos e da próstata, por meio de exame ultrassonográfico, de cães alimentados com três diferentes dietas, analisar a qualidade do sêmen fresco e congelado ao longo dos tratamentos; verificar possíveis correlações entre rações fornecidas e variáveis reprodutivas analisadas. Oito cães da raça Beagle, adultos, foram selecionados e condicionados a colheita de sêmen. Três grupos de cães receberam rações comerciais distintas, a cada cinco meses. Transcorrido esse período, as rações foram trocadas de modo que, ao final de 15 meses, todos os grupos receberam três dietas diferentes. A cada 15 dias, o sêmen foi colhido e avaliado e, a cada troca de ração, foram realizadas avaliações ultrassonográficas de próstata e testículos, além da congelação do sêmen. As médias das variáveis, em cada grupo, nos três períodos, foram analisadas por meio de comparação emparelhada (teste t). Não existiu diferença (p>0,05) das análises andrológicas frente as rações utilizadas, de modo que os tipos de rações utilizadas não interferiram positiva ou negativamente na qualidade seminal dos animais tratados / Abstract: The accelerate raise of the production of food for pets worldwide sometimes do not lead to increased food quality. Alimentary imbalances may be directly associated to several bone and metabolic diseases. Testicular degeneration and infertility may also be observed. Regarding the influence of the nutritional imbalance on the reproductive performance, the aim of the present study was: evaluate the quality of canine fresh semen and testis an prostate by means ultrasonography treated with three different types of commercial food for dogs. Eight adult Beagles were selected and conditioned to semen sampling. Three groups of dogs received three different types of dog food, every fivemonth intervals. After this period, the kind of dog food was changed. In the end of 15 months, all dogs were fed with all the types of dog food. Semen samples were obtained every 15-days interval and semen analysis were performed. The mean values of the variables were evaluated in the three periods by means of the t test. There was no significant difference (p>0,05) regarding the semen analysis among the different types of dog food. The types of dog food did not influence the quality of the semen of the animals in the present study during each experimental period / Doutor

The goal of the thesis is to evaluate the haematologic indicators of red blood cells depending to the physiological condition and yield of cattle, sheep and dogs. The evaluation of qualitative parameters of red blood cells included 59 dairy cows, 57 meat cows, 62 sheep and 73 dogs. The evaluation of the haematologic parameters of cattle was performed based on the production (yield) type and by season; that of sheep, by season, age and sex of lambs; and that of dogs, by sex, age and size - weight. The yield types showed statistically significant difference between the number or erythrocytes, haematocrit, haemoglobin, MCV and MCH concentration. The dairy cows showed seasonal fluctuations of the number of erythrocytes, haematocrit and haemoglobin; the meat cows showed fluctuations of the haemoglobin, MCH and MCHC values. The age influenced higher values of erythrocytes in sheep and lower values of MCV and MCH in lambs. The sex of lambs did not lead to significant differences. The evaluation of dogs by age showed differences of haematocrit and haemoglobin values. The dog size (weight) and the sex did not have demonstrable influence on haematologic values. Anaemia was found in 36 % dairy cows (it did not occur in meat cattle) and in 2 sheep (3 %). By most authors, anaemia was not found in dogs.

Esta dissertação teve como objetivo determinar parâmetros farmacocinéticos da metadona utilizada por via oral em diferentes formas farmacêuticas. A metadona foi administrada i.v. e v.o. (líquido e comprimido) (0,5 mg/kg) em cães da raça Cocker Spaniel Inglês. Foi também utilizada metadona comprimido (0,5 mg/kg) associada a cetoconazol (10 mg/kg, duas doses). As concentrações séricas resultantes foram determinadas por LC-MS/MS, empregando-se metodologia validada. Os perfis plasmáticos individuais foram avaliados por abordagem não compartimental e compartimental, utilizando os softwares Excel® 2003 e Scientist® 2.01, respectivamente, para a determinação dos parâmetros farmacocinéticos, que foram comparados estatisticamente pelo teste Wilcoxon Rank-Sum e Kruskal-Wallis (α = 0,05). Os perfis plasmáticos da metadona i.v. foram descritos pelo modelo de dois compartimentos e o da metadona oral, pelo modelo de um compartimento. Após a administração da metadona oral, todos os grupos apresentaram concentrações séricas, com exceção do grupo que utilizou o comprimido de metadona isoladamente. O grupo que utilizou solução de metadona apresentou concentrações significativamente superiores ao grupo que recebeu comprimido de metadona isolado, com detecção de níveis do fármaco aos 5 min. O grupo associado ao cetoconazol apresentou o fenômeno de duplo pico plasmático sérico. Estes resultados nos permitem sugerir que a utilização de comprimido de metadona apresenta reduzida absorção, alcançando níveis séricos não quantificáveis pela metodologia empregada. No entanto, quando co-administrada com cetoconazol ou como solução, concentrações séricas foram detectadas, o que nos permite concluir que a utilização de metadona em solução se apresenta como uma boa alternativa para a administração oral deste fármaco em cães. / To determine pharmacokinetic parameters of methadone used in different formulations and routes of administration in dogs. Methadone alone was administered iv, bucal, esophagic and v.o. (liquid and tablet) (0.5 mg/kg) in dogs English Cocker Spaniel. It was also used methadone tablet (0.5 mg/kg) associated with ketoconazole (10 mg/kg, two doses). The resulting serum concentrations were determined by LC-MS/MS, using a validated methodology. The serum concentrations were evaluated by noncompartmental and compartmental model using the software Excel ® Scientist ® 2003 and 2.01, respectively, to determine the pharmacokinetic parameters, which were statistically compared using the Wilcoxon Rank-Sum and Kruskal-Wallis ( α=0.05). The plasma profiles of methadone i.v. are described by the compartmental model and the oral methadone for a noncompartimental model. After administration of methadone, all groups presented serum concentrations but the group that used the pill alone. The group using the methadone solution had significantly higher concentrations when compared with the group that received methadone isolated v.o., and the drug was detected in 5 min. The group associated with ketoconazole showed the phenomenon of double peak plasma. The tablet formulation of methadone reduced the absorption to values not measurable by the method used, however, when co-administered with ketoconazole or as a liquid formulation, serum concentrations were detected. The liquid formulation is presented as a potential alternative to oral administration of methadone in dogs.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-25T18:00:25Z No. of bitstreams: 1 Manuela da Silva Solcà. Uso do PCR...pdf: 11332317 bytes, checksum: 657938584a2c80bcdcc02a52c2d8fa4d (MD5) / Made available in DSpace on 2013-10-25T18:00:25Z (GMT). No. of bitstreams: 1 Manuela da Silva Solcà. Uso do PCR...pdf: 11332317 bytes, checksum: 657938584a2c80bcdcc02a52c2d8fa4d (MD5) Previous issue date: 2012 / Universidade Federal da Bahia. Faculdade de Medicina da Bahia. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / No Brasil, os cães são considerados como o principal reservatório doméstico para Leishmania infantum (sin. L. chagasi). Desta forma, o diagnóstico da leishmaniose visceral canina (LVC) deve ser rápido e preciso. Este trabalho visa comparar a performance da reação em cadeia da polimerase (PCR) em detectar o DNA do parasito em diferentes tecidos para diagnóstico da LVC. Com este intuito, na primeira parte do estudo, foi padronizado um protocolo de PCR convencional (cPCR), para detecção do DNA do minicírculo do cinetoplasto (kDNA) de Leishmania sp., em 45 fragmentos esplênicos caninos. As mesmas amostras foram avaliadas utilizando-se um protocolo de PCR quantitativa (qPCR) tendo como alvo o gene da subunidade do RNA ribossomal (SSU rRNA) de Leishmania sp. Também foi comparada a eficácia do diagnóstico para LVC pelas técnicas moleculares e convencionais como a cultura e o ELISA. A cPCR apresentou sensibilidade mais elevada para detecção de DNA de Leishmania sp. (88,9%), comparada à qPCR (83,3%). Possivelmente o melhor desempenho da cPCR foi devido ao maior números de cópias do kDNA no genoma da Leishmania sp. Diante dos promissores resultados apresentados pela cPCR tendo o kDNA como alvo, na segunda parte do estudo foi padronizado um novo protocolo de qPCR com este mesmo alvo, objetivando-se aumentar a sensibilidade da técnica. Foram selecionados aleatoriamente 61 cães errantes e classificados de acordo com o número de sinais clínicos associados à LVC apresentados. Todos os cães foram eutanasiados, e durante a necropsia, foram coletados fragmentos de linfonodo, aspirado esplênico, medula óssea e sangue. Também foram realizadas culturas esplênicas e ELISA. A qPCR foi empregada para a avalição da taxa de detecção do DNA do parasito e carga parasitária nos diferentes tecidos. As diferenças entre a carga parasitária de cada tecido foram avaliadas pelo teste de Friedman (p ≤ 0,05). Para inclusão dos tecidos nas análises dos resultados de qPCR, foi avaliada a integridade do material genético de cada amostra. Desta forma, 52 animais apresentaram resultados que atendiam aos critérios de seleção para baço, sangue e linfonodo, e destes, 24 animais também atendiam aos critérios para medula óssea. Utilizando-se a qPCR e considerando pelo menos um dos tecidos avaliados, foi detectado o DNA do parasito em todos os cães. A qPCR detectou DNA do parasito em 98,1% dos aspirados esplênicos, 80,8% das amostras sanguíneas, 53,8% dos linfonodos e 41,7% dos aspirados de medula óssea. A carga parasitária foi melhor detectada nos aspirados esplênicos, em relação ao linfonodo, nos animais oligossintomáticos e polissintomáticos (p ≤ 0,05). O aspirado esplênico foi o tecido com maior taxa de detecção do DNA de Leishmania sp. pela qPCR. No entanto, não foi achada diferença estatística entre a carga parasitária do aspirado esplênico e do sangue. Desta forma, a amostra sanguínea, por ser a segunda amostra de melhor taxa de detecção, e por necessitar uma coleta menos invasiva, foi considerada como uma amostra alternativa válida para a detecção do DNA de Leishmania sp. em cães sintomáticos, utilizando a qPCR. / Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn. L. chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The aim of the present study was to compare the performance of the Polymerase Chain Reaction (PCR) to detect parasite DNA in different clinical sample for CVL diagnosis. For this purpose, in the first stage of the study, a conventional PCR (cPCR) protocol was standardize to detect the presence of Leishmania sp. kinetoplast minicircle DNA (kDNA) in 45 canine spleen fragments. The same samples were evaluated using a quantitative PCR (qPCR) technique targeting the Leishmania sp. sub-unit of the ribossomal RNA (SSU rRNA) gene. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. The cPCR presented the highest sensitivity for Leishmania sp. DNA detection (88,9%), when compared to qPCR was (83.3%). Possibly the cPCR best performance was due to a higher copies number of the kDNA in the Leishmania sp. genome. Given the promising results presented by the cPCR targeting the kDNA, a new qPCR protocol with the same target was standardized in the second stage of the study, aiming increase the technique sensitivity. Sixty-one stray dogs were randomly selected and classified according to the number of clinical signs of CVL. All dogs were euthanized and lymph node fragments and splenic, bone marrow and blood aspirates were obtained during necropsies. ELISA and parasite culture of spleen aspirates were performed to confirm parasite infection. The qPCR was used to determine the parasite DNA detection rate and the parasite load in the clinical samples. Differences between parasite loads of each tissue were evaluated using Friedman test (p ≤ 0.05). In order to include the samples in the qPCR data analysis, the DNA integrity of each sample was analyzed. This way, 52 dogs fulfilled the selection criteria for DNA results for spleen, blood and lymph nodes, with 24 of these dogs also fulfilling the criteria for bone marrow results. Using qPCR, all the 52 dogs showed positivity, considering at least one of the tissues evaluated. Positivity in qPCR was detected in 98.1%8 of the splenic aspirates, 80.8% of blood samples, 53.8% of lymph node fragments and 41.7% of bone marrow samples. Using qPCR, parasite DNA was better detected in splenic aspirates in comparison with lymph node in both polysymptomatic and oligosymptomatic (p ≤ 0.05) dogs. Splenic aspirates have shown to be the tissue with highest Leishmania sp. DNA detection rate using qPCR, however no statistical difference was found between blood and splenic aspirate to detect. Thus, the blood sample, being the second sample with best DNA detection rate and requiring a less invasive collection, was considered a valid alternative sample for Leishmania sp. DNA detection in symptomatic dogs using the qPCR.

The central aim of this thesis is to critically examine the values associated with dogs in Victorian British art and visual culture. It studies the redefining and restructuring of the domestic dog as it was conceptualized in visual culture and the art market. It proposes that the dog was strongly associated with social values and moral debates which often occurred within a visual arena, including exhibitions, illustrated newspapers, and prints. Consequently, visual representations of the dog can be seen as an important means through which to study Victorian culture and society. Historians have agreed that the Victorian period was a significant turning point for how we perceive the dog. Harriet Ritvo, Michael Worboys and Neil Pemberton cite the Victorian period as founding or popularizing many recognisable canine constructs; such as competitive breeding; a widespread acceptance of dogs as pets; and the association of particular breeds with particular classes of people. Phillip Howell defines the Victorian period as the point at which the domestic dog was conceptually established. The figurative domestic dog did not simply exist in the home but was part of the home; an embodiment of its core (often middle class) values. As such, the domestic dog became the standard by which all other dogs were perceived and the focal point for related social debates. Yet most studies concerning the Victorian dog overlook the contribution of visual culture to these cultural developments. William Secord compiled an extensive catalogue of Victorian dog artwork and Diana Donald examined Landseer and the dog as an artistic model yet neither have fully situated the dog within a broader Victorian social environment, nor was their intention to critically examine the dog’s signification within the larger visual landscape. Chapter One provides this overview, while subsequent chapters provide studies of key canine motifs and the manner in which they operated in art and visual culture. Underpinning this thesis is a concern with the Victorian moral values and ideals of domesticity in urban environments. These values and their relation to the dog are explored through the framework of the social history of art. Seen through this methodology, this thesis allows the relationship between canine debates, social concerns, and visual representations to be understood. It will argue that the figure of the dog had a significant role to play both socially and visually within Victorian society and propose a reappraisal of the dog in art historical study.

The efficiency of five natural antioxidants (curcumin, cranberry, pomegranate, grape seed extract (GSE) and açai berry) in reducing lipid oxidation in dog food was tested in comparison to the synthetic antioxidant butylated hydroxyanisole (BHA). Lipid oxidation was evaluated after 12 days of storage at 55C and thiobarbituric acid-reactive substances (TBARS) was measured as an indicator of lipid oxidation. In project one, the natural antioxidants were added at 0.2% and BHA at 0.02% of the food (DM basis) and samples were collected on day 12 and analyzed for TBARS. Compared with the control treatment, TBARS values were lower (p<0.01) for four antioxidant treatments (curcumin, cranberry, pomegranate and GSE) but not for the açai berry treatment (p<0.39). The four antioxidants showed similar efficacy at lowering lipid oxidation as BHA and therefore may have the potential to substitute BHA in dog food. In project two, we evaluated the effects of GSE and curcumin at two inclusion rates (0.1 and 0.2% of food DM) on TBARS and omega-3 fatty acid (FA) content over 12 days of storage at 55C. By day 12, our results showed no significant differences in TBARS values between the BHA and the 0.1% GSE treatment, however BHA was still more effective than the 0.1% GSE as the differences in fold increase in TBARS were lower for BHA (19.4%) than for the 0.1% GSE (75.5%) treatment. Omega-3 FA loss tended (P>0.11) to be greater at the lower inclusion rate which correlated with the increased TBARS values at the 0.1% when compared to the 0.2% inclusion rate. Curcumin and GSE were most effective at maintaining omega-3 FA content at the 0.2% inclusion rate and showed no significant differences from the BHA treatment. In conclusion, BHA in dog food can be effectively substituted by GSE, cranberry, curcumin and pomegranate at the inclusion rate of 0.2% of food DM.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-11T16:05:45Z No. of bitstreams: 1 Barbara Maria Nascimento de Oliveira Produção... 2016.pdf: 2363764 bytes, checksum: ec48ade14eccf109aecb436eec7cd66a (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-11T16:05:58Z (GMT) No. of bitstreams: 1 Barbara Maria Nascimento de Oliveira Produção... 2016.pdf: 2363764 bytes, checksum: ec48ade14eccf109aecb436eec7cd66a (MD5) / Made available in DSpace on 2016-05-11T16:05:58Z (GMT). No. of bitstreams: 1 Barbara Maria Nascimento de Oliveira Produção... 2016.pdf: 2363764 bytes, checksum: ec48ade14eccf109aecb436eec7cd66a (MD5) Previous issue date: 2016 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO. A interleucina 7 (IL-7) é uma citocina pleiotrópica produzida principalmente por células estromais da medula óssea, células epiteliais tímicas e intestinais. IL-7 e que promove o desenvolvimento de linfócitos T e B e diferenciação de células T memória, especialmente CD4+. Por isso, diversos autores têm estudado a capacidade de IL-7 promover a restauração da população de linfócitos em situações de linfopenia e resposta imune de memória. Cães que desenvolvem leishmaniose visceral e recebem tratamento especfico apresentam cura clínica. No entanto, após a interrupção do tratamento, a maioria dos animais exibe recaída da doença, mesmo na ausência de reinfecção. É possível que esse fenômeno esteja associado a uma dificuldade de estabelecimento de linfócitos T de memória específicos para Leishmania em cães. Em nosso laboratório, tentativas prévias foram realizadas para produzir a IL-7 canina em Escherichia coli, no entanto, o rendimento da proteína biologicamente ativa foi pequeno. OBJETIVO. Por isso, resolveu-se avaliar a viabilidade de produzir IL-7 do sistema baculovírus-células de inseto. MATERIAL E MÉTODOS. No presente trabalho, uma construção de DNA foi concebida contendo uma sequência de nucleotídeos que codificam o peptídeo sinal da proteína GP64 do baculovírus da Autographa californica (AcMNPV), a IL-7 canina (proteína madura) com códons otimizados para a tradução em Trichoplusia ni, um espaçador com 23 aminoácidos e uma cauda de seis histidinas. A construção de DNA elaborada foi sintetizada quimicamente, inserida em um plasmídeo de clonagem (pUC57-GP64caIL-7), subclonada em um plasmídeo carreador (pFastBac1- GP64caIL-7), adequado para o sistema baculovirus-célula de inseto, e transferida para o cromossoma artificial do baculovírus recombinante (bacmídeo). RESULTADOS. A produção de IL-7 recombinante canina (rcaIL-7) em células de inseto infectadas com baculovírus recombinante foi otimizada. Em seguida, a rcaIL-7 foi produzida em células High-five cultivadas em suspensão utilizando multiplicidade de infecção (MOI) de 5 e tempo de infecção (TOI) de 48 h. A proteína recombinantes foi purificada por cromatografia de afinidade em Sepharose-níquel, obtendo-se um rendimento médio de 5,5 mg por litro de cultivo. CONCLUSÃO. RcaIL-7 purificada, avaliada a 40 ng/mL, mostrou-se capaz de promover a proliferação de células mononucleares de sangue periférico de cães sadios, sem estímulo prévio ou concomitante, indicando que a proteína foi produzida biologicamente ativa. Futuros estudos serão realizados para avaliar a capacidade imunomoduladora da rcaIL-7 em cães e determinar a utilidade dessa citocinas no tratamento de enfermidades caninas, como por exemplo, na leishmaniose visceral canina. / INTRODUCTION. The interleukin 7 is a pleiotropic cytokine produced mainly by bone marrow stromal cells, thymus and intestinal epithelial cells. IL-7 promotes the development of T and B limphocytes and differentiation T cells to memory cells, especially T CD4+ cells. For these reasons, several authors have studied the ability of IL- 7 to promote restoration of the lymphocyte population in cases of lymphopenia and memory imune responses. Dogs which develop visceral leishmaniasis and are treated with specific drugs develop only transient clinical cure. After treatment withdrawal, most animals show disease relapse, even in the absence of reinfection. One possible explanation for the lack of control of the infection after treatment in dogs is a difficulty to develop/maintain specific memory T cells. Therefore, it is possible that effective treatments for canine visceral leishmaniasis be developed by manipulating the immune system. Previously, in our laboratory, it was attemped to produce recombinant canine IL- 7 (rcaIL-7) in Escherichia coli, however there was a low yield of biologically active protein. OBJECTIVE. In the current project, the baculovírus-insect cell system was evaluated to produce rcaIL-7. MATERIAL AND METHODS. In this study, a construction of DNA was designed to encode nucleotides of Autographa californica GP64 signal peptide, canine IL-7 (mature protein) with optimized codons for Trichoplusia ni, a 23-amino acid spacer and a six histidine tail. The DNA construct was chemically synthesized, inserted into a cloning plasmid (pUC57-GP64caIL-7), subcloned into a carrier plasmid (pFastBac1-GP64caIL-7), suited to the baculovirus-insect cell expression system, and transfered to an artificial chromosome of the recombinant baculovirus (bacmid). RESULTS. Recombinant protein expression was optimizatized, then, the rcaIL-7 was produced in a cell culture in suspension using multiplicity of infection (MOI) 5 and time of infection (TOI) 48 hours. Subsequently rcaIL-7 was purified by Nickel Sepharose-Affinity chromatography, obtaining an average yield of 5.5 mg per liter of culture. CONCLUSION. The purified recombinant rcaIL-7 at 40 ng/mL was able to induce prolifereation of peripheral blood mononuclear cells of heath adult dog, suggesting it was biologically active. Given the results, the rcaIL-7 may be used in further studies to assess its ability to modulate canine immune system, that would be useful for the development immunotherapy