Establishing blood serum profiles is important for understanding animal disease ecology and nutrition, the effects of capture and immobilization, and general physiological variation among individuals and populations. Elk (Cervus elaphus nelsoni) have been successfully translocated to several states for reintroduction or population augmentation, including most recently in Kentucky, where over the course of three years (2011-13) wild elk were captured, temporarily quarantined, and subsequently moved to Missouri and Virginia to establish populations in those states. I used this opportunity to collect a variety of biological samples, including blood from which I present and compare serological profiles for a variety of elk age and sex classes. Additionally, I took the opportunity to investigate the effects of the translocation process on some morphological and physiological parameters of elk. Quarantine and processing of elk can be stressful for animals and lead to injury or death. I characterized how elk respond to this process by measuring biochemical serum values along with various physiological parameters at 5 handling events from the time of capture until final transport to recipient states. Changes observed in parameters between paired workups were indicative of the physical exertion and stress associated with handling activities.

[pt] Embora o papel do vanádio no organismo ainda não tenha sido bem definido, sabemos que ele inibe a atividade das ATPases em eritrócitos e células tubulares renais, afetando a função diurética dos rins, o metabolismo de leucócitos e macrófagos, a função do músculo cardíaco, causando efeitos insulina-like em doses farmacológicas. O vanádio é de grande importância para a indústria, é utilizado como catalisador, na produção de vidros e aço. Os trabalhadores em usinas de produção de aço e indústrias que utilizam combustíveis fósseis como fonte de energia, são expostos a quantidades significativas de vanádio, sendo importante o monitoramento dos seus níveis. No presente trabalho foi desenvolvido um método simples e direto para determinação de vanádio em soro usando a espectrometria de absorção atômica em forno de grafite, otimizado por um planejamento composto central. As temperaturas de pirólise e atomização foram de 1240 e 2600 oC, e calibração externa pode ser realizada com soluções de calibração preparadas em HNO3 2,5 % v/v, mostrando-se linear até, pelo menos, 3 ug L(-1). O pré-tratamento da amostra consistiu tão somente de uma diluição (2+1) no mesmo meio, e os limites de detecção e quantificação encontrados foram, respectivamente, 0,3 e 1,0 ug L(-1). A velocidade analítica foi de 8 amostras h-1, para análises em triplicata. Nos estudos de recuperação foram obtidas valores médios de 97,2%; 99,2% e 105,3%, para concentrações de 1, 2 e 3 ug.L(-1), respectivamente. Houve excelente concordância entre os valores encontrados e o certificado na análise de um material de referência certificado. Nos estudos da repetitividade, o maior coeficiente de variação encontrado foi 10,3% na amostra adicionada de 1 ug L(-1). Nos estudos da precisão intermediária, o maior coeficiente de variação foi 5,6% na amostra adicionada de 2 ug L(-1). As incertezas combinadas obtidas foram 1,65x10-1; 2,72x10-1 e 3,72x10-1, para concentrações de 1, 2 e 3 ug L(-1), respectivamente. O tempo de vida do tubo de grafite nas condições otimizadas foi de, aproximadamente, 150 ciclos. O método foi considerado adequado para a determinação rotineira de vanádio em soro em laboratórios clínicos. / [en] Although the role of vanadium in the body has not been yet well defined, it is known that it inhibits the ATPase activity in erythrocytes and renal tubular cells, affecting the diuretic function of the kidneys, the metabolism of leukocytes and macrophages, the function of the heart muscle and causes insulin-like effects in pharmacological doses. Vanadium is of great importance to the industry, and is mostly used as catalyst, and in the production of glass and steel. Workers of the steel industries as well as those in plants that use large amounts of fossil fuels are potentially exposed to large amounts of vanadium, and it is important to monitor their serum levels. In the present study it was developed a simple and direct method for determination of vanadium in serum using graphite furnace atomic absortion spectrometry optimized by a central composite design. External calibration was possible, using calibration solutions prepared in 2 % v/v HNO3. The sample was 2+1 diluted in the same medium and the calibration curve was found linear up to at least 3 ug L(-1). The limits of detection and quantification were respectively 0.3 and 1.0 ug L(-1), in the original sample. Average recoveries were 97.2%, 99.2% and 105.3% for concentrations of 1, 2 and 3 ug L(-1), respectively. Excellent agreement was found between found and certified values in the analysis of a certified reference material. The sample throughput was 8 h-1 (n=3). In the study of repeatability, the largest coefficient of variation was 10.3% in the sample spiked with 1 ug L(-1) of vanadium. In the study of the intermediate precision the higher coefficient of variation was 5.6% in the sample spiked with 2 μg.L-1. The combined uncertainties obtained were 1.65x10-1, 2.72x10-1 and 3.72x10-1, for concentrations of 1, 2 and 3 ug L(-1), respectively. The graphite tube life time in the optimized conditions was 150 cycles, approximately. The method was considered adequate to the routine determination of vanadium in serum in clinical laboratories.

Masters of Science / Invasive pneumococcal disease (IPD), which includes potentially fatal conditions such as meningitis, septicaemia and pneumonia poses a threat in children aged <5 years, pneumonia being the leading cause of child mortality worldwide. Even though capsular polysaccharides are the main antigens involved in the immunity to encapsulated bacteria, it was found that in children in that age group, the immune system was unresponsive. Conjugate vaccines however induce immunologic memory and provide long-term protective immunity. Therefore the aim of this project was to develop novel conjugation strategies towards a pneumococcal conjugate vaccines and focuses mainly on the serotypes that are a burden to the African continent. The chemistry involved in developing a conjugate vaccine is of importance beacuse while some polysaccharides contain chemical grouping which can be conveniently utilized for conjugation, many medically important ones require derivatization before they can be coupled to protein. Derivatization of which can be achieved through various strategies, important to note is through hypervalent iodine oxidants. Two hypervalent iodine reagents, O-Methyl substituted-1-hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide (Me-IBX)and modified 1-hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide (mIBX)were successfully synthesized in preparation for the use in polysaccharide, polyribitol phosphate, (PRP) oxidation. The polysaccharide to be oxidised was first size reduced by microfluidisation to allow maximum oxidation. However, the extent to which oxidisation was achieved was not enough to conjugate the polysaccharide to the protein of preference, Bovine Serum Albumin, (BSA).

Lipid soluble carotenoids are micronutrients present in human milk that serve as precursors of vitamin A and also play an important role protecting cells from damage arising from photooxidative processes and reactive oxygen species. Fatty acids comprise about 3-5% of human milk and are mainly present as triglycerides. They are a major energy source for the infant and are necessary to support cell growth required for normal development and maturation of critical organs. Transport of carotenoids into milk has been little studied and there has been no previous investigation of the relationship of carotenoid transport with that of individual fatty acid secretion into milk. In the present study, levels of the carotenoids, lutein, zeaxanthin, b-cryptoxanthin, and b-carotene, in maternal serum, infant cord blood, and milk obtained from 74 Peruvian mothers were measured by HPLC methods. The fat content and fatty acid profile of maternal milk were determined by GC-FID and confirmed by GC-MS. Twenty nine fatty acids were identified and quantified after conversion to methyl esters. Statistical analysis was employed to investigate potential trends and relationships among the carotenoids in all three fluids as well as between carotenoids and fatty acids present. Concentrations of lutein in maternal serum and milk as well as maternal serum and infant cord blood were highly correlated (r =0.43, p

The feasibility of Fourier transform infrared (FTIR)-imaging spectroscopy as a tool to discriminate samples from patients suffering from benign prostatic hyperplasia (BPH) and prostate cancer (CaP) samples in blood serum was investigated. Prostate cancer is known to be an age related disease, with the risk of developing the disease dramatically increasing in men past forty years old. Currently the PSA blood test is notoriously unreliable and is non specific for CaP thus leading to overtreatment of the disease. It is important therefore to develop diagnostic method that is non-invasive, reliable, and specific for CaP.In order to achieve the objective of establishing a robust protocol, which could be applied to a clinical study, obtaining optimal sample preparation for the FTIR analysis of serum smears, had to be achieved. A protocol was developed to prepare the serum samples prior to their FTIR analysis. First, the samples were centrifuged with ultrafiltration devices of different sizes to obtain several fractions which were then smeared to obtain thin films of serum. The spectra from the larger (>100 kDa components) and medium (containing the 10–100 kDa components) fractions were utilised for both a pilot and a clinical study, while the spectra from the smaller fractions (containing the 3–10 and <3 kDa components) were affected by fringing and could therefore not be used. A major novelty of this project involved the application of FTIR-imaging to the analysis of serum smears. The use of the Focal Plane Array detector system enabled the collection of a spectral image containing 16,384 spectra, on which a Quality Testing and pre-processing techniques were applied to select the “good spectra” and reject the spectra that failed the Quality Test. Several types of substrates were assessed to determine the most appropriate for the analysis of the smears and it was established that the spectra obtained from the serum smeared on CaF2 windows gave the most reproducible results. 5 BPH and 5 CaP samples were analysed for the pilot study following the developed protocol. While no clear separation was observed in the Principal Component Analysis (PCA) plots between the BPH and the cancerous samples, a trend emerged throughout the results, with the CaP samples clustering together and the BPH samples scattered around them. A larger clinical study was conducted with 60 BPH samples and 60 CaP samples. PCA was applied on the “good spectra” and while the over 100 kDa fraction did not show a clear separation between the two types of samples, the 10–100 kDa fraction showed a distinct classification between the BPH and CaP samples. An artificial neural network was then applied to create a model using patients from the database used for the PCA analysis to determine whether the discrimination between the two types of samples could be increased or highlight different classification trends. For the >100 kDa fraction, the sensitivity value was calculated to be 97.8% and the specificity value was calculated to be 44.3% while the sensitivity and the specificity value for the 10 to 100 kDa fraction were calculated to be 78.9% and 60% respectively. A complementary study using mass spectrometry was carried out on healthy and diseased samples to identify the components contained within the different fractions and determine whether they could be correlated with the components identified from the spectral features of the FTIR data. While no quantitative information was obtained from this study, the components found in the different fractions were identified, confirming the results of the FTIR studies.

In this study the heat-induced gelation of two (readily available) proteins, which contain disulphide bonds, has been investigated over a range of protein concentrations in the presence and absence of the presence of the reductant, dithiothreitol at neutral pH. The proteins selected in this study were: β-Lactoglobulin and bovine serum albumin. These proteins have different number of disulphide bonds and possess different protein secondary structures. The influences of the reductant and protein concentration on their heat-induced gelation were explored to see whether the proteins were able to form protein hydrogels and that the mechanical properties of the resulting protein hydrogels were controllable. The tilting test tube method revealed that both proteins formed macroscopic hydrogels, at protein concentrations above the critical gelation concentration and that the critical gelation concentration was constantly lower in the presence of the reductant. Micro-DSC revealed that both proteins had completely denatured upon heating and that the denaturation temperature and enthalpy were significantly lower in the presence of the reductant. IR spectroscopy revealed that both proteins undergo major secondary structure transitions that resulted in the formation of fibers that are rich in β-sheet structure upon heating and that the protein lost some secondary structure before any heating and gained more β-sheet structure in the presence of the reductant. Both proteins had partially denatured before any heating in the presence of the reductant and that β-LG underwent aggregation that was accompanied by the loss of native β-sheet structure and the formation of intermolecular β-sheet structure, while that BSA underwent aggregation that was accompanied by the loss of native α-helix structure and the formation of intermolecular β-sheet structure. Cryo-TEM revealed that both proteins formed fibers (10 nm in diameter) that exist as single entities at low protein concentrations and become entangled into macroscopic networks, at protein concentrations above the critical gelation concentration and that more fibers and denser macroscopic networks were formed in the presence of the reductant. Oscillatory rheology revealed that both proteins formed macroscopic networks exhibit viscoelastic behaviour and that their elastic modulus had increased in the presence of the reductant and with increasing protein concentration.

Orientador: Flavia Maria Netto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T01:03:16Z (GMT). No. of bitstreams: 1 Paz_JanaiCristianeSantosNascimento_M.pdf: 1327387 bytes, checksum: 694e3264c81a1a8b6a29d69943a20d23 (MD5) Previous issue date: 2004 / Resumo: A influência do pH (3,5 e 7,0); da temperatura de aquecimento (80 e 95 ºC) e da concentração de proteína (8 a 14%) na obtenção de géis de isolados protéicos comerciais de soja (IPS) e de soro de leite (IPSL) foi estudada em sistemas simples e mistos, nas proporções de IPS : IPSL 100 : 0; 80 : 20; 60 : 40; 40 : 60; 20 : 80; 0 : 100. Os géis simples e mistos formados em pH 3,5 foram do tipo coágulo, opacos, com grande exudação de água e quando a concentração de IPSL foi maior que a de IPS, os géis apresentaram separação de fase dos dois sistemas protéicos. Os géis preparados em pH 7,0, com 12 e 14% de proteína e temperatura de 80ºC eram extremamente moles e, com 12% de proteína e temperatura de aquecimento de 95ºC, eram formados géis incapazes de manter a sua forma. Os géis simples e mistos preparados em pH 7,0, com 14% de proteína, e temperatura de aquecimento 95ºC eram firmes e elásticos, sendo que nos géis mistos essas características tiveram intensidade intermediária a dos géis simples de IPS e IPSL. As propriedades físico-químicas, estruturais e funcionais dos géis foram estudadas pela análise da solubilidade dos componentes protéicos dos géis em diferentes meios de extração, caracterização dessas frações solúveis por SDS-PAGE e da microestrutura por microscopia diferencial de varredura. A capacidade de retenção de água (CRA) e os parâmetros de textura dureza, coesividade e elasticidade também foram determinados. Os resultados mostraram que houve aumento na dureza dos géis com o aumento da proporção de IPSL na mistura em ambos os pHs estudados e em pH 7,0, géis com dureza igual (p = 0,05) à do gel de IPSL foi obtido quando a substituição foi de 20%. A mistura IPS:IPSL também favoreceu o aumento da coesividade dos géis de pH 7,0 e da elasticidade dos géis de pH 3,5, quando comparados ao gel simples de IPS. Os géis de filamentos finos e estrutura compacta, obtidos em pH 7,0, apresentaram maior CRA que os géis de estrutura grosseira obtidos em pH 3,5. A substituição parcial de IPS por IPSL na obtenção de géis com pH 3,5 favoreceu a formação de uma microestrutura mais compacta, menos porosa, que a do gel simples de IPS. A analise de solubilidade indicou que os géis foram mantidos principalmente por interações hidrofóbicas e pontes de hidrogênio em ambos os pHs estudados. O perfil indica que a a-lactalbumina, b-lactoglobulina, proteínas do soro de leite, e o polipeptídeo ácido da fração 11S, foram estabilizados principalmente por interações eletrostáticas. A fração 7S e o polipeptídeo básico da fração 11S, foram mantidos principalmente por ponte de hidrogênio e interações hidrofóbicas / Abstract: The influence of pH (3.5 and 7.0); heating temperature (80 and 95ºC) and protein concentration (8 to 14%) on gelation properties of commercial soy protein isolate (SPI), whey protein isolate (WPI) was tested in isolation and when mixed in ratios 100:0; 80:20; 60:40; 20:80; 0:100 SPI:WPI. The simple and mixed gels obtained in pH 3.5 were clot gels, opaque, with high loss of water, and when the concentration of WPI was larger than the concentration of SPI, the gels showed phase separation of the two protein systems. In pH7.0 with protein concentration of 12 and 14% and 80ºC, the gels were very soft and when the protein concentration was of 12% and the heating temperature was 95ºC the gels were unable to keep their shape. In pH 7.0, 14%, 95ºC, the simple and mixed gels were firm and elastic, and the mixed gels showed these characteristics in an intermediate intensity when compared to simple gel obtained from SPI or WPI. These gels¿ physical-chemical, structural, and functional properties were studied by determination of solubility of the gel proteinic components, characterization of the soluble fractions by SDS-PAGE and of the microstructure by scanning electron microscopy (SEM). The water-holding capacity (WHC) and parameters of texture (hardness, cohesiveness and elasticity) were also determined. The results showed that the hardness increased when the amount of WPI was higher than the amount of SPI in the mixture in both pH studied, and in pH 7.0 the gels as hard as (p=0.05) the simple gel of WPI were obtained when the substitution of SPI to WPI was of 20%. The mixture SPI:WPI favored the increase of cohesiveness of the gels with pH 7.0 and the elasticity of the gels with pH 3.5, when compared to the simple gel of SPI. The gels that showed fine strand and compact structure, pH 7.0, had higher WHC than the gels with coarse structure, pH 3.5. The partial change of SPI for WPI in the gels pH 3.5 made the microstructure more compact and less porous than the microstructure of the simple gel of SPI. Solubility assays indicated that the main responsible forces for the maintenance of the gel structure in these pHs are hydrophobic and H bindings. It is possible that the a-lactalbumin, b-lactoglobulin, whey prtein, and the acid polypeptide of the 11S globulin was maintenanced main for eletrostatic forces. The 7S globulin and the basic polypeptide of the 11S globulin was maintenanced main for hydrophobic and H binding / Mestrado / Mestre em Alimentos e Nutrição

Orientador: Aloisio Jose Antunes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T16:03:54Z (GMT). No. of bitstreams: 1 Alleoni_AnaClaudiaCarraro_D.pdf: 4141640 bytes, checksum: db9a912da57044569b27e9e031edb1c0 (MD5) Previous issue date: 2003 / Resumo: As proteínas da clara do ovo de galinha têm sido extensivamente usadas como ingredientes em alimentos processados, devido a suas propriedades funcionais, tais como gelatinização e formação de espuma. Durante o armazenamento, podem ocorrer mudanças nas características físicas, microbiológicas, químicas e funcionais dos ovos, que dependem das condições de armazenamento. As vantagens de utilizar filmes e coberturas comestíveis podem ser justificadas pela manutenção das propriedades funcionais dos alimentos, através da diminuição da perda de umidade e da diminuição do transporte de gases (02 e CO2). A aplicação de cobertura em ovos com casca reduz a perda de peso e mantém a qualidade interna do produto. Os objetivos foram quantificar a perda de peso, os valores da unidade Haugh, o pH e os teores de s-ovalbumina do albume, além de determinar alterações na estabilidade da espuma da clara, no perfil de textura e na umidade espremível de géis da clara de ovos frescos e de ovos com e sem cobertura à base de concentrado protéico de soro de leite, armazenados a 25°C, por 3,7, 10, 14,21 e 28 dias. Durante todo o período de estocagem houve decréscimo dos valores de unidade Haugh e perda de peso, tanto para os ovos com cobertura como para os ovos sem cobertura. Já o pH do albume aumentou. A variação dos valores nos ovos com cobertura foi menor do que para os ovos sem cobertura. Os valores da unidade Haugh para ovos com cobertura foram semelhantes aos de ovos armazenados sob refrigeração, encontrados em referências bibliográficas, no mesmo período de armazenamento. O volume de líquido drenado foi maior (P < 0,01) em ovos sem cobertura do que em ovos com cobertura armazenados a 25°C, em todos os períodos de armazenamento. A diferença no terceiro dia de armazenamento foi de 59% entre os ovos com cobertura e ovos sem cobertura, e no vigésimo oitavo dia foi 202%. Durante o período de armazenamento, ocorreu um aumento do pH e do volume do líquido drenado nos ovos sem cobertura. O teor de s-ovalbumina foi maior em ovos sem cobertura, comparados aos ovos com cobertura, em todos os períodos de armazenamento (P<0,01). Após três dias, os ovos sem cobertura apresentaram aumento de 33% em relação aos ovos com cobertura. Já com 28 dias de armazenamento essa diferença passou para 205%. Em ovos com cobertura armazenados a 25°C, o pH ficou na faixa de 8, e o teor de s-ovalbumina permaneceu na faixa de 20%. No vigésimo oitavo dia de armazenamento o teor chegou a 27%. Houve correlação positiva entre o teor de s-ovalbumina com o volume de líquido drenado (R2 = 0,92 para ovos com cobertura e R2 = 0,94 para ovos sem cobertura). Quando aumentou o teor de s-ovalbumina, aumenta o volume de líquido drenado, e diminuiu a estabilidade da espuma. A dureza do gel do albume de ovos sem cobertura foi maior do que a de ovos com cobertura (P < 0,01), dentro de todos os períodos de armazenamento. Não houve efeito do tempo de armazenamento na dureza dos géis dos ovos sem cobertura. Em ovos cobertos, a regressão linear explicou 60% do comportamento da dureza em relação ao período de armazenamento. No caso da elasticidade, não houve interação entre período de armazenamento e a cobertura. Houve diferença entre as médias dentro de cada período, mas não durante o armazenamento. A maior elasticidade foi dos géis de ovos sem cobertura, comparados com os géis de ovos com cobertura. O índice de coesividade e a mastigabilidade de géis de ovos sem cobertura foi maior que o de géis de ovos com cobertura, em todos os períodos de armazenamento. A percentagem de umidade espremível (UE) de géis de clara de ovos com cobertura foi maior do que a de ovos sem cobertura em todo o período de estocagem / Abstract: Egg white proteins are extensively used as processed food because of their functional properties, as gelation and foam formation. Depending on the storage conditions, some physical, microbiological, chemical and functional changes may occur. Functional food properties may be preserved when foods are coated by edible films, since both the loss of moisture and the transport of O2 and CO2 are reduced. The objectives of this work were quantify the weight loss, the Haugh unit and the pH contents of s-ovalbumin in the albumen, determining changes in the stability of egg white foam, in the texture profile and in the expressible moisture of fresh egg white gels and eggs coated or not by whey protein concentrate 0NPc), under six storage periods (3, 7, 10, 14,21 and 28 days), at 25°C. During the storage period, independently of the coating, values of Haugh unit, weight loss and albumen pH decreased, and the differences among the values from the first to the last period were lower in the coated eggs. The Haugh unit values for coated eggs were similar to those found in bibliographic references when the same storage period is considered. For the six storage periods, the volumes of drained liquid in non-coated eggs were higher (P< 0.01). In the 3rd day the difference between the coated and the non-coated egg was 59%, and after 28 days that difference increased up to 202%. In the non-coated eggs, values of pH and volume of drained liquid increased during the storage. The content of s-ovalbumin was higher in the non-coated than in the coated eggs, independently of the storage period (P < 0.01). After 3 days, the content of s-ovalbumin was 38% higher than in the coated eggs, and after 28 days the difference between the values was 205%. In the coated eggs, stored at 25°C, the pH was around 8, and the content of s-ovalbumin remained in 20%; after 28 days, the value increased to 27%. A positive correlation was found between the content of s-ovalbumin and the volume of drained liquid (R2 = 0.92 for coated eggs, and R2 = 0.94 for non-coated eggs). When the content of s-ovalbumin increased, the volume of drained liquid also increased, and the foam stability decreased. Hardness of the albumen gel of non-coated eggs was higher than the coated eggs, during the whole storage period. For the non-coated eggs, the storage period did not affect the hardness of the gels, but for the coated eggs, a linear regression explained 60% of the behavior of the hardness as a function of the storage period. When considering the gel elasticity, no interaction was found between the storage period and the egg coating. Non-coated eggs exhibited gels with higher springiness than coated eggs. The same was found for the cohesiveness index and the gel chewiness, independently of the storage periods. However, the opposite was observed for the percentage of expressible moisture, where the values for coated eggs were higher than for non-coated eggs / Doutorado / Doutor em Alimentos e Nutrição

Duas patologias principais estão associadas ao HTLV-1: a leucemia/linfoma de células T do adulto (ATLL) e a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). O mecanismo pelo qual ocorre o desenvolvimento destas doenças em apenas 1 a 5% dos indivíduos infectados é desconhecido. Sabe-se que diversos fatores estão associados ao desenvolvimento destas patologias, como carga proviral, características celulares, genéticas e imunológicas do hospedeiro. No entanto, tais mecanismos não foram totalmente elucidados e novas abordagens de estudos são necessárias para a compreensão da patogênese viral. Nesse sentido, vários estudos relatam o papel dos exossomos no desenvolvimento de infecções virais como HIV, EBV e HCV através do transporte de componentes incluindo proteínas, RNAm e miRNA. A possibilidade da transferência do conteúdo viral por essas vesículas para células não infectadas na ausência de vírus é um mecanismo intrigante. Dessa forma, esse trabalho teve como objetivo isolar os exossomos diretamente do soro de indivíduos infectados pelo HTLV-1 e comparar o conteúdo dessas vesículas quanto à expressão dos genes virais tax e HBZ entre os grupos assintomático e sintomático. Para tanto, os exossomos foram isolados por precipitação polimérica (Exoquick(TM)) e caracterizados por Western Blot (expressão dos marcadores CD9, CD81, Alix e citocromo c) e Análise de Rastreamento de Nanopartículas (tamanho e concentração). Além disso, o conteúdo dos exossomos foi analisado por PCR em tempo real para quantificação dos genes virais regulatórios tax e HBZ. Por fim, no intuito de estabelecer o papel dos exossomos como biomarcadores para o desenvolvimento da HAM/TSP, os níveis de expressão destes genes foram associados à carga proviral do HTLV-1. Os transcritos de RNAm de tax e HBZ foram detectados apenas em exossomos isolados do soro dos indivíduos sintomáticos, com ausência nos assintomáticos. Além disso, a expressão de tax e HBZ nos exossomos dos indivíduos infectados pelo HTLV-1 foi correlacionada positivamente com a CPV. Dessa forma, os resultados sugerem que os exossomos podem desempenhar importante papel na patogênese da mielopatia, através do transporte de genes virais de uma célula infectada para não-infectada. No entanto, mais estudos que relacionam o papel dos exossomos na infecção pelo HTLV-1 são necessários. / Two major pathologies has been associated with HTLV-1 infection: adult T- cell leukemia/lymphoma (ATLL) and HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM / TSP). The mechanism which is responsible for the appearance of these diseases in only 1 to 5% of the infected individuals is unknown. Is known that several factors has been related to the development of these diseases, such as proviral load and cellular, genetic and immunological characteristics of the host. However, these mechanisms have not been fully elucidated and new approaches are necessary to understand the viral pathogenesis. Thus, several studies have reported the involvement of exosomes in the development of the HIV, HCV, and EBV viral infections carrying components including proteins, mRNA and miRNA. The possibility of transfer of viral content via these vesicles to uninfected cells in the absence of virus is an intriguing mechanism. Therefore, this proposal aimed to isolate exosomes from serum of HTLV-1 infected patients and compare the content of these vesicles regarding viral gene expression of tax and HBZ between asymptomatic and symptomatic group. For this, the exosomes were isolated by polymeric precipitation (Exoquick(TM)) and characterized by Western Blot (expression of markers CD9, CD81, and Alix) and Nanoparticle tracking Analysis (size and concentration). Furthermore, the exosomes content were analyzed by real-time PCR for the quantification of tax and HBZ regulatory viral genes. Finally, to establish the role of exosomes like biomarkers to HAM/TSP development, levels of these genes were associated with proviral load of HTLV-1. Viral mRNA transcripts including tax and HBZ were found only in exosomes isolated from serum of symptomatic individuals and absence in assymptomatic. Furthermore, tax and HBZ expression in exosomes from HTLV-1 infected individuals were positive correlated with proviral load. Thus, the results suggests that exosomes can able to develop important role in myelopathy pathogenesis carrying viral genes of infected cell to uninfected cell. However, more studies that relate the role of exosomes in HTLV-1 are needed

The leishmaniase is a serious public health problem in the world, demanding effective measures for its control and treatment. The patient's immune response in visceral leishmaniasis has a fundamental role in the prognosis of this infection inflammation, which can be fatal if untreated. So that chemotherapy can result in healing, it is important that the immune system is able and necessary stimulus to the development of appropriate cytokine profile and disease resistance to fighting agent.Thus, the purpose of this study was to evaluate the levels serum of patients on the adjuvant action of N-acetyl-L-cysteine (NAC) in the chemotherapy of human visceral leishmaniasis, made with pentavalent antimony. From an intervention study, clinical trial, blinded, randomized, 60 patients were investigated at the University Hospital with the diagnosis of visceral leishmaniasis. These patients were divided into two groups of 30: Study Group - which made use of pentavalent antimony standard dose supplemented with N-acetyl-L-cysteine (NAC) and the control group - which only used a standard dose of antimony. This study was approved by the ethics committee in search of UFS under CAAE 0151.0.107.000-07 number. The patients selected randomly and were assessed for clinical and laboratory parameters. To evaluate the rate of the patients were performed serum levels of cytokines IFN-γ, TNF-α, IL-10, IL-12p40 and of molecule sCD40L before, during and after treatment using the Luminex 100 analyzer. The results suggest that the addition of NAC to conventional therapy improves the immune response of patients, especially in lowering serum levels of IL-10, IL-12p40 and elevated sDC40L. This study showed that although both groups had healed up on the last day of treatment, the study group developed a response indicative of early healing, sugesting that the NAC has acted in a manner adjunct to pentavalent antimony, being also proposed the use of the sCD40L molecule as a prognostic marker for visceral leishmaniasis. / Para que a quimioterapia possa resultar em cura, é importante que o sistema imune tenha condições e estímulos necessários ao desenvolvimento do perfil de citocinas adequado à resistência à doença e ao combate do agente etiológico. Estudos demonstram que o N-acetil-L-cisteína (NAC), uma substância antioxidante, contriubi para a imunoregulação de determinadas doenças. Assim, o objetivo deste trabalho foi avaliar os níveis séricos dos pacientes quanto à ação adjuvante do N-acetil-L-cisteína (NAC) na quimioterapia da leishmaniose visceral humana, feita com antimônio pentavalente. A partir de um estudo de intervenção, tipo ensaio clínico, cego, randomizado, foram investigados 60 pacientes do Hospital Universitário com diagnóstico positivo para leishmaniose visceral aguda. Esses pacientes foram distribuídos em dois grupos de 30: Grupo Estudo que fez uso do antimônio pentavalente dose padrão complementado com o N-acetil-L-cisteína (NAC) e o Grupo Controle - que fez uso apenas do antimônio dose padrão. Este trabalho foi aprovado pelo comitê de ética em pesquisa da UFS sob número CAAE 0151.0.107.000-07. Os pacientes selecionados e randomizados foram avaliados quanto aos parâmetros laboratoriais. Para avaliar as taxas de citocinas dos pacientes, foram realizadas dosagens dos níveis séricos de IFN-γ, TNF-α, IL-10, IL-12p40 e IL-12p70, sendo também dosada a molécula sCD40L antes, durante e após o tratamento, com uso do analisador LUMINEX 100. Os resultados sugerem que a adição do NAC ao tratamento convencional melhora as taxas das citocinas dos pacientes, sobretudo a diminuição dos níveis séricos de IL-10, IL-12p40 e na elevação de sDC40L. Este estudo mostrou que, apesar de ambos os grupos apresentarem-se curados no último dia do tratamento, o grupo estudo desenvolveu uma resposta indicativa de cura mais precocemente, sugerindo que o NAC tenha atuado de forma adjuvante ao antimônio pentavalente, sendo proposto ainda o uso da molecula sCD40L como um marcador prognóstico para a leishmaniose visceral.