Em alguns casos, pacientes que sobreviveram a uma sepse grave podem desenvolver um quadro de imunossupressão, caracterizado pela expansão dos linfócitos T reguladores (Tregs). Porém, apesar de inúmeros avanços, os mecanismos associados à expansão das Tregs, ainda não estão completamente esclarecidos. Nesse sentido, trabalhos recentes demonstraram que a atividade da enzima Indoleamina 2,3-Dioxigenase (IDO), responsável pela formação da quinurenina a partir da degradação do aminoácido essencial triptofano, está relacionada à diferenciação das Tregs e com o desenvolvimento de um quadro de tolerância. Dessa forma, o objetivo deste estudo foi investigar o papel da IDO no desenvolvimento da imunossupressão induzida pela sepse. Os resultados demonstraram um aumento da expressão proteica e da atividade enzimática da IDO no baço de camundongos que sobreviveram à sepse grave. Para avaliar o desenvolvimento da imunossupressão, os camundongos foram desafiados com células do melanoma B16-F10. A inibição farmacológica da IDO promoveu redução do crescimento tumoral nos camundongos que sobreviveram à sepse, por um mecanismo dependente da redução na diferenciação das Tregs e das células C11b+ Ly6G+ no baço e da ativação de células CD8+ produtoras de IFN- ?, no linfonodo drenate da região tumoral. Adicionalmente, foi demonstrado que, o receptor de hidrocarbonetos de arila (AhR) está associado ao aumento da expressão da IDO, nos camundongos que sobreviveram à sepse. Ainda, os resultados demonstraram que células CD11C+ são as principais responsáveis por expressar a IDO no baço de camundongos que sobreviveram à sepse. Por fim, células CD11C+ isoladas do baço de camundongos que sobreviveram a sepse, foram mais efetivas em induzir a diferenciação das Tregs quando comparadas a células CD11C+ provenientes de camundongos naive. Em conjunto, os resultados sugerem que a ativação do AhR pela quinurenina é importante para expressão da IDO nas células CD11C+ encontradas no baço de camundongos que sobreviveram à sepse, o que por sua vez, está associado com a expansão das Tregs e com o desenvolvimento do quadro de imunossupressão. / Immunosuppression has been shown to be one long-term sequels of severe sepsis, which is mainly characterized by the expansion of regulatory T cells (Tregs). However, the mechanisms underlying Tregs expansion after sepsis remain poor understood. Indoleamine 2,3-dioxygenase (IDO), an enzyme that initiates the kynurenine pathway of tryptophan degradation, has been implicated in promoting Tregs generation. Therefore, we propose to investigate the role of IDO in the development of sepsis-induced immunosuppression. The results presented here demonstrated that there is an increase in both IDO protein expression and IDO enzymatic activity in spleen of sepsis-surviving mice. Employing a melanoma mouse model as a second challenge in sepsis-surviving mice, we found that pharmacological inhibition of IDO suppressed the enhanced tumor growth observed in sepsis-surviving mice. Importantly, inhibition of IDO decreased the expansion of Tregs in sepsis-surviving mice, leading to reduced CD11b+ Ly6G+ cells frequency in spleen and activation of INF-? production by CD8+ in draining lymph, after tumor challenge. Moreover, the results suggest that aryl hydrocarbon receptor (AhR) is associated with increased IDO expression found in sepsis-surviving mice. Furthermore, we identified that a CD11C+ population of cells are expressing IDO in spleen of sepsis surviving mice. In addition, CD11C+ cells isolated from spleen of sepsis-surviving mice, presented a higher capacity to induce a regulatory phenotype in naïve CD4+ CD25- T than CD11C+ isolated from naïve mice. Taken together, our results suggest that AhR activation by kynurenine during acute phase of sepsis is important to IDO expression in a specific CD11C+. This new sepsis-induced CD11C+ IDO+ population is important to Treg cells expansion and immunosuppression development in sepsis-surviving mice.

Electron transfer, ET, and proton-coupled electron transfer, PCET, reactions are central to biological reactions involving catalysis, energy conversion and energy storage. The movement of electrons and protons in either a sequential or concerted manner are coupled in a series of elementary reaction steps in respiration and photosynthesis to harvest and convert energy consumed in foodstuffs or by absorption of light into high energy chemi-cal bonds in the form of ATP. These electron transfer processes may be modulated by conformational dynamics within the protein matrix or at the protein-protein interface, the energetics of which are still not well understood. Photoacoustic calorimetry is an estab-lished method of obtaining time-resolved reaction enthalpy and volume changes on the nanosecond to microsecond timescale. Photoacoustic calorimetry is used here to probe 1) the energetics and volume changes for ET between the self-assembled anionic uroporphy-rin:cytochrome c complex and the role of the observed volume changes in modulating ET within the complex, 2) the enthalpy and volume change for the excited state PCET reac-tion of a tyramine functionalized ruthenium(II) bis-(2,2'-bipyridine)(4-carboxy-4'-methyl-2,2'-bipyrine) meant to be a model for the tyrosine PCET chemistry carried out by cyto-chrome c oxidase and photosystem II, 3) the enthalpy and volume changes related to car-bon monoxide and tryptophan migration in heme tryptophan catabolic enzyme indoleam-ine 2,3-dioxygenase.

A artrite reumatoide (AR) é a artropatia inflamatória mais prevalente no mundo, de etiologia multifatorial e fenótipos heterogêneos. Busca-se, além de definir fatores etiológicos, compreender as interações entre mecanismos envolvidos na fisiopatologia da AR. Entre estes, fatores genéticos, tanto genes do antígeno leucocitário humano (HLA), especialmente a presença do epítopo compartilhado (Shared epitope - SE) do HLA-DRB1, como genes não-HLA, e fatores ambientais e epigenéticos têm sido associados à doença. Assim, a identificação de novos fatores relacionados à etiopatogenia da AR e suas possíveis associações com características clínicas motivaram esse estudo. Um estudo caso-controle foi desenhado e dividido em duas etapas. Para a primeira etapa, foi obtido plasma de 18 indivíduos de AR e 18 voluntários saudáveis de Ribeirão Preto, no qual foram identificados quinurenina (Kyn), Trp, serotonina (5-HT) e taxa Kyn/Trp (KTR) por cromatografia líquida de ultra-eficiência (CLUE) acoplada a espectrômetro de massas sequencial (CLUE-DAD-EM/EM). Na segunda etapa, de estudo genético, uma coorte formada por 328 indivíduos com AR e por 234 voluntários saudáveis de Ribeirão Preto e de Porto Alegre foi avaliada quanto ao polimorfismo do gene da enzima indoleamine 2,3-dioxigenase 1 (IDO1). Foram obtidos dados clínicos e epidemiológicos e coletadas amostras de sangue periférico para extração de DNA pelo método de salting-out. Em seguida, tipificação HLA e reação em cadeia de polimerase (RCP) das variantes da IDO1, rs7820268, rs3739319, rs61753677, rs35059413, rs35099072 e rs9298586, foram realizadas. A positividade para fator reumatoide (FR) em indivíduos com AR foi associada ao tabagismo (p= 0.0002) e ao SE (p < 0.0001), e para anticorpo antipeptídeo citrulinado cíclico (anti-CCP), associado ao SE (p < 0.0001). Quando combinadas a presença de SE e a de tabagismo, houve associação estatisticamente significante para FR (p < 0.001) e para anti-CCP (p = 0.03). Foram observadas menores concentrações plasmáticas de 5-HT em indivíduos com AR quando comparados a voluntários saudáveis (p =0.006), mas sem diferença para níveis de Trp, Kyn e KTR. Para estes, diferenças apareceram quando avaliados subgrupos. Em indivíduos com AR sem tratamento com drogas modificadoras do curso da doença (DMCDs), os valores plasmáticos de Trp foram menores quando comparados aos em terapia (p = 0.0016), enquanto em pacientes com AR tabagistas os valores de Kyn e KTR foram menores que em pacientes não tabagistas (p = 0.039 e p = 0.032, respectivamente). Não foram identificadas associações estatisticamente significantes entre as variantes genéticas estudadas e o risco de desenvolver AR, nem entre os polimorfismos da IDO1 estudados e a concentração plasmática de Trp, Kyn e 5-HT e KTR. Este estudo não identificou relação das variantes do gene da IDO1 com suscetibilidade para AR. Assim, novos estudos são necessários para que possam ser explicadas as associações encontradas na via das Kyns e na 5-HT em etiopatogenia da AR. / Rheumatoid arthritis (RA) is the most prevalent inflammatory arthropathy in the world, with multifactorial etiology and heterogeneous phenotypes. Besides defining etiological factors, it is sought to understand the interactions between mechanisms in RA pathophysiology. About these, genetic factors, both human leucocity antigen (HLA) genes, especially the HLA-DRB1 Shared epitope (SE) presence, and not-HLA genes, and environmental and epigenetics factors have been associated with the disease. Therefore, the aim of this study was to identify new possible associations between RA clinical features and its etiopathogenesis. A case-control study was designed and it was divides in two phases. The first phase, it was obtained plasma of 18 RA patients and 18 healthy controls from Ribeirão Preto to identify the kynurenine (Kyn), Trp and serotonin (5-HT) concentrations and Kyn/Trp ratio (KTR) by ultra-high performance liquid chromatography coupled to sequential mass spectrometer. The second phase was a genetic study that evaluated a cohort of 328 RA patients and 234 healthy volunteers from Ribeirão Preto and Porto Alegre about the indoleamine 2,3-dioxygenase 1 (IDO1) gene polymorphism. Clinical and epidemiological data were obtained and peripheral blood samples were collected to DNA extraction by salting-out method. Then, HLA typification and polymerase chain reaction to identify IDO1 genetic variants rs7820268, rs3739319, rs61753677, rs35059413, rs35099072 and rs9298586 were performed. Rheumatoid factor (RF) positivity was associated to smoking (p = 0.0002) and SE (p < 0.0001), and cyclic citrullinated peptide autoantibodies (anti-CCP) positivity was associated to SE (p < 0.0001). When SE presence and smoking were combined, there was statistically significant association to RF (p < 0.001) and anti-CCP (p = 0.03). We observed lower plasma 5-HT concentrations in RA patients than in healthy volunteers (p = 0.006), but no significant difference to Trp, Kyn and KTR levels. For these, differences were observed when subgroups were evaluated. In RA patients not using disease modifying antirheumatic drugs (DMARDs) the plasma Trp levels were lower than RApatients using DMARDs, while the plasma Kyn concentrations and KTR in smokers RA patients were lower than nonsmokers RA patients (p = 0.039 and p = 0.032 respectively). We did not indetify statistically significant associations neither between studied genetic variants and risk to develop RA nor between IDO1 polymorphisms and plasma Trp, Kyn, 5HT concentrations and KTR. This study did not identify relation between IDO1 genetic variants with susceptibility to RA. Therefore, new studies are necessary to explain the searched associations between Kyns pathway and 5-HT in RA etiopathogenenesis.

Macrófagos recuperados de peritôneo de camundongos 48 horas após administração de concanavalina A (Con A) são ativados e possuem um conteúdo maior de mieloperoxidase (MPO) do que macrófagos residentes. Este aumento pode ser observado por immunobloting e pelo aumento da atividade peroxidásica. Esta atividade foi avaliada pela quimiluminescência desencadeada por homogenato de macrófagos e peróxido de hidrogênio, durante a oxidação de luminol e melatonina. Os macrófagos contendo MPO são capazes de gerar ácido hipocloroso quando estimulados com acetato de forbol miristato (PMA). O aumento do conteúdo de MPO em macrófagos é um processo que independe de interferon-&#947; (IFN-&#947;), uma vez que camundongos IFN-&#947;-\"knockout\" têm uma atividade ainda maior que camundongos \"wild type\". O contato entre macrófagos e neutrófilos in vivo, não é necessário para o incremento observado, visto que macrófagos de camundongos tratados com anticorpo anti-granulócitos preservam a atividade peroxidásica. Este achado é uma evidência de que em algumas condições a ativação de macrófagos é acompanhada por um aumento da atividade peroxidásica. Dentre o amplo espectro de ação da MPO, este processo pode ter um papel especial na inflamação. Também é de especial interesse o fato de macrófagos serem capazes de oxidar melatonina, um hormônio com vários efeitos imunomodulatórios. / Macrophages recovered from mice peritoneum 48 after concanavalin-A (Con A) administration are primed and have a higher content of myeloperoxidase (MPO) than resident cells. The increase of MPO is accompanied by an increment in peroxidase activity, evaluated by the chemiluminescence during the oxidation of luminol and melatonin triggered by macrophages homogenates plus hydrogen peroxide. MPO present in macrophages are able to generate hypochlorous acid when macrophages are stimulated with phorbol myristate acetate. Interferon-&#947; (IFN-&#947;) does not participate in the process that lead macrophages to become enriched in MPO, since IFN-&#947; knockout mice have an even higher peroxidase activity compared to the wild type. The contact of macrophages with neutrophil in vivo seems to be not necessary for the observed increment in peroxidase since macrophages recovered from mice treated with antigranulocyte antibody preserves the peroxidase activity. These finding provides evidences that, at least in some conditions, macrophage activation is accompanied by an increment in peroxidase activity. Given the broad spectrum of action of MPO, this process might play a special role in inflammation. Also of special interest is the finding that macrophages are able to oxidize melatonin, a hormone with several immunomodulatory effects.

O tubarão-azul (Prionace glauca) é uma espécie que apresenta desenvolvimento vivíparo placentário em que o saco vitelino se desenvolve ao longo da gestação tornando-se uma placenta que executa função matrotrófica. A Indoleamina 2,3-dioxigenase (IDO) é uma proteína encontrada em mamíferos nos quais participa, além de outras funções, da tolerância materno-fetal, sendo também encontrada em peixes ósseos. Assim, a proposta deste trabalho foi verificar a expressão da IDO na interface materno-fetal de Prionace glauca e descrever sua localização. Para tanto, material placentário/uterino e embriológico de três fases distintas da gestação (pré-placenta, meia gestação e fim da gestação) de fêmeas de P. glauca foram coletados e processados para a imuno-histoquímica. Os resultados mostraram a presença da IDO ao longo do desenvolvimento do saco vitelino/placenta, na ectoderme nas três fases e na endoderme apenas nas duas primeiras fases. No epitélio uterino observou-se a marcação da IDO nas duas últimas fases. Esses tecidos de interface seriam locais de maior contato entre a mãe e o concepto, fato que poderia levar à indução de uma resposta imunológica contra o concepto semi-alogenêico. A soma destes fatores poderia contribuir como um indício de uma possível atuação da IDO como mecanismo da tolerância materno-fetal na interface placentária de Chondrichtyes, como relatado em mamíferos eutérios / The blue shark (Prionace glauca) is a viviparous placentary species in which the yolk sac develops along pregnancy turning into a placenta with a matrotrofic role. The indoleamine 2 3-dioxygenase (IDO) is a protein usually described in mammals, which, among other functions, participates on the maternal-fetal tolerance process. Although it has also been reported in bony fish, no information is available regarding its function. Therefore, the purpose of this study was to investigate the expression of IDO in blue shark maternal-fetal interface and describe its distribution. Thus, placental / uterine and embryonic materials from three different stages (pre-placenta, middle and late gestation) of pregnant P. glauca females were processed for immunohistochemistry. The results showed IDO labelling during the yolk sac / placenta development in ectoderm along the three development phases and at endoderm only at phases I and II. In uterine epithelium, IDO was observed in the last two phases. These interface tissues are major contact areas between the mother ant the conceptus, that would induce an immunological response against the semialogeneic conceptus.The sum of these factors may contribute as an indication to the possible IDO role as a mechanism of maternal-fetal tolerance in Chondrichtyes placentary interface, as described in eutherian mammals

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-17T21:35:19Z No. of bitstreams: 1 Mario Cezar Saffi Junior.pdf: 600477 bytes, checksum: 389162272a6d7d9eb1d1a097d21a6d9a (MD5) / Made available in DSpace on 2016-05-17T21:35:19Z (GMT). No. of bitstreams: 1 Mario Cezar Saffi Junior.pdf: 600477 bytes, checksum: 389162272a6d7d9eb1d1a097d21a6d9a (MD5) Previous issue date: 2015-01-22 / The cervical cancer is the first cancer of the female genital tract in Brazil and HPV is essential factor for carcinogenesis. The Brazilian program tracking proposes conventional cervical cytology as the primary method to detect cervical cancer, despite its low sensitivity. Risk factors associated with the spread of HPV are despised and not rely on a biomolecular tool that can increase the program offered by the Ministry of Health. The aim of this study was to determine whether the risk factors for cervical cancer may contribute to the conventional cervical cytology to increase diagnostic sensitivity and assess whether the mRNA indoleamine 2,3 dioxygenase (IDO) and mRNA CDKN2A / p16 may increase the diagnostic yield of this neoplasm. The logistic regression analysis was based on clinical variables (risk factors), cytological and biomolecular to seek an association with pathological results. The proportion of explained variance (PVE) for each variable studied was calculated by the formula omega, whereas the sensitivity, specificity, positive predictive value and negative predictive value were calculated by the formulas of Galen and Gambino. We conclude that oral contraceptive showed greater predictive power of high-grade lesions compared to other risk factors, and that both the IDO mRNA as CDKN2A mRNA / p16 may help screening of cervical cancer, either when used alone, or in conjunction with conventional cervical cytology, increasing their sensitivity and maintaining a considerable specificity. / O câncer de colo uterino apresenta-se como a principal neoplasia do trato genital feminino no Brasil, sendo o HPV fator essencial para a carcinogênese. O programa brasileiro de rastreamento propõe a citologia oncológica cervical convencional como principal método para detectar o câncer do colo uterino, apesar da sua baixa sensibilidade. Os fatores de risco associados ao contágio do HPV são desprezados e não contamos com uma ferramenta biomolecular que possa incrementar o programa oferecido pelo Ministério da Saúde. O objetivo desse trabalho foi verificar se os fatores de risco para o câncer de colo uterino podem contribuir com a citologia oncológica cervical convencional para aumentar a sensibilidade diagnóstica e avaliar se o RNAm Indoleamine 2,3 dioxigenase (IDO) e o RNAm CDKN2A/p16 podem incrementar a capacidade diagnóstica dessa neoplasia. A análise de regressão logística foi baseada nas variáveis clínicas (fatores de risco), citológicas e biomoleculares a fim de buscar uma associação com o resultado anatomopatológico. A proporção de variação explicada (PVE) por cada uma das variáveis estudada foi calculada pela fórmula ômega, enquanto que a sensibilidade, especificidade, valor preditivo positivo e o valor preditivo negativo foram calculados pelas fórmulas de Galen e Gambino. Concluímos que o uso do contraceptivo oral mostrou um maior poder de predição de lesões de alto grau em relação aos demais fatores de risco, e que tanto a RNAm IDO quanto o RNAm CDKN2A/p16 poderem auxiliar no rastreamento do câncer de colo uterino, seja quando usados de forma isolada, seja conjuntamente com a citologia cervical convencional, elevando sua sensibilidade e mantendo uma considerável especificidade.

Il existe plusieurs défis au développement d’une thérapie visant à stimuler l’immunité cellulaire. Dans la prévention contre certains virus et en immunothérapie du cancer, l’induction de lymphocytes T spécifiques est cependant primordiale. Dans la première partie de l’étude, nous avons porté notre attention sur la compréhension de la présentation croisée par le complexe majeur d’histocompatibilité de classe I (CMH I) médiée par des particules pseudo-virales (VLP) composées de la protéine de surface de potexvirus à laquelle nous avons ajouté un épitope de la protéine M1 du virus de l’influenza ou un épitope de la protéine gp100 du mélanome. Cette VLP se caractérise par sa capacité à stimuler, sans l’aide d’adjuvant, le système immunitaire et de présenter de façon croisée l’épitope inséré dans sa protéine de surface et ce, indépendamment de l’activité du protéasome. Nous avons, tout d’abord, comparé les propriétés de présentation antigénique croisée des VLP formées du virus de la mosaïque de la malva (MaMV) à celles des VLP du virus de la mosaïque de la papaye (PapMV). Les résultats confirment que ces propriétés sont partagées par plusieurs membres de la famille des potexvirus malgré des divergences de séquences (Hanafi et al. Vaccine 2010). De plus, nous avons procédé à des expériences pour préciser le mécanisme menant à la présentation de l’épitope inséré dans les VLP de PapMV. Les résultats nous confirment une voie vacuolaire dépendante de l’activité de la cathepsine S et de l’acidification des lysosomes pour l’apprêtement antigénique. L’induction de l’autophagie par les VLP semble également nécessaire à la présentation croisée par les VLP de PapMV. Nous avons donc établi un nouveau mécanisme de présentation croisée vacuolaire dépendant de l’autophagie (Hanafi et al. soumis Autophagy). En second lieu, en immunothérapie du cancer, il est aussi important de contrôler les mécanismes d’évasion immunitaire mis en branle par la tumeur. Nous avons spécifiquement étudié l’enzyme immunosuppressive indoleamine 2,3-dioxygénase (IDO) (revue de la littérature dans les tumeurs humaines; Hanafi et al. Clin. Can. Res 2011) et son inhibition dans les cellules tumorales. Pour ce faire, nous avons tenté d’inhiber son expression par la fludarabine, agent chimiothérapeutique précédemment étudié pour son activité inhibitrice de l’activation de STAT1 (signal transducers and activators of transcription 1). Étonnamment, nos résultats ont montré l’inhibition d’IDO dans les cellules tumorales par la fludarabine, indépendamment de l’inhibition de la phosphorylation de STAT1. Nous avons démontré que le mécanisme d’action dépendait plutôt de l’induction de la dégradation d’IDO par le protéasome (Hanafi et al. PlosOne 2014). Les travaux présentés dans cette thèse ont donc portés autant sur la compréhension d’une nouvelle plateforme de vaccination pouvant médier l’activation de lymphocytes T CD8+ cytotoxiques et sur le contrôle d’une immunosuppression établie par les cellules tumorales pour évader au système immunitaire. Ces deux grandes stratégies sont à considérer en immunothérapie du cancer et la combinaison avec d’autres thérapies déjà existantes pourra permettre une meilleure réponse clinique. / There are several challenges to the development of therapies aimed at stimulating cellular immunity. In viral infection prevention and in cancer immunotherapy, the induction of specific T lymphocytes is, however, of paramount importance. In the first part of this study, we focused our attention on understanding the major histocompatibility complex class I (MHC I) cross-presentation mediated by virus-like particles (VLP) composed of potexvirus coat protein, in which we had inserted an epitope from the M1 protein of the Influenza virus or an epitope from gp100, a tumour antigen of melanoma. This particular VLP is characterized by its ability to stimulate the immune system with no adjuvant and its cross-presentation of the inserted epitope independently of proteasome activity. First, we compared the antigenic cross-presentation properties of Malva mosaic virus (MaMV) VLPs to that of Papaya mosaic virus (PapMV) VLPs. The results confirm that cross-presentation mechanisms are shared among different members of the potexvirus family despite marked differences in their sequences (Hanafi et al. Vaccine 2010). Furthermore, we have conducted experiments to clarify the mechanism leading to the cross-presentation of the inserted epitope in PapMV VLPs. The results confirm a vacuolar pathway dependent on cathepsin S activity and on lysosomal acidification for antigen presentation. Autophagy induction by VLPs is also important to PapMV VLP antigen cross-presentation. We have herein described a new vacuolar MHC I cross-presentation pathway dependent on autophagy (Hanafi et al. in preparation). Secondly, in cancer immunotherapy, it is crucial to control immune evasion mechanisms that are initiated by the tumour. We have specifically studied the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) (human cancer literature review in Hanafi et al. Clin. Can. Res. 2011), and its inhibition in tumour cells. To this end, we inhibited its expression using fludarabine, a chemotherapeutic agent previously studied for its inhibitory effect on STAT1 (signal transducers and activators of transcription 1) phosphorylation. Surprisingly, our results demonstrate that IDO inhibition in cancer cells by fludarabine was independent of STAT1 phosphorylation. We showed that the mechanism of action was rather dependent on the induction of IDO degradation by the proteasome (Hanafi et al. PlosOne 2014). The work presented in this thesis provides a better understanding of how a new vaccine platform can mediate cytotoxic CD8+ T lymphocytes activation and the control of the problem of immunosuppression by tumour cells for the evading of the immune system. These two main strategies are to key for the consideration of cancer immunotherapy in combination with other existing therapies, as these should allow a better clinical response to cancer treatment.

Neste estudo avaliamos o papel do metabolismo do triptofano (Trp) na homeostasia, na vaginose bacteriana e nas lesões cervicais associadas ao HPV. A importância do metabolismo do Trp se deve a sua ação na proliferação de microrganismos e de células do sistema imune. O consumo de triptofano tem sido identificado como uma forma de controlar o crescimento bacteriano limitando a infecção. Por outro lado, a oxidação de Trp produz quinurenina (QUIN), que tem papel chave na tolerância imunológica. A formação de QUIN se dá através das enzimas indoleamina 2,3-dioxigenase (IDO) e triptofano 2,3- dioxigenase (TDO). A mais estudada delas no âmbito das infecções/ imuno escape é a enzima IDO. Mais recentemente, tem-se dado ênfase ao papel da TDO no câncer. Nesta dissertação, o interesse foi avaliar a expressão da IDO no epitélio cervicovaginal de mulheres com vaginose bacteriana e de IDO e TDO em amostras cervicais de mulheres com diferentes graus de lesão cervical associada ao HPV. Foram incluídas 165 mulheres atendidas no CAISM/UNICAMP, as quais foram divididas em dois grupos: grupo caso composto por mulheres com lesão de baixo ou alto grau e carcinoma invasor (n=42) e grupo controle composto por mulheres com citologia oncológica normal, independente de apresentar infecção genital (n=123). IDO foi avaliada por imunocitoquímica em citologia em base líquida e IDO e TDO em biópsias cervicais. Mulheres com vaginose bacteriana apresentaram expressão aumentada de IDO em células escamosas em comparação às mulheres sem vaginose bacteriana (OR=7.41; IC 95%= 2.50 a 21.4; p <0.0001). No epitélio vaginal normal com ou sem infecção por HPV houve uma expressão leve de IDO em células escamosas. Na presença de lesões ou carcinoma, houve um aumento no número de células escamosas displásicas e de leucócitos IDO-positivos; aumento de IDO também pôde ser observada em culturas de pele organotípicas transduzidas com as oncoproteínas E6/ E7 do HPV16. Nas lesões cervicais, assim como visto para a IDO, a TDO esteve expressa em leucócitos, especialmente os infiltrados na região estromal e na parede dos vasos sanguíneos. A expressão basal de IDO no epitélio cervical normal e sua regulação positiva na infecção por HPV e lesões associadas sugerem a participação do metabolismo do Trp nos mecanismos imunossupressores envolvidos na doença. Embora o papel do IDO já tenha sido abordada anteriormente, até onde sabemos esta é a primeira evidência da expressão de TDO no epitélio vaginal, na neoplasia intraepitelial cervical e carcinoma de células escamosas. Ainda, em leucócitos, especialmente aqueles com morfologia típica de polimorfonucleares, parecem ser importantes fontes de IDO na cérvix uterina. / In this study we evaluated the role of tryptophan (Trp) metabolism in cervix homeostasis, bacterial vaginosis and HPV-associated lesions. The importance of Trp metabolism is due to its action on microorganisms and immune cells. Tryptophan consumption has been identified as a way to controlling bacterial growth limiting infection. On the other hand, the oxidation of Trp produces kynurenine (Kyn) which plays a key role in immunological tolerance. The formation of Kyn occurs through the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). IDO is the most studied of them within the context of infections / immune escape. More recently, TDO has also been considered in studies of cancer progression. In this thesis, we were interested in cervicovaginal epithelium IDO expression in women with bacterial vaginosis and of IDO and TDO in cervical samples of women with different degrees of cervical lesion associated with HPV. A total of 165 women attended at CAISM/UNICAMP were divided into two groups: a case group composed of women with low or high grade lesions and invasive carcinoma (n = 42) and a control group composed of women with normal cytology, independent to present genital infection (n =123). IDO was evaluated by immunocytochemistry in liquid-based cytology and IDO and TDO in cervical biopsies. Women with bacterial vaginosis had increased IDO expression in squamous cells compared to women without bacterial vaginosis (OR = 7.41, 95% CI = 2.50- 21.74; p<0.0001). In normal vaginal epithelium with or without HPV infection there was a mild IDO expression in squamous cells. In the presence of cervical intraepithelial lesions or squamous cell carcinoma, there was an increase in the number of IDO-positive dysplastic squamous cells and leukocytes; increase in IDO can also be observed in organotypic skin cultures transduced with HPV-16 E6/E7 oncoproteins. In cervical lesions, as observed for IDO, TDO was expressed in leukocytes, especially infiltrates in the stromal region and in the wall of blood vessels. The basal expression of IDO in the normal cervical epithelium and its positive regulation in HPV infection and associated lesions suggests the participation of Trp metabolism in the immunosuppressive mechanisms involved in the disease. Although some previous data have already considered the role of IDO, as far as we know this is the first evidence of the participation of TDO in the vaginal epithelium, cervical intraepithelial neoplasia and squamous cell carcinoma. In addition, in leukocytes, especially those with a typical polymorphonuclear morphology, appear to be important sources of IDO in the uterine cervix.

Introduction: Coronary artery disease (CAD) has been associated with depressive symptoms and deficits in cognitive performance, both of which have been associated with poorer medical prognoses and poorer psychosocial outcomes. Physical activity can improve cognitive and depressive symptoms, and, for those with CAD, improve medical prognoses. It was hypothesized that depressive symptoms and poorer cognitive performance would be associated with poorer cardiopulmonary fitness in patients with CAD, and that these sequelae would be associated prospectively with noncompletion of cardiac rehabilitation (CR). The benefits of physical activity are thought to result, in part, from decreased inflammatory activity and increased adaptive neural plasticity, to which the ratio of kynurenine to tryptophan (K/T) and brain derived neurotrophic factor (BDNF), respectively, in peripheral blood may pertain. Methods and Results: In a cohort study of patients entering CR, depressive symptoms (Center for Epidemiological Studies Depression scale; CES-D scores) were associated with cardiopulmonary fitness (peak volume of oxygen uptake; VO2Peak) during an exercise stress test (B=-.404, p=.001, n=366). The VO2Peak was also associated with performance across multiple cognitive domains, but most strongly with performance on tests involving executive function, attention and psychomotor processing speed (β=.322, p=.002 for composite score, n=81) in a cohort of patients entering CR. In prospective cohort studies, Major Depressive Disorder (adjusted hazard ratio [HR] 2.5, 95% confidence interval [CI] 1.3–4.7, n=195) and poorer performance on a verbal memory test (HR 0.86, 95% CI 0.77-0.96, p=.009, n=131) predicted non-completion of CR. In patients undertaking CR, higher serum K/T ratios were associated with CES-D scores (β=.322, p=.002, n=95) and with VO2Peak (β=-.391, p<.001, n=95), and in a cohort of patients entering CR (n=88), serum concentrations of BDNF were associated with psychomotor processing speed (F1,87=9.620, p=.003), overall cognitive status (Mini Mental Status Exam) scores (F1,87=15.406, p<.0005) and VO2Peak (β=.305, p=.013). Conclusions: Depressive symptoms and poorer cognitive performance are clinically important in patients with CAD entering CR and they are both associated with poorer cardiopulmonary fitness. Poorer cardiopulmonary fitness was also associated with higher K/T ratios and with lower BDNF concentrations in serum, which predicted depressive symptoms and poorer cognitive performance, respectively.

Introduction: Coronary artery disease (CAD) has been associated with depressive symptoms and deficits in cognitive performance, both of which have been associated with poorer medical prognoses and poorer psychosocial outcomes. Physical activity can improve cognitive and depressive symptoms, and, for those with CAD, improve medical prognoses. It was hypothesized that depressive symptoms and poorer cognitive performance would be associated with poorer cardiopulmonary fitness in patients with CAD, and that these sequelae would be associated prospectively with noncompletion of cardiac rehabilitation (CR). The benefits of physical activity are thought to result, in part, from decreased inflammatory activity and increased adaptive neural plasticity, to which the ratio of kynurenine to tryptophan (K/T) and brain derived neurotrophic factor (BDNF), respectively, in peripheral blood may pertain. Methods and Results: In a cohort study of patients entering CR, depressive symptoms (Center for Epidemiological Studies Depression scale; CES-D scores) were associated with cardiopulmonary fitness (peak volume of oxygen uptake; VO2Peak) during an exercise stress test (B=-.404, p=.001, n=366). The VO2Peak was also associated with performance across multiple cognitive domains, but most strongly with performance on tests involving executive function, attention and psychomotor processing speed (β=.322, p=.002 for composite score, n=81) in a cohort of patients entering CR. In prospective cohort studies, Major Depressive Disorder (adjusted hazard ratio [HR] 2.5, 95% confidence interval [CI] 1.3–4.7, n=195) and poorer performance on a verbal memory test (HR 0.86, 95% CI 0.77-0.96, p=.009, n=131) predicted non-completion of CR. In patients undertaking CR, higher serum K/T ratios were associated with CES-D scores (β=.322, p=.002, n=95) and with VO2Peak (β=-.391, p<.001, n=95), and in a cohort of patients entering CR (n=88), serum concentrations of BDNF were associated with psychomotor processing speed (F1,87=9.620, p=.003), overall cognitive status (Mini Mental Status Exam) scores (F1,87=15.406, p<.0005) and VO2Peak (β=.305, p=.013). Conclusions: Depressive symptoms and poorer cognitive performance are clinically important in patients with CAD entering CR and they are both associated with poorer cardiopulmonary fitness. Poorer cardiopulmonary fitness was also associated with higher K/T ratios and with lower BDNF concentrations in serum, which predicted depressive symptoms and poorer cognitive performance, respectively.