Mental disorders present difficulties in the research of their mechanisms, considering the high levels of comorbidity and the lack of specific neuroscience data to evaluate them. Estipulating deficit circuits in the disorders and the best treatment is a complex task, given the limited comprehension of the factors that correlate to the disorders. The utilization of biomarkers has proved an efficient and reliable alternative to provide precise diagnosis. Among the biomarkers, the Error-Related Negativity component, an event-related cortical potential, has presented high indexes of stability and validity in correlating to anxiety, obsessive, and mood-related mental disorders. The present dissertation evaluated irritability traits in a community sample, using a Flanker task, that has consistently elicited Error-Related Negativity according to the literature. Our results corroborate literature and found a frontocentral negativity, that peaked around 100ms after the commission of an error in the Flanker Task. However, our manipulation of negative feedback did not support literature, and ERN amplitudes were less enhanced post negative feedback. The relationship between irritability and ERN remains unclear. Future studies should, therefore, address these questionings.

L’allergie ou l’hypersensibilité de type I est une réponse inappropriée du système immunitaire à une substance étrangère à l’organisme, nommée « allergène ». L’immunothérapie allergénique (ITA) est actuellement le seul traitement sur le marché qui permet de traiter l’étiologie de la maladie allergique par opposition aux traitements symptomatiques qui diminuent temporairement les manifestations allergiques. Son action consiste à réduire la sensibilité de l’organisme vis-à-vis de l’allergène en modulant progressivement la réponse immunitaire dirigée contre ce dernier. L’objectif de cette thèse était de définir des biomarqueurs d’efficacité clinique utilisables dans le cadre des traitements de l’ITA. La stratégie de recherche est basée sur une hypothèse qui consiste à suggérer que les cellules dendritiques (DCs) sont impliquées dans le succès de l’immunothérapie. En particulier, nous supposons que le traitement induit une baisse des DCs de type 2 (DC2), qui induisent des lymphocytes T auxiliaires de type 2 (TH2), et une augmentation des DCs régulatrices (DCreg), qui induisent des lymphocytes T régulateurs. La première partie de cette thèse a consisté à mettre au point des conditions de culture induisant des DC2. Pour cela, un criblage de molécules biologiques et pharmacologiques a été entrepris sur les DCs dérivées des monocytes afin d’induire in vitro des DC2 et a conduit à la mise au point d’un mélange de plusieurs molécules, dont certaines sont impliquées dans les mécanismes de l’allergie. Le phénotype des DC2 obtenu a été étudié ainsi que la polarisation des lymphocytes T induite après co-cultures en comparaison avec des DCs de type 1 (DC1) et des DCreg.La deuxième partie de cette thèse a consisté à analyser, à l’échelle moléculaire, les différents types de DCs induites (DC1, DC2 et DCreg). Pour cela, deux techniques ont été utilisées, une analyse transcriptomique par puces à ADN et une analyse protéomique par spectrométrie de masse sans marquage, pour comparer le transcriptome et le protéome des DCs induites. Le différentiel d’expression des marqueurs les plus pertinents a été validé au niveau transcriptionnel et protéique.Dans la troisième partie de cette thèse, le suivi des marqueurs dans des cellules du sang de patients allergiques traités ou non par ITA lors d’une étude clinique randomisée, contrôlée, en double aveugle, a permis de définir six nouveaux candidats biomarqueurs d’efficacité de l’immunothérapie, dont trois spécifiques des DC2 et trois autres spécifiques des DCreg. Ces marqueurs pourront être suivis lors des traitements d’ITA pour distinguer les patients répondeurs des non-répondeurs. / Allergy or type I hypersensitivity is an inappropriate response of the immune system to a foreign substance in the body, called "allergen". Allergen immunotherapy (AIT) is currently the only treatment on the market that can handle the etiology of allergic disease versus symptomatic treatments that temporarily reduce allergic manifestations. Its action is to reduce the sensitivity of the body against allergens.The aim of this thesis was to define biomarkers of clinical efficacy of AIT. The research strategy is based on the following hypothesis: dendritic cells (DCs) are involved in the success of immunotherapy. In particular, we assume that the treatment induces a decrease in DCs type 2 (DC2), which induce type 2 helper T cells, and an increase of regulatory DCs (DCreg), which induce regulatory T cells.First, we defined optimal culture conditions inducing the polarization of in vitro immature monocyte-derived DCs (MoDCs) toward a DC2 pattern. After screening several biological and pharmaceutical agents, we selected a cocktail of six molecules with some of them are pro-allergenic molecules. The phenotype of those DC2 cells and the CD4+ T cell polarization induced after coculture were characterized extensively in comparison with type 1 DC (DC1) and DCreg.In a second part, we compared the transcriptomes and the proteomes of MoDCs polarized into DC1, DC2 and DCreg by using cDNA microarrays together with label-free mass spectrometry. The differential expression of the most relevant markers was confirmed at the transcriptional and protein level. In the third part, markers were also followed in the peripheral blood from allergic patients enrolled in a randomized, double-blind, placebo-controlled AIT study. The expression of three DC2 markers was down-regulated and of three DCreg markers was up-regulated in patients who responded to the treatment and correlated with clinical efficacy. These markers could be used as follow-up read-outs of AIT efficacy in order of to discriminate responders from nonresponders.

Les dernières avancées dans le domaine de l’imagerie par résonance magnétique permettent aujourd’hui de mieux comprendre l’anatomie et le fonctionnement du cerveau humain. L’IRM s’avère d’ailleurs aujourd’hui un outil clé pour la recherche de biomarqueurs d’imagerie dans la plupart des pathologies cérébrales. Nous nous sommes intéressés dans le cadre de cette thèse à l’étude de la connectivité anatomique des noyaux gris centraux, structures impliquées dans de nombreuses boucles cortico-sous-cortico-corticales, et dont l’atteinte est à l’origine de troubles moteurs à l’instar de la maladie de Huntington, du syndrome Gilles de la Tourette et de la maladie de Parkinson. Nous avons pour cela effectué plusieurs développements méthodologiques qui permettent de segmenter les noyaux gris centraux et d’inférer leur connectivité anatomique. Tout d’abord, nous avons développé une méthode de segmentation des noyaux gris centraux à partir de différents contrastes et capable de s’adapter à des cas pathologiques présentant une forte modification de ces structures. Ensuite, nous avons développé des méthodes robustes d’analyse et de sélection des fibres reliant les différentes structures cérébrales, obtenues à l’aide de méthodes de tractographie par IRM du processus de diffusion cérébrale. Ces nouvelles méthodes de sélection présentent l’avantage de tenir compte d’a priori anatomiques, et fournissent ainsi des résultats plus proches de la réalité que les résultats obtenus dans la littérature. Nous avons également développé une méthodologie permettant de construire des cartes de connectivité surfaciques afin de projeter les connexions des noyaux gris centraux sur la surface corticale et de comparer le profil de connectivité corticale des noyaux gris au sein d’une population et entre populations. Enfin, nous avons utilisé ces outils pour étudier les modifications putatives de la connectivité anatomique des noyaux gris centraux dans la maladie de Huntington et dans le syndrome Gilles de la Tourette. / The recent advances in magnetic resonance imaging helped understanding brain anatomy and function. Today, MR imaging is a key tool for inferring imaging-based biomarkers for most neuropathologies. In this work, we focused on the anatomical connectivity of the basal ganglia which are involved in several cortico-subcortical loops and which dysfunction is the origin of motor disorders like Huntington and Parkinson diseases and Gilles de la Tourette syndrome. We developed several tools allowing the segmentation of the basal ganglia and inferring their anatomical connectivity. First, we developed a method for deep nuclei segmentation using several contrasts and that was adapted for pathological cases presenting high modifications in the morphology of these structures. Second, we developed robust methods for the analysis and the selection of the fiber tracts linking different brain structures and obtained using dMRI and tractography methods. These novel tools have the advantage of taking into account anatomical prior knowledge. Therefore the obtained results are closer to the real anatomy than those obtained using the tools available in the literature. We also developed surface connectivity maps that project the cortical connections of the deep nuclei directly on the cortical surface and that allow the comparison of the connectivity profile of the deep nuclei between different subjects and different groups. Finally, we used these tools to study the putative modifications of the anatomical connectivity of the deep nuclei in the Huntington disease and Gilles de la Tourette syndrome.

O carcinoma basocelular (CBC) é uma neoplasia cutânea maligna de baixo potencial metastatizante, originada das células da camada basal da epiderme. Sua importância clínico-epidemiológica pode ser constatada pelo fato de constituir a neoplasia maligna mais comum na espécie humana, cujo principal fator etiológico é a exposição à radiação ultravioleta. Apesar da baixa incidência de metástases, a neoplasia pode adotar um comportamento localmente agressivo, com comprometimento de estruturas profundas e de forte apelo estético, como ocorre na região periocular. Uma das complicações advindas de sua infiltração neste sítio anatômico consiste na invasão de tecidos orbitários cujo tratamento é a exenteração, conduta mutiladora que consiste na retirada do globo ocular e das partes moles da órbita acometida. O objetivo deste estudo foi avaliar os casos de CBC com invasão orbitária que foram submetidos à exenteração no Hospital de Clínicas de Ribeirão Preto, no período de 1992 a 2012, para a possível identificação de fatores clínicos e morfológicos que possam predizer uma evolução desfavorável da neoplasia. Foi realizada uma coleta de dados clínicos, epidemiológicos e histopatológicos dos casos submetidos à exenteração a partir dos prontuários médicos dos pacientes. As lâminas referentes aos exames anátomo-patológicos foram revistas e foi realizado estudo imuno-histoquimico para os marcadores p53, bcl-2, actina de músculo liso e metaloproteinase-1. O grupo controle foi composto por pacientes com diagnóstico da neoplasia em topografia periocular, sem sinais de invasão orbitária. Para os casos com invasão orbitária o número de casos positivos marcados para p53 (0,21) e para actina de músculo liso (0,21) foi significantemente menor que o número de casos positivos para bcl-2 (0,63) e MMP-1 (0,58) (p= 0,0331). Entretanto, o número de casos positivos para bcl-2 (0,63) foi significantemente maior que o número de casos marcados por MMP-1 (0,58) (p=0,0126). Para os tumores sem invasão orbitária, o número de casos positivos para p53 (0,31) e actina (0,31) foi significantemente menor que o número de casos positivos para bcl-2 (0,63) eMMP-1 (0,50) (p=0,0273). Os resultados indicam que a invasão orbitaria por carcinoma basocelular palpebral ocorre com maior frequência no sexo masculino, em pacientes com longa história clínica de múltiplas lesões e submetidos a múltiplos procedimentos. Além disso, os marcadores estudados aparentemente não podem predizer um comportamento mais agressivo do tumor. / Basal cell carcinoma (BCC) is a malignant skin cancer of low metastasizing potential originated from the basal cells of the epidermis. Its clinical and epidemiological importance is evidenced by the fact that it is the most common malignancy in humans and it has as the main etiological factor the exposure to ultraviolet radiation. Despite the low incidence of metastases, the cancer can adopt a locally aggressive behavior with involvement of deep structures and it can have a strong aesthetic appeal, as in the periocular region. One of the complications arising from its infiltration in this anatomical site consists of orbital tissue invasion whose treatment is exenteration, a mutilating procedure consisting of the removal of the eyeball and the soft tissue of the affected orbit. The aim of this study was to evaluate the cases of BCC with orbital invasion that underwent exenteration at the Clinics Hospital of Ribeirão Preto Medical School, University of Sao Paulo, from 1992 through 2012, for possible identification of clinical and morphological factors that can predict an unfavorable evolution of the tumor. The clinical data were obtained from the patients\' charts and we have reviewed all the slides from exenteration specimens and performed immunohistochemical studies with p53, bcl-2, smooth muscle actin and metalloproteinase-1(MMP-1). The control group consisted of age-matched patients with eyelid basal cell carcinomas without orbital invasion. For cases with orbital invasion the number of positive cases labeled for p53 (0.21) and actin (0.21) was significantly lower than the number of positive cases for bcl-2 (0.63) and MMP -1 (0.58) (p = 0.0331). However, the number of positive cases for bcl-2 (0.63) was significantly greater than the proportion of positive cases for MMP-1 (0.58) (p = 0.0126). For cases without orbital invasion the number of positive cases for p53 (0.31) and actin (0.31) was significantly lower than the number of positive cases for bcl-2 (0.63) and MMP-1 (0.50) (p = 0.0273), even though the number of positive cases marked for MMP-1 (0.50) was not significantly different from number of positive cases for bcl-2 (0.63) (p = 0.059). The results indicate that orbital invasion of basal cell carcinoma of the eyelid was more frequent in male sex and that the patients have usually a long history of multiple lesions and were submitted to several procedures. In addition, our results suggest these markers can not predict an aggressive behavior for basal cell carcinomas of the periocular region.

O exercício físico tem sido proposto como uma terapia não farmacológica eficaz para a prevenção e tratamento de neoplasias, com destaque ao câncer de cólon. O presente estudo visa investigar o papel da glândula pineal sobre carcinogênese colônica experimental de ratos submetidos ao exercício físico Dessa forma, propusemos investigar os biomarcadores colônicos de câncer, foco de cripta aberrante (FCA), antígeno nuclear de proliferação nuclear (PCNA), expressão de ciclooxigenase-2 (COX-2) e Caspase 3. Além de parâmetros oxidativos hepáticos de peroxidaçao lipídica e gluationa reduzida (GSH). O Estudo foi conduzido através dos grupos experimentais controle (C), luz contínua (L), Exercício (E) e luz contínua associada ao Exercício (LE) e os mesmos grupos expostos ao carcinógeno químico 1,2-Dimetilhidrazina (DMH). Exercício físico foi realizado pelo modelo de natação, 5 dias por semana durante 10 semanas. Após o periodo de treinamento, os animais foram sacrificados, sendo coletadas amostras de sangue e fígado e colón para análises. O estudo do fígado revelou uma significativa influência do DMH na modulação dos parâmetros oxidativos. Já as análises colônicas dos FCAs e do PCNA mostraram-se controlados pelo exercício físico realizado em condições de normais do ritmo circadiano ao passo que em condições de desbalanço fisiológico da glândula pineal pela exposição luz constante, esses mesmos efeitos foram revertidos em comparação ao demais grupos (LED>LD>D>ED=p<0.001), aumento este acompanhado pela redução dos níveis plasmáticos de melatonina neste grupo. Em conclusão, nossos dados alertam para a forte influência da glândula pineal sobre o complexo de adapatações do exercício físico no tecido colônico de ratos expostos a um carcinógeno químico. / Physical activity has been proposed as a nonpharmacologic therapy for the prevention and treatment of cancer patients, with emphasis on colon cancer. The current study aims to investigate the role of the pineal gland on experimental colon carcinogenesis in rats submitted to physical exercise. We proposed to analyze cancer colonics biomarkers, Aberrant Foci Crypt (ACFs), proliferating cell nuclear antigen (PCNA) and Caspase-3, inflammation parameters cyclooxigenase-2 (COX-2) and liver oxidative enzymes reduced glutathione (GSH) and lipid peroxidation (MDA). Experimental design was constructed by control group (C), continuous light (L), exercise (E) and continuous light plus exercise (LE) and the same groups with chemical carcinogen 1,2 dimethyl-hydrazine (DMH). Exercise training groups were performed swimming exercise 5 d-wk1 for 10 wk. After training period rats were sacrificed. Blood samples and liver were collected to analyses and colon was processed for histological and immunohistochemistry examination. The study of the liver revealed a significant effect of DMH in the modulation of oxidative parameters. Since the analysis of colonic FCAs and PCNA were shown to be controlled by exercise carried out with a normal circadian rhythm while in conditions of physiological imbalance of the pineal gland by constant light exposure, these effects were reversed in comparison to other groups (LED> LD> D> ED = p <0.001), increase accompanied by reduced plasma levels of melatonin in this group. In conclusion, our data call attention to the strong influence of the pineal gland on the complex adaptations of physical exercise on colonic mucosa of rats exposed to a chemical carcinogen.

A poluição atmosférica interfere diretamente com a saúde populacional. Há evidências de que os animais de companhia representam sentinelas para fatores ambientais onde o homem e os demais animais estariam expostos. Animais podem ser usados como indicadores de poluição ambiental. O desenvolvimento industrial e urbano tem originado, em todo o mundo, um aumento crescente da emissão de poluentes atmosféricos. A complexa mistura de gases, partículas sólidas e substâncias químicas presentes no ar (&quot;indoor&quot; e &quot;outdoor&quot;) podem gerar a genotoxicidade, levando as alterações citogenéticas, mutações ou produção de adutos de DNA. O trabalho tem o objetivo de avaliar moforlogicamente 36 cães coletando o epitélio olfatório e pulmonar de cães saudáveis oriundos das diferentes regiões da cidade de São Paulo (zona Leste, Oeste, Norte e Sul). Analisou-se ainda, a quebra do DNA pela técnica do teste do Cometa (Eletroforese em gel de célula isolada) e Micronúcleo, marcar o aduto 8´hydroxy 2´deoxyguanosine pelo teste do Elisa (Enzyme linked immunosorbent assay), associando-os com o índice de poluição atmosférica (PM10), fornecidos pela CETESB. Não houve resultado significante na avaliação feita em escore de 0 a 3 pelo exame histopatológico e não há relação entre o índice de Material particulado Atmosférico e a lesão de DNA pelo Teste do Cometa no epitélio olfatório. Não há correlação entre idade e a lesão de DNA pelo Teste do Cometa no epitélio olfatório e pelo epitélio pulmonar. Há correlação significativa entre o índice de Material particulado Atmosférico e a lesão de DNA no epitélio pulmonar pelo Teste do Cometa. A utilização do epitélio pulmonar em cães no teste do COMETA demonstrou resultado significante, podendo ser indicado como um bioindicador do ar atmosférico. Este projeto mostrou uma tendência da zona Sul ser uma região menos poluída, onde há menor alteração histopatológica pulmonar e menor lesão de DNA em cães comparada à outras regiões com maior índice de material particulado. O teste do Micronúcleo foi padronizado, porém não mostrou resultado significante. O teste Elisa teve resultados opostos ao teste Cometa, sugerindo sua relação com o índice de ozônio. / The atmospheric pollution directly interferes with the population health. Evidences have been shown that pets represent sentinels for environmental factors which the man and the other animals would be susceptible. Animals can be used as indicatives of the environmental pollution. The industrial and urban developments have originated, in the whole world, an increase of the emission of atmospheric pollutants. The complex indoor &quot;mixture of gases, solid particles and chemical substances in the air can induce genotoxicity, leading the citogenetic alterations, mutations or production of DNA adducts. Thus, this work has the objective to evaluate morforlogically the olfactory and pulmonary epitelia of 36 healthy dogs of the different regions of the city of São Paulo (zone East, West, North and South). The DNA breaks was analyzed by the Comet assay (Eletroforese in gel of isolated cell) and Micronucleus. The analysis of the 8´hydroxy 2´deoxyguanosine adduct was performed by Elisa test (Enzyme linked immunosorbent assay). The parameters were associated with the index of atmospheric pollution (PM10), supplied for the CETESB. The histopathological evaluation of the epithelium included the graduation of 0 to 3. No relationship with the index of atmospheric particulated material with the level of DNA breaks by the Comet assay in the olfactory epithelium was obtained. It does not have correlation between age and the injury of DNA for the Test of the Comet in the olfactory epithelium and neither for the pulmonary epithelium. It has significant correlation between the index of atmospheric particulate material and the injury of DNA in the pulmonary epithelium for the Comet assay. The use of the pulmonary epithelium in dogs in the COMET assay resulted significant, being able to be indicated as a bioindicador of atmospheric air. This project showed that the South region of the Sao Paulo city is apparently a less polluted region. The animals from that region show minor pulmonary alterations and lesser injury of DNA, in comparison with dogs from other regions. The micronucleus test did not show significant results. The Elisa test showed opposite results when compared to the Comet assay, suggesting its relation with the ozone index.

A Síndrome de Sézary (SS) é um dos tipos mais comuns de linfoma cutâneo de células T (CTCL). Esta síndrome é caracterizada por eritrodermia, linfadenopatia generalizada e presença de células tumorais na pele, linfonodos e sangue periférico. A imunidade do hospedeiro está comprometida nesta síndrome, contudo, estudos relacionados à imunidade inata, particularmente sobre as células Natural Killer (NK) são mais escassos. As células NK exercem um papel essencial na resposta imune antitumoral. A proposta deste estudo é caracterizar o perfil fenotípico associado à ativação e inibição das células NK e a capacidade de secreção de citocinas em resposta aos agonistas de receptores Toll-like (TLR), TLR4, TLR7/TLR8 e TLR9 em pacientes com SS. Foram selecionados 14 pacientes com SS (7 homens, 7 mulheres), com 48-85 anos de idade provenientes do Ambulatório de Linfomas Cutâneos do Hospital das Clínicas da Faculdade de Medicina da USP, e um grupo controle com 22 indivíduos sadios (11 homens, 11 mulheres, idade de 48-75). Os resultados evidenciaram diminuição percentual de células NK CD56dim do sangue periférico dos pacientes SS, bem como baixa expressão de marcadores de ativação, NKG2D e NKG2C. A forma solúvel dos ligantes de NKG2D, MICB e MICA solúvel (sMICA/MICB), foi detectada em níveis séricos elevados para sMICB, e sMICA foi detectado em apenas 2 pacientes. Houve uma correlação negativa entre os níveis de sMICB e o percentual de células NK CD56brightCD16 NKG2D+. O perfil de secreção de citocinas das células NK, mostra, já na condição sem estímulo, um aumento percentual de TNF e CD107a em células NKCD56dim de pacientes com SS. Após o estímulo com os agonistas de TLR7/TLR8 houve um aumento na produção de CD107a e TNF nas células NKCD56bright e de TNF para as células NKCD56dim. Na produção de citocinas induzidas por agonistas de TLRs nos sobrenadantes de células mononucleares, foi observado que os agonistas TLR2 e TLR4 foram capazes de estimular a produção de IL-6, TNF e IL-10 enquanto o agonista de TLR7/8 mostrou um efeito adjuvante para a secreção de IFN-alfa, IFN-y e IL-10. Além disso, foi observado aumento de expressão de RNAm para TLR2 em células mononucleares avaliada por PCR em tempo real, como também para os fatores relacionados à resposta antiviral, como interferon tipo I (IFN- alfa), receptor de IFN-beta e interferon tipo III (IFN- ) em pacientes com SS. Quanto aos níveis séricos de citocinas, foi detectado aumento de IL-5 (citocina Th2), IL-6 (pró-inflamatória) e IL-10 (reguladora) nos SS, não diferindo quanto aos níveis de IL-1beta, IL-2, IL-17a, TNF e TGF-beta em relação ao grupo controle. Os resultados mostram que há uma diminuição percentual de células NK CD56dim, alteração da expressão dos receptores de ativação e elevação de ligantes solúveis que podem bloquear a ativação das células NK nos pacientes com SS. Além disto, as células NK exibem diminuição da produção de IFN-alfa, que contribuem na diminuição da resposta Th1. Há uma baixa responsividade das células NK aos ligantes de TLRs. Contudo, há um efeito adjuvante dos ligantes de TLRs em células mononucleares dos pacientes com SS, sendo que os agonistas de TLR2/4 e TLR7/8 foram capazes de restaurar parcialmente a produção de citocinas. Os resultados evidenciam que nos pacientes com SS, apesar da redução percentual das células NK, essas células são funcionais, mas que podem ser bloqueadas pelos ligantes solúveis de receptores de ativação. O uso de ligantes de TLRs pode ser uma estratégia para potencializar a produção de citocinas na Síndrome de Sézary / Sézary syndrome (SS) is one of the most common types of cutaneous T-cell lymphoma (CTCL). This syndrome is characterized by erythroderma, generalized lymphadenopathy and presence of tumor cells in the skin, lymph nodes and peripheral blood. Immunity of the host is compromised in syndrome, a study has related an innate immunity, particularly in Natural Killer (NK) cells are more scarce. NK cells play an essential role in the antitumor immune response. The aim of this study is to characterize the phenotypic profile associated with the activation and inhibition of NK cells and cytokine secretion capacity in response to the TLR4, TLR7 / TLR8 and TLR9 receptor agonists in SS patients. METHODS: We selected 14 patients with SS (7 men, 7 women, 48-85 years old) from the Cutaneous Lymphoma Outpatient Clinic of the Medical School of USP, and a control group with 22healthy individuals (11 men , 11 women, 48-75 years old). The results showed a decrease in CD56dim NK cells in the peripheral blood of SS patients, as well as low expression of activation markers, NKG2D and NKG2C. The soluble form of NKG2D, MICB and soluble MICA (sMICA/MICB) ligands was detected at high levels in serum for sMICB, and sMICA was detected in only 2 patients. There was a negative correlation between sMICB levels and the percentage of NK cells CD56brightCD16 NKG2D+. The cytokine secretion profile of NK cells, in the unstimulated condition, showed an increase of TNF and CD107a in NKCD56dim cells of SS patients. Upon stimulation of TLR7 / TLR8 agonists, CD107a and TNF production was higher in NKCD56bright cellsandforTNF in CD56dim cells.The agonists TLR2 and TLR4 in NK cells were able to stimulate the production of IL-6, TNF and IL-10,, while TLR7/8 agonist showed an adjuvant effect for secretion of IFN-alfa, IFN-y e IL-10. In addition, increased expression of TLR2 mRNA was observed in mononuclear cells evaluated by real-time PCR, simirlaly for factors related to antiviral response, such as interferon type I (IFN-alfa), IFN-Type III receptor (IFN - ) in patients with SS. Forserum levels of cytokines, IL- 5 (cytokine Th2), IL-6 (proinflammatory) and IL-10 (regulatory) were detectedhigher in SS, not differing in levels of IL-1beta, IL-2 , IL-17a, TNF and TGF-beta in relation to the control group. The results show that, there is a percentage decreased of CD56dim NK cells, alteration in activation receptor expression and elevation of soluble ligands that block theactivation of NK cells in SS patients. In addition, NK cells exhibit decreased IFN-? production, thatcontribute to the decrease in Th1 response. There is low responsiveness of NK cells to TLR ligands. However, there is an adjuvant effect of TLR ligands on mononuclear cells of SS patients, with TLR2/4 and TLR7 / 8 agonists is able to partially restore cytokine production. The results show that patients with SS, despite the percentage reduction of NK cells, these cells are functional, but can be blocked by soluble ligands of activation receptors. The use of TLR ligands may be a strategy to potentiate a production of cytokines in Sézary Syndrome

<p>Aluminum (Al) is the third most abundant metal on the earth’s surface. An association of Al with Alzheimer’s disease has been suggested, but, given limited human evidence, is controversial. It is possible that exploration of long-term, or cumulative, exposure to Al will help clarify this debate. Therefore, our study hypothesis was that occupational exposure to Al, particularly long-term exposure, is associated with adverse cognitive and olfactory outcomes. </p><p>Biomarkers are important tools in epidemiologic research; however, the reliability and quality of various biomarkers may vary. Therefore, in Chapter 2, we reviewed current evidence on Al biomarkers with the goal to synthesize current understanding regarding their utility for use in research. Our review evaluated 78 papers which discussed various biomarkers of Al exposure. Limited evidence is currently available for Al in nails, hair, breastmilk, saliva and semen. Although they have more daily variation than other biomarkers, blood Al concentrations may differentiate between exposed and non-exposed groups. Unlike blood Al, Al concentrations in both urine and bone are not subject to daily fluctuations. Bone Al appears able to quantify long term Al exposure. Therefore, the use of <i>in vivo</i>neutron activation analysis (IVNAA), a novel bone biomarker, can potentially represent cumulative Al exposure. </p><p>In Chapter 3, we evaluated the hypothesis that the correlation between bone and CEI measurements will be higher than the correlation between bone with fingernail. A cross-sectional cohort of 61 ferroalloy and manufacturing factory workers from Zunyi, China were used in this secondary analysis. Correlations of bone Al with other Al measures (fingernail and cumulative exposure index (CEI)) were assessed for 43 of the factory workers who had bone Al samples. Fingernail samples were analyzed using inductively coupled plasma mass spectrometry. Bone measurements were taken with a compact IVNAA developed by our research team. CEI was calculated as CEI_i=, where C_iis estimated Al exposure based on job title (low/medium/high) and Y_iis the years employed. The CEI was calculated for the prior 5, 10, 15, 20 years and lifetime work history. Median Al values were: 34.4 mg/g (IQR=46) fingernail; 15 mg/g (IQR=28) bone; and 26 (IQR=21) total CEI. Adjusting for age and education, the linear regression model suggests that bone Al is reflective of 15 years of exposure based off the significant association between bone Al and 15-year CEI (b=0.91, <i>p</i><0.02). Other models with CEI were not statistically significant.</p><p>In Chapter 4, we evaluated the association between bone, CEI and fingernail Al measurements with olfactory and cognitive function. The relationship between cognitive and olfactory function was compared with bone Al (N=43), fingernail Al (FnAl) (N=56), 5 year Al CEI and lifetime Al CEI (N=61) measurements. Olfactory function was assessed with the University of Pennsylvania Smell Identification Test and cognitive function was assessed with the WHO/UCLA Auditory Verbal Learning Test (AVLT). Verbal fluency was assessed using Fruit Naming and Animal Naming. Additional variables were assessed via questionnaire. Linear regression models, adjusted for age, education, current alcohol consumption and current smoking status, showed a significant association between higher BnAl and higher recall on the AVLT interference list (b=0.04, 95% Confidence interval (CI)=0.01, 0.08) and higher 5-year CEI with higher recall on the fifth AVLT trial (b=-0.23, 95% CI=-0.45, -0.01). No significant association between FnAl and lifetime CEI with olfactory and cognitive function was observed. </p><p>In Chapter 5, we explored the potential for interactions of Al with another metal to impact the potential association of Al with cognitive and olfactory function. Interaction models used the natural log of fingernail Al with each of copper (Cu), manganese (Mn), lead (Pb) and zinc (Zn) for the 56 male workers. The linear regression models controlled for age, education current alcohol consumption, current smoking status, and all five fingernail metals. Higher ln(FnMn) was associated with lower recall for several AVLT trials. The interaction term for ln(FnAl) and ln(FnZn) was significantly associated with AVLT Trial 1 (β= 1.99, 95% CI=0.07, 3.93), AVLT Trial 5 (β= 2.71, 95% CI= 0.02, 5.41) and AVLT average (β=2.11, 95% CI=0.01, 4.21). </p>Overall, this research presents valuable information regarding noninvasive, <i>in vivo</i>assessment of BnAl and its relationship with other Al biomarkers and measures of cognitive function. To the best of our knowledge, this is the largest study to use INVAA BnAl measurements to quantify long-term aluminum exposures within an occupational population, the first to compare BnAl with an estimate of cumulative Al exposure. Additionally, we are not aware of prior studies which have examined the interaction of fingernail metals, including Al, with olfactory and cognitive tests. Our results suggest BnAl is significantly associated with the prior 15-years of Al exposure and that IVNAA to assess BnAl can be used in field epidemiology studies. For our study, there was limited evidence of an association of BnAl and 5-year CEI with cognitive function. Most comparisons did not show any evidence of an association of Al with cognitive or olfactory function, but a statistically significant interaction between Al and Zn with some measures of cognitive function was observed. It is recommended that our findings be confirmed with larger studies.

O objetivo do presente estudo foi investigar a estratégia nutricional neonatal das raias Rhinoptera bonasus e Rhinoptera brasiliensis, analisando suas interações tróficas e dependência do recurso materno durante as fases iniciais da vida. Lipídios, ácidos graxos (FA) e isótopos estáveis (SI) foram analisados em diferentes tecidos, como ferramentas para avaliar as alterações durante o crescimento em raias jovens-de-ano (YOY), divididas em dois grupos, YOY I (<=50 cm) e YOY II (50-70 cm). O registro de YOY em três anos consecutivos (2015, 2016 e 2017) e a permanência dos filhotes durante o ano, confirmam que a região de Bertioga é uma área de berçário para as espécies R. bonasus e possivelmente para R. brasiliensis. Em R. bonasus, as concentrações plasmáticas de triacilglicerol e &#946;ß-hidroxibutirato não diferiram entre YOY I e II. No entanto, a concentração plasmática de colesterol (CHOL) foi maior em YOY I quando comparado com YOY II. O perfil de FA foi similar no fígado, músculo e plasma de R. bonasus, com predomínio de FA polinsaturados (PUFA), seguidos por FA saturados (SFA) e monoinsaturados (MUFA). No músculo de R. bonasus ocorreu uma diminuição de DHA (ácido docosahexaenoico) e ARA (ácido araquidônico) e um aumento de EPA (ácido eicosapentaenoico) com o aumento do tamanho corporal, reduzindo a relação DHA/EPA (indicador de posição trófica). Diferenças na composição de FA do fígado e músculo entre R. bonasus e R. brasiliensis foram encontradas na somatória de SFA, MUFA e PUFA, sugerindo que as espécies apresentam diferentes dietas ou diferentes períodos de nascimento. Em R. bonasus os valores de &#948;15N diminuíram com o aumento do tamanho corpóreo, e os valores mais altos foram encontrados na nadadeira dorsal e hemácias (RBC) de YOY I comparados a YOY II. Em YOY I e II, o &#948;13C da nadadeira foi maior que nas RBC. Comparando-se as duas espécies, R. bonasus apresenta maior valor de &#948;15N e &#948;13C nas RBC que R. brasiliensis, indicando que particionam recursos alimentares, com diferentes níveis tróficos e/ou forrageiam em diferentes locais, apesar das duas espécies serem simpátricas na região de estudo. Esses dados combinados demonstram que estas raias investem significativamente nos jovens e, consequentemente, em melhores condições nutricionais no nascimento, pois os dados encontrados sugerem que filhotes não mostraram deficiência de FA essenciais (EFA), como observado em tubarões placentários. Valores elevados de CHOL, ARA e DHA em YOY I, e posterior redução com o aumento do tamanho corpóreo, sugere o uso destes substratos em processos metabólicos. Os dados sugerem ainda que animais maiores aumentam as habilidades de forrageamento, demonstradas pelo aumento plasmático de EFA, tais como EPA e DHA e uma diminuição de SFA em YOY II. Tais aspectos metabólicos ligados à estratégia nutricional neonatal são fundamentais para compreender os padrões de investimento materno e recursos energéticos necessários nas fases iniciais de vida de elasmobrânquios vivíparos / The aim of the present study was to investigate the neonatal nutritional strategy adopted by species R. bonasus and R. brasiliensis, related to its trophic interactions and maternal resource dependency during the early stages of life. Lipids, fatty acids (FA) and stable isotope (SI) were assessed in different tissues, as tools to evaluate biomarkers changes during growth in young-of-the-year (YOY) rays, divided into two stages, YOY I (<=50 cm) and YOY II (50-70 cm). The registration of YOY in three consecutive years (2015, 2016 and 2017) and the permanence of the YOY during the year, confirms that the region of Bertioga is a nursery area for R. bonasus and possibly to R. brasiliensis. In R. bonasus the plasma triglycerides and &#948;-hydroxybutyrate did not differ between YOY I and II. Plasma cholesterol (CHOL) was higher in YOY I than in YOY II. FA profiles were similar in liver, muscle and plasma, with a predominance of polyunsaturated FA (PUFA), followed by saturated FA (SFA) and monounsaturated FA (MUFA). In R. bonasus there was a reduction of C22:6n3 (DHA - docosahexaenoic acid) and C20:4n6 (ARA - arachidonic acid), and an increase in C20:5n3 (EPA - eicosapentaenoic acid) in muscle, with increasing body size, resulting in a decrease in DHA/EPA ratio (used as an indicator of trophic position). In terms of SI, &#948;15N values decreased with increasing body size with higher &#948;15N values found in dorsal fin and red blood cells (RBC) of YOY I compared to YOY II animals. In YOY I and II, the &#948;13C of fin tissue was higher than RBC. Comparing both species, R. bonasus presents higher &#948;15N and &#948;13C values in the RBC than R. brasiliensis, suggesting the partition of food resources, at different trophic levels and/or foraging in different locations, despite the two species being sympatric in the region. These combined data demonstrate that cownose rays significantly invest in their young and consequently better nutritional conditions at birth, since the data suggest that the pups did not show essential FA (EFA) deficiency, as observed in placental sharks. High values of DHA, ARA and CHOL in YOY I, and subsequent decrease with increasing body size, confirms the use of these substrates in metabolic processes. In addition, data indicate that larger animals have improved foraging skills, demonstrated by increased plasma levels of EFA, such as DHA, EPA and a decrease of &#931;SFA in YOY II. Such metabolic aspects linked to neonatal nutritional strategy are fundamental factors for understanding the patterns of maternal investment and energy resources required in the early life stages of viviparous elasmobranchs

O câncer de mama é uma doença extremamente heterogênea compreendendo diferentes subtipos moleculares que resultam em evoluções clínicas e condutas terapêuticas distintas. A maior gravidade desta patologia está associada a sua capacidade de formação de metástases Mudanças no padrão de expressão gênica têm sido associadas à manifestação do fenótipo metastático. Neste trabalho, utilizamos microarranjos de tecido (TMAs) para investigar a expressão de 8 biomarcadores candidatos (CIP4, PPIL1, ITGAV, AKAP14, MICA, FXYD1, ARPC3, ABG1) e avaliar seu potencial prognóstico em pacientes com carcinoma ductal invasivo da mama. Destes, ARPC3 PPIL1 e CIP4 mostraram associações estatisticamente significativas com a sobrevida câncer específica e/ou a probabilidade de desenvolvimento de metástases. Determinamos que a expressão aumentada de CIP4 nos tumores está associada a maior probabilidade de desenvolvimento de metástases. CIP4 é uma proteína adaptadora descrita na literatura como moduladora de migração e invasão celular e portanto selecionamos este candidato para caracterização funcional detalhada. Observamos que a expressão de CIP4 encontra-se aumentada em linhagens tumorais com características invasivas. A partir do silenciamento estável e regulado de CIP4 na linhagem metastática MDA-MB-231, determinamos que CIP4 modula positivamente a ativação de MAPK-p38 e a expressão de MMP2 , sugerindo que CIP4 participe em vias de sinalização importantes para a transição epitélio-mesenquima (EMT). O silenciamento de CIP4 resultou em uma redução de aproximadamente 50% da capacidade migratória e invasiva das células tumorais in vitro , e na diminuição da formação de metástases pulmonares in vivo. Coletivamente, nossos resultados indicam que CIP4 tem potencial como marcador de prognóstico assim como um possível alvo terapêutico no controle da disseminação de metástases nos tumores da mama. / Breast cancer is an extremely heterogeneous disease comprising different molecular subtypes that result in different clinical outcomes and therapeutic procedures. The severity of this disease is mainly associated with its ability to produce metastasis. Changes in gene expression profile have been associated with the manifestation of the metastatic phenotype. In this study, we used tissue microarrays (TMAs) to investigate the expression of 8 candidate biomarkers (CIP4, PPIL1, ITGAV, AKAP14, MICA, FXYD1, ARPC3 e ABG1) and to evaluate their prognostic potential in patients with invasive ductal breast carcinoma. Among these, ARPC3, PPIL1 and CIP4 showed statistically significant associations with cancer specific survival and/or the patient\'s probability to develop metastasis. We found that increased expression of CIP4 in tumors is associated with a higher probability of developing metastasis. CIP4 is an adaptor protein described in the literature as a modulator of cell migration and invasion and therefore we selected this candidate for detailed functional characterization. We observed that CIP4 expression is increased in tumor cell lines with invasive characteristics. Following the stable and regulated knockdown of CIP4 in the metastatic line MDA-MB-231, we determined that it modulates positively the activation of MAPK-p38 and the expression of MMP2, suggesting that CIP4 participates in important signaling pathways required for the epithelial mesenchymal transition (EMT). CIP4 silencing resulted in an approximate 50% reduction of the migratory and invasive capacity of tumor cells in vitro and decreased the generation of lung metastases in vivo. Collectively, our results indicate that CIP4 has potential as a prognostic marker as well as a potential therapeutic target to control the metastatic dissemination of breast tumors.