Conselho Nacional de Desenvolvimento Científico e Tecnológico / Chapter I - Toxoplasmosis is a zoonosis caused by the intracellular parasite Toxoplasma gondii, which infects a range of hosts, including about one-third of the world\'s human population. One of the most severe manifestations of this infection in humans corresponds to congenital toxoplasmosis, which occurs when there is placental transmission of the parasite to the fetus in cases of primary maternal infection during pregnancy. Congenital infection may cause abortions or fetal losses, as well as severe ocular and/or cerebral sequelae in newborns. The serological screening of pregnant women and newborns is mainly based on the detection of IgG and IgM antibodies to T. gondii and constitutes an important measure to be adopted in programs to control this infection, despite the limitations in the antibody detection and interpretation of results. Chapter II - A total of 173 pairs of serum samples from mothers with suspected primary toxoplasmosis during pregnancy and their newborns, obtained from the Department of Pediatrics and Neonatology of Clinical Hospital of the Federal University of Uberlândia (HC-UFU) from 2006 to June 2014, was analyzed by ELISA for the detection of IgG, IgM and IgA anti-T. gondii, and the results were correlated with clinical data obtained from research in the clinical records of each patient. It was concluded that (i) prenatal serological screening is very important for the identification of pregnant women exposed to toxoplasmosis during pregnancy; (ii) maternal treatment reduces congenital transmission of T. gondii; (iii) neonatal serologic screening, associated with analysis of clinical parameters, allows the identification of vertically infected newborns, mainly through simultaneous detection of IgM and IgA antibodies; and (iv) serological follow-up of newborns is important in clarifying doubtful situations, especially in cases of asymptomatic newborns that present suggestive serology of congenital infection. Chapter III - Different antigenic fractions derived from soluble antigen of tachyzoites of T. gondii (STAg) were obtained from sequential precipitation with increasing concentrations of ammonium sulfate and used in immunoblotting technique to detect IgG antibodies and its subclasses (IgG1, IgG2, IgG3 and IgG4) present in paired serum samples from mothers with presumptive serology of recent toxoplasmosis during pregnancy and their newborns. It was concluded that the use of antigenic fractions obtained from STAg precipitation in the diagnosis of human toxoplasmosis proved to be interesting to detect IgG and its subclasses, allowing differentiation between positive and negative samples, but it was not a good alternative for the diagnosis of congenital toxoplasmosis, presenting results considered inferior to the STAg, due to the lower frequency of recognized antigenic bands and the absence of differential recognition of antigens by sera of newborns. Chapter IV - The amino acid sequences of sixteen immunodominant antigens of T. gondii were used to perform B cell linear epitope prediction using a software-based approach. A total of 22 epitopes of antigens from surface (SRS), rhoptries (ROP), micronemes (MIC) and dense granules (GRA) of T. gondii were identified, and 15 residues from their amino acid sequences were used to synthesize peptides chemically linked to bovine serum albumin backbone, and the diagnostic performance of these synthetic peptides was evaluated in ELISA to detect specific IgG antibodies in sera of patients with suspected acute toxoplasmosis (G1) or chronic (G2). It was concluded that synthetic peptides designed from B cell linear epitope prediction constitute promising antigens in serological assays to diagnose toxoplasmosis and differentiate acute from chronic phases of toxoplasmosis, representing an alternative to the use of native or recombinant antigens. / Capítulo I - Toxoplasmose é uma zoonose causada pelo parasita intracelular Toxoplasma gondii, que infecta diferentes hospedeiros, incluindo cerca de um terço da população humana mundial. Uma das manifestações mais graves da infecção por este parasita em humanos corresponde à toxoplasmose congênita, quando há transmissão placentária do parasita para o feto durante infecção materna primária na gestação. A infecção congênita pode causar abortos ou perdas fetais, além de sequelas graves em recém-nascidos (RNs), principalmente oculares e/ou cerebrais. A triagem sorológica de gestantes e RNs, baseada principalmente na detecção de anticorpos IgG e IgM anti-T. gondii constitui-se em importante medida a ser adotada em programas de controle desta infecção, apesar das limitações na detecção dos anticorpos e na interpretação dos resultados. Capítulo II - Um total de 173 pares de amostras de soros de mães com suspeita de toxoplasmose primária na gestação e seus respectivos RNs, provenientes do Setor de Pediatria e Neonatologia do Hospital de Clínicas da Universidade Federal de Uberlândia (HC-UFU) no período de 2006 a junho de 2014, foi analisado por ELISA para a detecção de anticorpos IgG, IgM e IgA anti-T. gondii, e os resultados obtidos foram correlacionados com dados clínicos obtidos a partir de pesquisa nos prontuários clínicos de cada paciente. Concluiu-se que (i) a triagem sorológica pré-natal é de grande relevância na identificação de gestantes expostas à toxoplasmose durante a gestação; (ii) o tratamento materno reduz a transmissão congênita de T. gondii; (iii) a triagem sorológica neonatal, aliada à análise de parâmetros clínicos permite identificar RNs verticalmente infectados, principalmente por meio da detecção conjunta de anticorpos IgM e IgA; e (iv) o acompanhamento sorológico de RNs é importante no esclarecimento de situações duvidosas, principalmente em casos de RNs assintomáticos, mas com sorologia sugestiva de infecção congênita. Capítulo III - Diferentes frações antigênicas derivadas do antígeno solúvel de taquizoítas de T. gondii (STAg) foram obtidas a partir de precipitação sequencial em concentrações crescentes de sulfato de amônio e utilizadas na técnica de immunoblotting para a detecção de anticorpos IgG total e suas subclasses (IgG1, IgG2, IgG3 e IgG4) presentes em amostras pareadas de soros de mães com sorologia presuntiva de toxoplasmose recente na gestação e seus respectivos RNs. Concluiu-se que a utilização destas frações antigênicas de STAg no diagnóstico da toxoplasmose humana se mostrou interessante na detecção de anticorpos IgG e suas subclasses, permitindo diferenciação entre amostras positivas e negativas, mas não se mostrou uma boa alternativa no diagnóstico da toxoplasmose congênita, com resultados inferiores aos do STAg, em função da menor frequência de bandas antigênicas reconhecidas e da ausência de reconhecimento diferencial de antígenos pelos soros dos RNs, em relação aos soros maternos. Capítulo IV - As sequências de aminoácidos de 16 antígenos imunodominantes de T. gondii foram usadas para a predição de epítopos lineares de células B usando ferramentas de bioinformática. Um total de 22 epítopos de antígenos de superfície (SRS), roptrias (ROP), micronemas (MIC) e grânulos densos (GRA) de T. gondii foram identificados e utilizados para a síntese de peptídeos com 15 resíduos de aminoácidos, os quais foram quimicamente conjugados ao esqueleto proteico da albumina sérica bovina (BSA), e o desempenho diagnóstico destes peptídeos sintéticos foi avaliado por ELISA para a detecção de anticorpos IgG em soros de pacientes com suspeita de toxoplasmose aguda (G1) ou crônica (G2). Concluiu-se que peptídeos sintéticos obtidos a partir da predição de epítopos lineares de células B constituem antígenos promissores em ensaios sorológicos para o diagnóstico da toxoplasmose e para a diferenciação das fases aguda e crônica da infecção, representando uma alternativa ao uso de antígenos nativos ou recombinantes. / Doutor em Imunologia e Parasitologia Aplicadas

Application of Taenia saginata metacestodes as alternative antigen is an important alternative for neurocysticercosis (NC) serodiagnosis. The cross reaction with Echinococcus granulosus infection occurred in homologous and heterologous antigens, and could be avoid with different purified methods. This study analyzed antigen fractions obtained from crude saline extract of T. saginata metacestodes purified by affinity chromatography with the lectin jacalin (unbound and bound fraction), concanavalin A (unbound and bound fraction), concanavalin A using jacalina unbound fraction (unbound and bound fraction) and N-acetil (unbound and bound fraction). The fraction were tested for the detection of IgG antibodies by enzyme linked immunosorbent assay (ELISA) and immunoblot for the laboratory diagnosis of human NC. The application of T. saginata metacestodes as an alternative antigen for use in ELISA and WB tests compared with the metacestodes antigen of Taenia solium in CFS samples was also analyzed. Serum samples were obtained from 142 individuals: 40 were diagnosed with NC, 62 presented Taenia sp. and other parasitic diseases and 40 were apparently healthy individuals. The CSF samples were obtained from 35 patients with definitive neurocysticercosis; and 35 patients with other neurological disorder. Among the fractions, unbound concanavalin A demonstrated statically higher sensitivity and specificity by ELISA (90% and 93.1 %, respectively). By Immunoblot, the concanavalin unbound showed 100% of sensitivity and specificity, where only serum samples from patients with NC recognized the protein of 64-68 kDa, so this antigen fraction may be used as specific antigen for diagnosis of NC. The sensitivity and specificity of ELISA using antigen obtained from T. solium applied to CSF samples results of 100%. When the tests were conducted using T. saginata metacestodes, results were 100% and 94.3%, respectively. The 47-52, 64-68 and 70 kDa antigens were recognized by only CSF samples from patients with NC. The results indicated that T. saginata metacestodes can be used as alternative antigen for NC diagnosis using LCR samples. / A utilização de metacestódeos de Taenia saginata como antígeno alternativo constitui uma importante ferramenta no sorodiagnóstico da neurocisticercose humana (NC). A reatividade cruzada com indivíduos infectados por Echinococcus granulosus é comum em antígenos homólogos e heterólogos, podendo ser evitada com diferentes métodos de purificação. O presente estudo analisou as diferentes frações antigênicas obtidas, a partir do extrato salino total de metacestódeos de T. saginata, por cromatografia de afinidade em coluna de Jacalina (fração ligante e não ligante), de Concanavalina A (fração ligante e não ligante), de Concanavalina A utilizando a fração não ligante de Jacalina (fração ligante e não ligante) e Coluna de N-acetil (fração ligante e não ligante). As frações foram avaliadas quanto a detecção de anticorpos IgG anti-metacestódeos de Taenia solium nos testes ELISA e Immunoblotting. Foi avaliada a utilização do extrato salino total de metacestódeos de T. saginata como antígeno alternativo nos testes ELISA e Immunoblotting para detecção de anticorpos IgG no LCR. Foram obtidas 142 amostras de soro, sendo 40 de pacientes com diagnóstico definitivo de NC, 62 de indivíduos infectados por Taenia sp e por outros parasitos e 40 de indivíduos saudáveis. Foram coletadas 70 amostras de LCR, sendo 35 de pacientes com diagnóstico definitivo de NC e 35 de indivíduos com outras manifestações neurológicas. Entre todas as frações analisadas, a fração não ligante de Concanavalina A demonstrou maior sensibilidade e especificidade pelo teste ELISA em amostras de soro (90% e 93,1%, respectivamente). Pelo Immunoblotting esta mesma fração demonstrou 100% de sensibilidade e especificidade, sendo que apenas pacientes com NC reconheceram a banda especifica de 64-68 kDa, indicando que esta fração antigênica pode se usada como antígeno especifico no sorodiagnóstico da NC humana. A sensibilidade e especificidade do teste ELISA utilizando o antígeno homólogo no LCR humano foi de 100%. Quando este teste foi conduzido com o antígeno heterólogo obteve-se 100% de sensibilidade e 94,3% de especificidade. Na reação de Immunoblotting as bandas antigênicas de 47-52, 64-68 e 70 kDa foram reconhecidas exclusivamente no LCR de pacientes com NC. Os resultados conferem ao extrato salino de T. saginata sensibilidade e especificidade para ser utilizado como antígeno alternativo para o diagnostico da NC no LCR. / Doutor em Imunologia e Parasitologia Aplicadas

No / BACKGROUND: The tumor suppressors p14(ARF) (ARF) and p16(INK4A) (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene. METHODS: We examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided. RESULTS: We observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases. CONCLUSION: Genetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16.

El càncer de pàncrees és altament agressiu i representa la cinquena causa de mort al mon occidental. Anteriorment, en el nostre laboratori, vam identificar que el receptor tissular del plasminogen (tPA) hi està sobre-expressat i juga un paper important el la progressió tumoral. En la present tesi hem profunditzat en l'estudi del mecanisme molecular de tPA i seus receptors en aquest càncer. En primer lloc hem caracteritzat en detall la interacció de tPA amb Annexina A2 (principal receptor de tPA en endoteli i altament expressada en pàncrees) demostrant que les dades publicades sobre la seqüència responsable de la interacció no eren correctes. A més a més hem caracteritzat les proteïnes de lisats cel·lulars pancreàtics que interaccionen amb tPA mitjançant un assaig pull down i posterior anàlisi proteòmic. de tot identificant un conjunt de possibles lligands de tPA. D'entre aquests hem seleccionat galectina 1, una lectina que mai s'ha descrit que interaccioni amb tPA, per realitzar la caracterització bioquímica i funcional del seu paper com a nou lligand de tPA en càncer de pàncrees. / Pancreatic cancer is a highly aggressive disease and represents the fifth cause of death in occidental world. Our laboratory has previously reported tissue type plasminogen activator (tPA) over expression in this cancer and its role in tumoral progression. During the present thesis we have studied tPA and its molecular mechanism through its receptors in this tumor.We have first characterized tPA interaction with annexin A2 (its main receptor in endothelium and highly expressed in pancreas). Our results showed that published data about the sequence responsible of this interaction was not correct. We have also identified a set of new putative tPA receptors in pancreatic cell lisates using a pull down assay and proteomic analysis. One of the proteins identified was galectin 1, a lectin with not know relation with tPA. We performed a biochemical and functional characterization of the interaction between these two proteins in pancreatic cancer.

Insulin-like growth factors 1 and 2 (IGF-1/2) are single-chain peptides exerting homology (in both amino-acid sequence and tertiary structure) to insulin. The main function of these peptides is promoting celular growth, proliferation and differentiation. Both insulin and insulin-like growth factors mediate their function through membrane receptors - insulin receptor (isoforms A and B) and IGF-1 receptor. All these receptors are members of the tyrosinkinase family of receptors and they exert the same subunit and domain composition. The activation of insulin and IGF-1 receptors is tightly associated with activation of two intracellular signaling pathways. The PI3-K/Akt pathway is involved in the glucose transport to the cell, induction of proliferation or inhibition of apoptosis, while the Ras/MAPK pathway is involved mainly in the induction of cell growth and differentiation. Due to the structure similarity in both the ligands and receptors, every ligand can activate different receptors (with different potency) and the signaling pathways associated with these receptors. Thus, the functions of IGFs and insulin, the same as their receptors, are overlapping. The distinct function of the concrete ligand can be distinguished by the different tissue distribution of both isoforms of insulin receptor and...

Indiana University-Purdue University Indianapolis (IUPUI) / 17-β estradiol (E2) exerts protective effects in hypoxia-induced pulmonary hypertension (HPH) via endothelial cell estrogen receptor (ER)-dependent mechanisms. However, the effects of hypoxia on ER expression in the pulmonary-right ventricle (RV) axis remain unknown. Based on previous data suggesting a role of ER-β in mediating E2 protection, we hypothesized that hypoxia selectively up-regulates ER-β in the lung and pulmonary endothelial cells. In our Male Sprague-Dawley rat model, chronic hypoxia exposure (10% FiO2) resulted in a robust HPH phenotype associated with significant increases in ER- β but not ER-α protein in the lung via western blotting. More importantly, this hypoxia-induced ER-β increase was not replicated in the RV, left ventricle (LV) or in the liver. Hence, hypoxia-induced ER-β up-regulation appears to be lung-specific. Ex vivo, hypoxia exposure time-dependently up-regulated ER-β but not ER-α in cultured primary rat pulmonary artery endothelial cells (RPAECs) exposed to hypoxia (1% O2) for 4, 24 or 72h. Furthermore, the hypoxia induced ER-β protein abundance, while not accompanied by increases in its own transcript, was associated with ER-β nuclear translocation, suggesting increase in activity as well as post-transcriptional up-regulation of ER-β. Indeed, the requirement for ER-β activation was indicated in hypoxic ER-βKO mice where administration of E2 failed to inhibit hypoxia-induced pro-proliferative ERK1/2 signaling. Interestingly, HIF-1α accumulation was noted in lung tissue of hypoxic ER-βKO mice; consistent with previously reported negative feedback of ER-β on HIF-1α protein and transcriptional activation. In RAPECs, HIF-1 stabilization and overexpression did not replicate the effects of ER- β up-regulation seen in gas hypoxia; suggestive that HIF-1α is not sufficient for ER-β up- regulation. Similarly, HIF-1 inhibition with chetomin did not result in ER-β down-regulation. HIF-1α knockdown in RPAECs in hypoxic conditions is currently being investigated. Hypoxia increases ER- β, but not ER-α in the lung and lung vascular cells. Interpreted in context of beneficial effects of E2 on hypoxic PA and RV remodeling, our data suggest a protective role for ER-β in HPH. The mechanisms by which hypoxia increases ER-β appears to be post-transcriptional and HIF-1α independent. Elucidating hypoxia-related ER-β signaling pathways in PAECs may reveal novel therapeutic targets in HPH.

Indiana University-Purdue University Indianapolis (IUPUI) / Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.