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THESIS SUMMARY Food allergy is one of the frequent disorders and its incidence in paediatric as well as adult population is continuously rising, having doubled in the last two decades. Although wheat belongs to major food allergens and is a staple food in most diets, we have only little knowledge of wheat proteins causing IgE mediated hypersensitivity reaction. Diagnostic approaches of food allergy to wheat have a high sensitivity, but low specificity. Poor predictability and specificity may be associated with the insufficient purity of wheat extracts used in sIgE assays or with the lack of major allergens in these extracts. In the first step, we characterized 19 potential allergens recognised by IgE Abs of allergic patients, using proteomic techniques (1-DE, 2-DE, MALDI-TOF, QTOF and LCQDECA nLC-MS/MS ion trap technique). We identified these IgE-binding molecules such as: α-amylase inhibitors, β-amylase, profilin, serpin, β-D-glucan exohydrolase and 27K protein. To quantify sIgE in patient's sera we developed ELISA using the whole wheat extract and two commercially available α-amylase inhibitors. Second, we developed a procedure that allows isolation of wheat allergens from natural sources using Rotofor cell and HPLC. Twenty-seven potential wheat allergens have been successfully identified; of these, the...

Το σύνδρομο Hyper-IgE (HIES) είναι μια σπάνια πρωτοπαθής ανοσοανεπάρκεια η οποία χαρακτηρίζεται από υψηλά επίπεδα IgE στον ορό, υποτροπιάζουσες σταφυλοκοκκικές λοιμώξεις του δέρματος και επεισόδια πνευμονίας με σχηματισμό κύστεων. Επιπλέον, στο φαινότυπο του συνδρόμου περιλαμβάνονται και μη-ανοσολογικού τύπου ανωμαλίες όπως χαρακτηριστικό προσωπείο, υπερεκτασιμότητα των αρθρώσεων, σκολίωση, αυτόματα κατάγματα και διατήρηση των νεογιλών οδόντων. Προσφάτως, διαπιστώθηκε πως ετερόζυγες μεταλλάξεις στον μετγραφικό παράγοντα STAT3, ευθύνονται για την αυτοσωμική επικρατούσα μορφή του HIES. Στην παρούσα ερευνητική εργασία ταυτοποιήθηκε και χαρακτηρίστηκε μια νέα μετάλλαξη στην περιοχή δέσμευση του DNA (DNA-binding domain) του μεταγραφικού παράγοντα STAT3 σε έναν ασθενή με σύνδρομο Hyper IgE. Ανάλυση της αλληλουχίας του γονιδίου του STAT3 αποκάλυψε μια de novo ετερόζυγη αντικατάσταση βάσης από G (γουανίνη) σε A (αδενίνη), η οποία προκαλεί την αντικατάσταση στο επίπεδο της αμινοξικής αλληλουχίας του αμινοξέος γλυκίνη από το ασπαρτικό οξύ (G342D). Η ασθενής έχει φυσιολογικά επίπεδα της πρωτεΐνης STAT3, η οποία και εισέρχεται στον πυρήνα των κυττάρων κατόπιν ενεργοποίησης με ιντερλευκίνη-6 (IL-6). Παρoλ’ αυτά, μελέτη της ικανότητας της πρωτεΐνης STAT3 να δεσμεύεται στο DNA έδειξε πως η μετάλλαξη G342D επηρεάζει σημαντικά αυτή τη λειτουργία. Επιπλέον, ανάλυση με ποσοτική RT-PCR έδειξε πως η μετάλλαξη G342D αναστέλλει την STAT3-εξαρτώμενη επαγωγή του γονιδίου ROR γt, απαραίτητου γονιδίου για την διαφοροποίηση και ανάπτυξη των Th17 κυττάρων. Με βάση τα παραπάνω δεδομένα, φαίνετε πώς η νέα μετάλλαξη στο μεταγραφικό παράγοντα STAT3 επηρεάζει σημαντικά τη λειτουργικότητά του, και προκαλεί το σύνδρομο Hyper-IgE στην ασθενή. / The Hyper-IgE syndrome (HIES) is a rare primary immunodeficiency characterized by a highly elevated serum IgE, recurrent staphylococcal skin abscesses and cyst-forming pneumonia. Non-immunological abnormalities, including a distinctive facial appearance, hyperextensive joints, scoliosis, fracture following minor trauma, and the retention of primary teeth are also observed in many patients. Recently, it was shown that heterozygous mutations in signal transducer and activator of transcription 3 (STAT3), can cause autosomal-dominant HIES. Here we identify and characterize a novel mutation in the DNA binding domain of STAT3 in a patient with hyper-IgE syndrome. Sequence analysis revealed a de novo heterozygous transition of a G to A, causing a substitution of a glycine residue for an aspartic acid in the translated sequence (G342D). The patient has normal levels of STAT3, which is able to translocate to the nucleus upon IL-6 stimulation. However, enzyme-linked DNA–protein interaction analysis showed that the G342D mutation affects the binding ability of STAT3 to target DNA sequences. In addition, as shown by qRT-PCR, the mutation abrogates the STAT3 dependent transcription of the retinoid-related orphan receptor γt (ROR γt) gene, an indispensable transcription factor for the commitment of naive CD4+ T cells to the Th17 lineage. These data suggest that the novel G342D mutation affects the binding of STAT3 on DNA and the STAT3 dependent expression of ROR γt mRNA, leading to the HIES phenotype.

Objetivo: Avaliar as concentrações séricas do CA-125 e do CD-23 solúvel, durante dois períodos do ciclo menstrual (primeiro, segundo ou terceiro dia e o sétimo, oitavo ou nono dia), correlacionando-as a queixas clínicas, local de doença, estadiamento pela American Society for Reproductive Medicine e classificação histológica da endometriose pélvica. Pacientes e Métodos: No período de junho de 2007 a outubro de 2009, em estudo transversal, 102 mulheres, com queixas de infertilidade, dor pélvica refratária às terapêuticas clínicas, ou desejando laqueadura tubária, foram avaliadas por videolaparoscopia e divididas em um grupo caso, com endometriose (n=44), e outro controle, sem endometriose (n=58). Todas as pacientes foram submetidas a anamnese para investigação dos sintomas associados a endometriose e, antecedendo em até três meses da videolaparoscopia, a duas coletas de sangue venoso periférico para determinação da concentração do CA-125 e do CD-23 solúvel, por enzima-imunoensaio. Foram empregados os testes de Qui quadrado ou exato de Fisher para comparação de variáveis qualitativas. O teste t de Student, com análise de variância de Levene para amostras independentes, foi usado para diferença das médias de idade, intensidade dolorosa aferida pela escala visual analógica e concentrações dos marcadores, admitindo-se nível de significância igual a 0,05, para todos os testes. Resultados: Observamos que as concentrações médias do CA-125 foram significantemente maiores no grupo caso do que no grupo controle, em ambas as coletas na presença de dismenorréia severa e na ausência de infertilidade e de queixas urinárias cíclicas, mas apenas na segunda coleta naquelas com queixa de dor pélvica crônica e dispareunia de profundidade, permitindo diferenciação quando da presença de endometriose ovariana, grau III ou IV e padrões histológicos estromal ou glandular bem diferenciado. O CD-23 solúvel apresentou concentrações médias menores em pacientes com endometriose quando comparadas a mulheres sem a doença, porém sem atingir significância estatística, bem como essas concentrações foram menores na segunda coleta em pacientes sem endometriose com queixa de alterações intestinais cíclicas. Conclusões: Maiores concentrações do CA-125 em pacientes com endometriose reforçaram a afirmação de que esse marcador pode ser útil no diagnóstico e no manejo dessa doença. No entanto não se identificaram diferenças significantes na concentração desse marcador, em ambas as fases do ciclo menstrual, entre pacientes com endometriose em estádio inicial ou avançado, assim como entre aquelas com lesões histologicamente classificadas como diferenciadas ou indiferenciadas. O presente estudo trouxe uma análise diferenciada das concentrações médias do CD-23 solúvel e de suas associações com sintomas e local de doença / Objective: To evaluate serum concentrations of CA-125 and soluble CD-23, during two periods of the menstrual cycle (first, second or third day and the seventh, eighth or ninth day), correlating them to clinical complaints, site of disease, staging by the American Society for Reproductive Medicine and histological classification of pelvic endometriosis. Patients and Methods: From June 2007 to October 2009, within a cross-sectional study, 102 women with complaints of infertility, pelvic pain refractory to clinical therapies, or desiring for tubal ligation, were evaluated by videolaparoscopy and were divided into a case group, with endometriosis (n = 44), and a control group without endometriosis (n = 58). All patients underwent anamnesis for investigation of symptoms associated with endometriosis and, prior to three months to videolaparoscopy, they were submitted to two peripheral venous blood samples to measure CA-125 and soluble CD-23 concentrations by enzyme-linked immunosorbent assay. We utilized Chi square or Fisher exact test to compare qualitative variables. The t Student test, with Levene\'s variance analysis for independent samples, was used for mean difference in age, pain intensity measured by visual analogue scale and concentrations of markers, assuming a significance level of 0.05, for all tests. Results: We observed that average concentrations of CA-125 were significantly higher in case group than in control group in both samples, in the presence of severe dysmenorrhea and absence of infertility and cyclical urinary complaints, but only in the second collection, for women with chronic pelvic pain and deep dyspareunia, enabling differentiation in the presence of ovarian endometriosis grade III or IV and stromal or glandular well differentiated histological patterns. The soluble CD-23 showed lower average concentrations in patients with endometriosis compared to women without this disease, without statistical significance, and these concentrations were lower in the second collection for patients without endometriosis with complains of cyclical intestinal disorders. Conclusions: Higher concentrations of CA-125 in patients with endometriosis have reinforced the statement that this marker may be useful in the diagnosis and management of this disease. However we did not identify significant differences in concentration of this marker, in both phases of the menstrual cycle, between patients with endometriosis in early or advanced stage, and between those with lesions histologically classified as differentiated or undifferentiated. This study brought a differentiated analysis of the average concentrations of soluble CD-23, and of their associations with symptoms and site of disease.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-20T19:05:22Z No. of bitstreams: 1 alessasinsingerbrugiolo.pdf: 7024795 bytes, checksum: 9af11871ea610d9074a4267774a8aea9 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:25:08Z (GMT) No. of bitstreams: 1 alessasinsingerbrugiolo.pdf: 7024795 bytes, checksum: 9af11871ea610d9074a4267774a8aea9 (MD5) / Made available in DSpace on 2016-09-26T20:25:08Z (GMT). No. of bitstreams: 1 alessasinsingerbrugiolo.pdf: 7024795 bytes, checksum: 9af11871ea610d9074a4267774a8aea9 (MD5) Previous issue date: 2010-03-11 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A asma é uma doença caracterizada por obstrução intermitente das vias aéreas e inflamação crônica e afeta cerca de 300 milhões de pessoas em todo o mundo. É considerada um grave problema de saúde, trazendo consigo um grande encargo econômico. A resposta imunológica na asma é predominantemente de linfócitos Th2, com altos níveis de IgE total e alérgeno-específica e eosinofilia brônquica. O tratamento da asma visa ao controle da doença e os medicamentos utilizados atualmente apresentam efeitos colaterais sistêmicos e, em geral, não são eficazes nos casos de asma de difícil controle. Neste trabalho, foi investigado o efeito do extrato aquoso de Echinodorus grandiflorus, planta utilizada pela população por suas propriedades diurética e anti-inflamatória, no modelo de alergia pulmonar no qual camundongos BALB/c foram sensibilizados e desafiados com OVA. O tratamento com o extrato por via oral reduziu marcadamente o número de neutrófilos, linfócitos, macrófagos e eosinófilos no lavado bronco-alveolar (LBA), a atividade da peroxidase eosinofílica (EPO) no tecido pulmonar, os níveis de IgE específica para OVA no soro, os níveis de CCL11 e a expressão gênica de IL-4 e IL-13 no tecido pulmonar e a expressão de CD25 em células do LBA. Entretanto, o tratamento não alterou o infiltrado inflamatório e o número de eosinófilos no tecido pulmonar, os níveis das quimiocinas CCL2 e CCL5, das citocinas IL-4, IL-13, IL-10 e IL-17A e a expressão gênica de Foxp3 no tecido pulmonar e a expressão de CD28 e CD152 nas células do LBA. Esses resultados sugerem que o extrato aquoso de E. grandiflorus é capaz de modular alguns aspectos da inflamação pulmonar alérgica e pode ser útil no tratamento da asma. / Asthma is a disease characterized by intermittent obstruction of the airways and chronic inflammation and affects about 300 million people around the world. It is considered a serious health problem, bringing a large economic burden. The immune response in asthma is predominantly Th2 lymphocytes, with high levels of total and allergen-specific IgE and bronchial eosinophilia. Asthma treatment is aimed at controlling the disease and the drugs used currently have systemic side effects and generally are not effective in cases of asthma are difficult to control. In this study we investigated the effect of aqueous extract of Echinodorus grandiflorus, plant used in folk medicine for its diuretic and anti-inflammatory properties, in the model of lung allergy in which BALB/c mice were sensitized and challenged with OVA. Oral treatment with the extract markedly reduced the number of neutrophils, lymphocytes, macrophages and eosinophils in bronchoalveolar lavage (BAL), the activity of eosinophil peroxidase (EPO) in lung tissue, levels of OVA-specific IgE in serum, levels of CCL11 in lung tissue, the gene expression of IL-4 and IL-13 in lung tissue and the expression of CD25 on BAL cells. However, the treatment did not alter the inflammatory infiltrate and the number of eosinophils in lung tissue, levels of chemokines CCL2 and CCL5, levels of cytokines IL-4, IL-13, IL-10 and IL-17A and gene expression of Foxp3 in the lung tissue and the expression of CD28 and CD152 on BAL cells. These results suggest that the aqueous extract of E. grandiflorus is able to modulate some aspects of allergic pulmonary inflammation and may be useful in the treatment of asthma.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-17T12:32:21Z No. of bitstreams: 1 micheliaantoniadonascimentogusmao.pdf: 2641235 bytes, checksum: 154c338e19fb47129f176bdceb0cf92e (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: on 2017-08-24T11:26:04Z (GMT) / Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-24T15:14:14Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-30T14:21:34Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2017-08-30T14:21:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-17 / A esquistossomose é uma doença tropical negligenciada que acomete milhões de pessoas no mundo. A falta de vacina para essa doença helmíntica impulsiona a busca por moléculas antigênicas oriundas dos parasitos do gênero Schistosoma. Dentre as moléculas consideradas como promissoras, se encontra a ATP difosfohidrolase de Schistosoma mansoni. O parasito possui duas isoformas de desta enzima que são denominadas SmATPDases e são expressas em todos as fases do ciclo de vida deste helminto. A apirase de batata é uma proteína vegetal pertencente à mesma família de enzimas do parasito, compartilha epitopos com as isoformas de SmATPDases. Dessa forma, o objetivo deste trabalho foi realizar a caracterização imunológica da apirase de batata em modelo de infecção murina experimental com base nessa homologia protéica. A proteína vegetal foi inoculada em camundongos C57BL/6 que posteriormente foram infectados com cercárias de S. mansoni. A proteína vegetal induziu níveis significativos de anticorpos IgG e IgG1 após a imunização, os quais se mantiveram elevados na análise 60 dias após a infecção. A cultura de esplenócitos dos animais imunizados respondeu ao estímulo com a proteína vegetal, induzindo a produção de níveis significativos de IFN-γ, IL-10 e IL-5, mas não de IL-13. O processo de imunização não conferiu proteção, sendo incapaz de promover a redução da carga parasitária. O efeito imunomodulador da imunização foi observado através de uma redução da área afetada pela reação granulomatosa hepática nos animais imunizados. Em relação à ligação de anticorpos IgE, a apirase de batata mostrou reatividade significativa com estes anticorpos presentes em amostras do plasma de pacientes com esquistossomose. Em ensaios de Western blotting, os anticorpos IgE foram capazes de reconhecer três proteínas presentes no homogeneizado de vermes adultos do helminto, com 91, 63 e 55 kDa. Foi possível inferir que todas as bandas reveladas correspondem a isoformas de SmAPTDases com distintos processamentos pós-tradução, tais como glicosilação e proteólise. Comprovando esta reatividade, foi possível verificar que a imunização dos animais (C57BL/6) com a proteína vegetal foi capaz de induzir a produção significativa de anticorpos IgE detectáveis em técnicas de ELISA e Western blotting. Como a apirase de batata possui identidade com a NTPDase 1 (CD39) de mamíferos sugerimos que os pacientes possuam anticorpos naturais contra NTPDases envolvidos na manutenção da homeostase do organismo. O conjunto de dados obtidos mostra que a apirase de batata possui um perfil imunoregulatório na esquistossomose, podendo contribuir para a redução da morbidade, induzindo a produção de anticorpos IgE, tidos como protetores na esquistossomose. / Schistosomiasis is a neglected tropical disease that affects millions of people around the world. The lack of vaccine for this helminth disease prompts a search for antigenic molecules from the parasites of the genus Schistosoma. Among the molecules considered promising are an ATP diphosphohydrolase from Schistosoma mansoni. The parasite has two enzyme isoforms which are termed SmATPDases and are expressed in all as life cycle phases of this helminth. The potato apyrase is a plant protein belonging to the same family of parasite enzymes, shared epitopes with SmATPDase isoforms. Thus, the objective of this work was to perform an immunological characterization of potato apyrase in a model of experimental murine infection based on this protein homology. Plant protein was inoculated into C57BL / 6 mice that were infected with S. mansoni cercariae. Plant protein induced significant levels of IgG and IgG1 antibodies after immunization, which remained high for analysis 60 days after infection. The solenocytes culture of the immunized animals responded to the stimulus with a plant protein, inducing a significant production of IFN-γ, IL-10 and IL-5, but not IL-13. The immunization process did not provide protection, being unable to promote a reduction of the parasitic load. The immunomodulatory effect of immunization was observed through a reduction of the area affected by the hepatic granulomatous reaction in the immunized animals. Concerning the binding of IgE antibodies, potato apyrase showed significant reactivity with these antibodies present in plasma samples from patients with schistosomiasis. In Western blotting assays, IgE antibodies were able to recognize three non-homogenized languages of adult helminth worms at 91, 63 and 55 kDa. It was possible to infer that all the bands revealed correspond to SmAPTDase isoforms with various post-translational processing, such as glycosylation and proteolysis. (C57BL / 6) with a plant protein capable of inducing a significant production of IgE antibodies detected in ELISA and Western blotting techniques. As potato apyrase has an identity with a mammalian NTPDase 1 (CD39), we suggest that patients have an antibody against the NTPDases involved in maintaining the body's homeostasis. The obtained data show that the potato harvest has an immunoregulatory profile in schistosomiasis, and may contribute to the reduction of morbidity, inducing a production of IgE antibodies, considered as protectors in schistosomiasis.

Objetivo: Avaliar as concentrações séricas do CA-125 e do CD-23 solúvel, durante dois períodos do ciclo menstrual (primeiro, segundo ou terceiro dia e o sétimo, oitavo ou nono dia), correlacionando-as a queixas clínicas, local de doença, estadiamento pela American Society for Reproductive Medicine e classificação histológica da endometriose pélvica. Pacientes e Métodos: No período de junho de 2007 a outubro de 2009, em estudo transversal, 102 mulheres, com queixas de infertilidade, dor pélvica refratária às terapêuticas clínicas, ou desejando laqueadura tubária, foram avaliadas por videolaparoscopia e divididas em um grupo caso, com endometriose (n=44), e outro controle, sem endometriose (n=58). Todas as pacientes foram submetidas a anamnese para investigação dos sintomas associados a endometriose e, antecedendo em até três meses da videolaparoscopia, a duas coletas de sangue venoso periférico para determinação da concentração do CA-125 e do CD-23 solúvel, por enzima-imunoensaio. Foram empregados os testes de Qui quadrado ou exato de Fisher para comparação de variáveis qualitativas. O teste t de Student, com análise de variância de Levene para amostras independentes, foi usado para diferença das médias de idade, intensidade dolorosa aferida pela escala visual analógica e concentrações dos marcadores, admitindo-se nível de significância igual a 0,05, para todos os testes. Resultados: Observamos que as concentrações médias do CA-125 foram significantemente maiores no grupo caso do que no grupo controle, em ambas as coletas na presença de dismenorréia severa e na ausência de infertilidade e de queixas urinárias cíclicas, mas apenas na segunda coleta naquelas com queixa de dor pélvica crônica e dispareunia de profundidade, permitindo diferenciação quando da presença de endometriose ovariana, grau III ou IV e padrões histológicos estromal ou glandular bem diferenciado. O CD-23 solúvel apresentou concentrações médias menores em pacientes com endometriose quando comparadas a mulheres sem a doença, porém sem atingir significância estatística, bem como essas concentrações foram menores na segunda coleta em pacientes sem endometriose com queixa de alterações intestinais cíclicas. Conclusões: Maiores concentrações do CA-125 em pacientes com endometriose reforçaram a afirmação de que esse marcador pode ser útil no diagnóstico e no manejo dessa doença. No entanto não se identificaram diferenças significantes na concentração desse marcador, em ambas as fases do ciclo menstrual, entre pacientes com endometriose em estádio inicial ou avançado, assim como entre aquelas com lesões histologicamente classificadas como diferenciadas ou indiferenciadas. O presente estudo trouxe uma análise diferenciada das concentrações médias do CD-23 solúvel e de suas associações com sintomas e local de doença / Objective: To evaluate serum concentrations of CA-125 and soluble CD-23, during two periods of the menstrual cycle (first, second or third day and the seventh, eighth or ninth day), correlating them to clinical complaints, site of disease, staging by the American Society for Reproductive Medicine and histological classification of pelvic endometriosis. Patients and Methods: From June 2007 to October 2009, within a cross-sectional study, 102 women with complaints of infertility, pelvic pain refractory to clinical therapies, or desiring for tubal ligation, were evaluated by videolaparoscopy and were divided into a case group, with endometriosis (n = 44), and a control group without endometriosis (n = 58). All patients underwent anamnesis for investigation of symptoms associated with endometriosis and, prior to three months to videolaparoscopy, they were submitted to two peripheral venous blood samples to measure CA-125 and soluble CD-23 concentrations by enzyme-linked immunosorbent assay. We utilized Chi square or Fisher exact test to compare qualitative variables. The t Student test, with Levene\'s variance analysis for independent samples, was used for mean difference in age, pain intensity measured by visual analogue scale and concentrations of markers, assuming a significance level of 0.05, for all tests. Results: We observed that average concentrations of CA-125 were significantly higher in case group than in control group in both samples, in the presence of severe dysmenorrhea and absence of infertility and cyclical urinary complaints, but only in the second collection, for women with chronic pelvic pain and deep dyspareunia, enabling differentiation in the presence of ovarian endometriosis grade III or IV and stromal or glandular well differentiated histological patterns. The soluble CD-23 showed lower average concentrations in patients with endometriosis compared to women without this disease, without statistical significance, and these concentrations were lower in the second collection for patients without endometriosis with complains of cyclical intestinal disorders. Conclusions: Higher concentrations of CA-125 in patients with endometriosis have reinforced the statement that this marker may be useful in the diagnosis and management of this disease. However we did not identify significant differences in concentration of this marker, in both phases of the menstrual cycle, between patients with endometriosis in early or advanced stage, and between those with lesions histologically classified as differentiated or undifferentiated. This study brought a differentiated analysis of the average concentrations of soluble CD-23, and of their associations with symptoms and site of disease.