Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-03Bitstream added on 2014-06-13T20:29:34Z : No. of bitstreams: 1 pinto_jras_me_rcla.pdf: 1057969 bytes, checksum: 2e29026d533c6190983ed253a062779d (MD5) / Os venenos dos insetos da ordem Hymenoptera contêm uma variedade de proteínas alergênicas. As ferroadas desses insetos podem induzir reações alérgicas e ocasionalmente, anafilaxias fatais. Há um crescente interesse nos componentes químicos dos venenos desses insetos, sobretudo no campo da alergia e imunologia clínica. As principais reações desses venenos são as reações inflamatórias e/ou imunológicas em suas vítimas, podendo ocorrer alguns efeitos sistêmicos. Dentre os Hymenoptera sociais, os venenos de abelhas e vespas têm sido extensivamente estudados e muitos de seus componentes moleculares já foram isolados e identificados. Fosfolipase, hialuronidase, antígeno-5 e serinoprotease são proteínas antigênicas de elevada massa molecular que, quando injetadas durante o ato de ferroar, iniciam uma resposta imune peculiar, sensibilizando alguns indivíduos. Porém, poucos estudos têm sido realizados para a identificação e o mapeamento dos epítopos desses alérgenos. O conhecimento sobre a interação molecular dos principais alérgenos destes venenos certamente deverá contribuir para o aperfeiçoamento dos diagnósticos de alergia, bem como para o desenvolvimento de tratamentos mais seletivos de reações alérgicas mediadas por IgE. No presente estudo identificamos e mapeamos os epítopos lineares de células-B do antígeno-5 do veneno da vespa social Polybia paulista. O antígeno-5 é conhecido como um dos principais alérgenos do veneno de Hymenoptera com massa molecular em torno de 23 kDa e função biológica desconhecida, porém muitos estudos demonstram a sua alergenicidade. Tendo conhecimento da sequência primária do antígeno-5, a identificação e o mapeamento dos epítopos foram realizados através da síntese múltipla de peptídeos, utilizando as técnicas do SPOT-Synthesis, imunodetecção e método indireto do ELISA com soro de pacientes sensíveis... / The venom insects of Hymenoptera order contain a variety of allergenic proteins. The stings of these insects can induce allergic reactions and occasionally fatal anaphylaxis. There is a growing interest in the knowledge about the chemical components of the venom of these insects, especially in the field of allergy and clinical immunology. The main reactions of these venoms are inflammation and / or the induction of immune process in the victims; sometimes systemic effects also may be observed. Among the social Hymenoptera, the venoms of bees and wasps have been extensively studied and many of their molecular components have been isolated and identified. Phospholipase, hyaluronidase, antigen-5 and serine protease are antigenic proteins of high molecular weight, which may initiate a peculiar immune response to sensitize some individuals. However, few studies have been performed for the identification and mapping of the epitopes these allergens. The knowledge about the molecular interaction of the major allergens of these venoms with IgG and/or IgE will certainly contribute for improving the diagnosis of allergy, as well as to develop more selective treatments of reactions IgE-mediated allergy. In this study we identified and mapped the linear epitopes of B-cells of the antigen-5 from the venom of the social wasp Polybia paulista. The antigen-5 is known as one of the major allergens from Hymenoptera venoms with molecular weight around 23 kDa and unknown biological function, but many studies show its allergenicity. The aim of this study was to identify and to map the linear epitopes of B-cells of this allergen. Taking into account the knowledge the primary sequence of antigen-5, the identification and mapping of epitopes was performed by using multiple synthesis of peptides with the combination of different protocols: SPOT-Synthesis, immunoblotting and indirect method of ELISA with the sera... (Complete abstract click electronic access below)

Submitted by Susimery Vila Nova (susimery.silva@ufpe.br) on 2015-04-10T19:19:57Z No. of bitstreams: 2 tese final para impressão 1.pdf Joacilda.pdf: 3160703 bytes, checksum: 77f3910f7f992488c2ed2eada2b36dcf (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-10T19:19:57Z (GMT). No. of bitstreams: 2 tese final para impressão 1.pdf Joacilda.pdf: 3160703 bytes, checksum: 77f3910f7f992488c2ed2eada2b36dcf (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-05-30 / Leucemia linfoide aguda (LLA) é o câncer pediátrico mais comum e etiologicamente não apresenta um modelo definido, uma vez que apresenta uma história natural biologicamente diversificada. Fatores têm sido relacionados ao sistema imunológico como risco/proteção ao desenvolvimento de LLA, considerando-se como plausíveis o papel do sistema imune frente às infecções na infância, e a inter-relação dessas infecções com mecanismos envolvendo as Hipóteses da Higiene e da Adrenal. A proposta desse estudo foi investigar uma possível associação entre alergia prévia, bem como a ativação do eixo adrenal, pautado na Hipótese da Adrenal, com o desenvolvimento de leucemia linfoide aguda na infância e adolescência. Trata-se de um estudo do tipo caso-controle não pareado de base hospitalar, cuja amostra foi constituída de 60 crianças e adolescentes com diagnóstico incidente de leucemia linfoide aguda não T, classificadas pela avaliação da medula óssea através do mielograma e imunofenotipagem e 120 controles selecionados com proporcionalidade com relação à idade e sexo a partir dos casos e oriunda dos estados de Pernambuco e Paraíba no nordeste brasileiro, entre 2008 e 2011. A coleta dos dados consistiu na aplicação de questionário estruturado para identificação de alergias como asma, rinite alérgica, antecedente de urticária e dermatite atópica, uso prévio de glicocorticoides, além de exame clínico e coleta de sangue para dosagem de IgE total, cortisol basal, ACTH, marcador de infecção prévia pelo EBV e parvovírus B19 através da dosagem de IgG. Outras variáveis como aleitamento materno, peso ao nascer, escolaridade materna, infecção materna na gestação e número de pessoas no domicílio também foram analisadas. Para a análise estatística foram utilizados Teste de associação de 2, Teste Exato de Fisher, Odds Ratio, Regressão Logística Binária e Regressão Logística Múltipla. Os resultados encontrados na análise univariada (p < 0,20) foram: Asma ( pvalor = 0,116), Rinite Alérgica (p-valor=0,032), Antecedente de Urticária (p-valor = 0,011), Dermatite Atópica (p-valor = 0,086), Nível Sérico Elevado de IgE Total (p-valor = 0,00), Nível Elevado de Cortisol Basal (p-valor = 0,004), Infecção Prévia pelo EBV (p-valor = 0,143), Infecção Prévia por Parvovírus B19 (p-valor = 0,068). Após o modelo ajustado através da análise de regressão logística múltipla persiste a significância de uma relação inversa entre Asma com p-valor = 0,044 e OR/IC 95% 0,14 (0,02 – 0,95), Nível Sérico Elevado de IgE Total com p-valor = 0,001 e OR/IC 95% 0,10 (0,02 – 0,41), além de Níveis Elevados de Cortisol apresentando p-valor 0,004 e OR/IC 95% 0,16 (0,04 – 0,56); Para infecção Prévia pelo Parvovírus B 19 o resultado expressa risco p-valor = 0,037 e OR/IC 95% 2,19 (1,05 – 4,57). Asma e Níveis Séricos Elevados de IgE Total e ainda Níveis Elevados de Cortisol Basal, parecem estar relacionados com modificações na resposta imune e como consequência promoveriam uma diminuição de clones leucêmicos, desempenhando um papel de proteção a crianças e adolescentes contra LLA. A infecção prévia pelo parvovírus B 19 está associada com aumento de risco de LLA.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-08-31T18:55:41Z No. of bitstreams: 2 Dissertação - Natallia Moreira Lopes Leão - 2015.pdf: 2760107 bytes, checksum: 7e60311bb25828c1bc39aa20617c0b2e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-01T12:07:41Z (GMT) No. of bitstreams: 2 Dissertação - Natallia Moreira Lopes Leão - 2015.pdf: 2760107 bytes, checksum: 7e60311bb25828c1bc39aa20617c0b2e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-01T12:07:41Z (GMT). No. of bitstreams: 2 Dissertação - Natallia Moreira Lopes Leão - 2015.pdf: 2760107 bytes, checksum: 7e60311bb25828c1bc39aa20617c0b2e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-07-02 / Outro / Leishmaniases comprise a disease group with a great epidemiological and clinical diversity. The detection of IgE can be an important tool for prognosis of American Tegumentary Leishmaniasis (LTA). The objective of this present work was to assess if the immunoglobulin E (IgE), anti - Leishmania can be used as biomarkers for prognosis in LTA. The standardization of an enzyme-linked immunosorbent assay (ELISA) was carried out applying antigenic extract of L. (V.) braziliensis to detect IgE specific for leishmania in LTA patients’ plasmas. It was determined that the dilution which differed positive from negative samples was 1/20 for plasma and 1/100 for peroxidase labeled anti-IgE antibody. Forty LTA patients were assisted in the laboratory of endemic diseases at AnuarAuad Hospital (HDT/GO), Goiânia - GO, between 2009 and 2012. Twenty-seven of those patients showed localized cutaneous leishmaniasis (LCL) and 1 showed mucosal leishmaniasis (LM). Twenty-seven of those patients were male and thirteen were female. The LCL patients’ ages varied between 18 to 65 years and LM patients’ ages varied from 25 to 61 years. The LCL patients showed 1 to 20 lesions whose sizes varied from 0,2 to 7 cm and the duration of the lesion diagnostic varied from 0,5 to 4 months, and in LM patients it varied from 0,5 to 120 months. The positive result of the AH, IDRM, IF and Elisa lab tests for IgG was 80%, 67,5%, 42,5% and 87,5% respectively. A cross - reactivity analysis was performed using samples of patients with blastomycosis, malaria, visceral leishmaniasis (LV), Chagas disease and toxoplasmosis and only one sample from an LV patient (10%) was positive, the test was 98,3% precise. The specific IgE detection occurred in 40% of the LTA patients. No correlation between the quantity of IgE and IDRM results was found. No correlation between the index of IgE and the number of lesions in LCL patients was observed. No association between clinical cure or no cure and the index of IgE reactivity was detected. A higher number of patients’ samples are necessary to obtain more accurate results. / As leishmanioses compreendem um grupo de doenças, com grande diversidade epidemiológica e clínica. A detecção de IgE pode constituir uma ferramenta valiosa para o prognóstico da Leishmaniose Tegumentar Americana (LTA). O objetivo do presente trabalho foi avaliar se Imunoglobulina E (IgE) anti - Leishmania podem ser utilizados como biomarcadores de prognóstico na LTA. Foi realizada a padronização do ensaio imunoenzimático (ELISA) utilizando extrato antigênico de L. (V.) braziliensis para detecção de IgE específica para leishmânia, nos plasmas de pacientes com LTA. Foi determinado que a diluição que discriminava amostras positivas de negativas era de 1/20 para o plasma e 1/100 para o anticorpo anti-IgE marcado com peroxidase. Dos 40 pacientes com LTA atendidos no ambulatório de endemias do Hospital Anuar Auad (HDT/GO), Goiânia-GO, entre 2009 e 2012, 27 apresentavam leishmaniose cutânea localizada (LCL) e 1 leishmaniose mucosa (LM). Destes pacientes, 27 eram do sexo masculino e 13, feminino. A idade dos pacientes com LCL variou entre 18 a 65 anos e com LM, de 25 a 61 anos. Os pacientes com LCL apresentavam de uma a 20 lesões, os tamanhos das lesões variaram entre 0,2 a 7 cm e o tempo das lesões ao diagnóstico variou de 0,5 a 4 meses e com LM de 0,5 a 120 meses. A positividade dos testes laboratoriais AH, IDRM, IF e Elisa para IgG foi de 80%, 67,5%, 42,5% e 87,5% respectivamente. Foi realizada análise de reatividade cruzada com amostras de pacientes com blastomicose, malária, leishmaniose visceral (LV), doença de chagas e toxoplasmose e apenas uma amostra de paciente com LV (10%) foi reagente, a especificidade do teste foi de 98,3%. A detecção de IgE específica ocorreu em 40% dos pacientes com LTA. Não houve correlação entre as quantidades de IgE e os resultados da IDRM. Não foi observada correlação entre os índices de IgE e o número de lesões em pacientes com LCL. Não foi detectada uma associação entre cura ou não cura clínica e os índices de reatividade de IgE. Um aumento do número de amostras de pacientes é necessário para melhorar a acurácia dos resultados.

Etablir le diagnostic d'une réaction d'hypersensibilité aiguë (RHA) peropératoire est difficile du fait de l'incidence supérieure des diagnostiques différentiels. Les facteurs de risques en sont mal établis. Il n'existe pas de signe clinique fiable aidant à un diagnostic rapide. Alors que 25% des explorations immunologiques classiques ne mettent pas en évidence un mécanisme IgE-médié, des études animales et humaines suggèrent un rôle des IgG dans ces RHA non caractérisées. Ce travail s'est basé sur deux cohortes de patients : la première pour étudier des phénotypes en lien avec l'apparition d'un bronchospasme, la seconde pour analyser des marqueurs diagnostiques au bloc opératoire et explorer un mécanisme alternatif impliquant les IgG. La survenue d'un bronchospasme au cours d'une RHA peropératoire n'est pas associée à un antécédent d'asthme mais au curare comme agent étiologique. La mise en évidence de la valeur diagnostique d'une hypocapnie inférieure à 20 mmHg dans le caractère sévère de la RHA devrait aider les cliniciens à l'établissement rapide du diagnostic. La présence d'IgG spécifiques anti-curares était associée à la survenue d'une RHA sévère suggérant une participation des IgG à la gravité de la RHA en association avec les IgE. En fonction du curare administré, le mécanisme de la RHA semble différent : implication d'anticorps (IgE et/ou IgG) dans 90% pour succinylcholine et rocuronium mais uniquement 50% pour atracurium suggérant une importante contribution de l'histamino-libération dans ces réactions. L'ensemble de ces travaux devraient permettre d'améliorer les performances diagnostiques en temps réel et à distance lors de la survenue d'une RHA peropératoire. / Diagnosis of intra-anesthetic acute hypersensitivity reactions (AHR) is challenging because of elevated incidences of differential diagnoses. Risk factors remain mostly unknown and there is no reliable clinical sign to help physicians in establishing a rapid diagnosis. In 25% of cases, immunologic exploration cannot identify the culprit agent through the exploration of the IgE-mediated pathway. Several animal and human studies suggest a role of IgG in the physiopathology of anaphylaxis, which could explain these uncharacterized AHR. This PhD has focused on two cohorts of patients: the first cohort allowed the exploration of phenotypic links in relation to bronchospasm occurrence; the second cohort analyzed clinical markers of severe AHR and the alternative pathway involving IgG against neuromuscular blocking agents (NMBA). Analysis of risk factors identified a NMBA as the culprit agent of the intra-anesthetic AHR to be the only factor statistically associated with bronchospasm. We propose that a hypocapnia below 20 mmHg may be a novel and useful tool for physicians for the rapid diagnosis of severe intra-anesthetic AHR. Among the second cohort of patients, NMBA-specific IgG were identified and associated with clinical severity, suggesting that they may participate in the severity of NMBA-AHR in association with IgE. The chemical structure of a given NMBA may dictate the mechanism of anaphylaxis to this particular NMBA: an IgE/IgG-pathway for succinylcholine and rocuronium, whereas atracurium may be rather linked to spontaneous histamine release mechanisms. Altogether, our results might improve diagnosis efficacy at the time of the AHR and during immunologic explorations.

The evolution of social behaviour has been studied using different frameworks based on game theory and quantitative genetics. While both approaches provide a conceptually clear explanation of evolution of social behaviour, both have been limited in their applicability to empirical systems, mainly due to difficulties in measuring model parameters. Here, I develop a new quantitative genetics approach to the study of the evolution of social behaviours based on indirect genetic effects (IGEs), which parameters can be readily determined by empirical studies. IGEs describe effects of an individual's genotype on phenotypes of social partners, which may indirectly affect their fitness. Unlike traditional quantitative genetics assuming a non-genetical, non-heritable environment, IGE models assume that part of the environment is social, provided by parents and other interacting partners, thus has a genetic basic and can be heritable. In this study I explore the effects of IGEs on the magnitude and range of phenotypic values in a focal individual. I show that social interactions may not only cause indirect genetic effects but can also modify direct genetic effects. I demonstrate that interactions can substantially alter group mean phenotype and variance. This may lead to scenarios in which between group phenotypic variation is much higher than within group variation despite similar underlying genetic properties of different groups. Further, I analyse how IGEs influence levels of selection and predictions about evolutionary trajectories. I show that IGEs can create selection pressure at the group level, leading to evolution of behaviours that would not evolve otherwise. Moreover, I demonstrate that IGEs may lead to differences in the direction of evolutionary response between genotypes and phenotypes. Building on these results, I show that IGE models can be translated to and are fully compatible with traditional kin and multilevel selection models. I express costs and benefits in IGE parameters and determine the conditions under which social interactions lead to the evolution of cooperative or harmful behaviours. Therefore, the model I propose combines the conceptual clarity of kin and multilevel selection models with the applicability of IGE models, which parameters can be empirically determined, facilitating the testing of model predictions. Finally, I show that the use of IGE models is strongly limited by the underlying assumption of linearity. I prove that the modelling of interaction dynamics leads to steady state solutions found by IGE models only under limited conditions. In this light, I discuss the relevance of results published previously and propose a solution of how this problem can be addressed.

Cannabinoid treatment increases Th2 activity and previous reports showed B cells express the highest level of CB2 mRNA relative to other immune cells suggesting that cannabinoids play a critical role in B cell activation and maturation. To examine the direct effect of cannabinoids on B cell antibody class switching, mouse splenic B cells were purified by negative selection and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective cannabinoid agonist, CP55940, or the CB1 selective agonist, methanandamide, or the CB2 selective agonist, JW015. The cultures were then analyzed at different times by flow cytometry for expression of B cell surface markers, such as CD19, CD138, CD40, MHCII, CD23, CD80, CD45R, immunoglobulins produced such as IgM, IgE, IgD, and IgG1, and Toll-like receptors such as TLR 2 and 4. Cells treated with CP55940 showed an increase in surface expression of IgE by day 5 in culture; methanandamide had no effect. CP55940 also induced an increase in secreted IgE in culture supernatants analyzed by ELISA. In addition, CB2 receptors were increased on B cells following stimulation with IL-4 and anti-CD40 and the class switching effect of CP55940 was attenuated by the CB2 antagonist, SR144528. We also observed that cannabinoid treatment of B cells modulates cell functions other than antibody class switching such as surface marker and TLR expression. CP55940 caused a significant increase in surface expression of TLR 4, but had no effect on other markers. Additional experiments with cannabinoid receptor selective agonists and antagonists suggested both CB1 and CB2 receptors were involved in the TLR effect. Receptor involvement and Gi coupling was supported by our findings that cannabinoids inhibit intracellular cAMP levels in forskolin stimulated B cells, and increasing intracellular cAMP with forskolin suppressed IgE antibody class switching in activated B cell cultures. These results suggest cannabinoids negatively regulate cAMP in B cells resulting in increased IgE. In conclusion, cannabinoids can directly affect the function of B cells by inducing antibody class switching to IgE and TLR4 expression through mechanisms involving CB1 and CB2 receptors suggesting the endocannabinoid system may be an important regulator of humoral immunity and the allergic response.

1. Abstract (EN) This thesis is collection of work focused mainly on the understanding of mast cell activation and its regulation by Orm1-like (ORMDL) proteins. ORMDL family is a group of endoplasmic reticulum (ER) membrane resident proteins that are highly conserved amongst mammalian species. ORMDL proteins can be found in diverse range of organisms from plants through fungi to animals. ORMDL proteins were first discovered in yeasts and the interest in these proteins skyrocketed after the discovery that ORMDL3 is associated with childhood onset asthma in genome wide association studies. Following research connected ORMDL3 also with allergic inflammation and inflammatory bowel disease. Since mast cells are mainly known for their role in allergy and allergen induced inflammation, we decided to investigate the role of ORMDL proteins in regulation of mast cell activation and signaling. In our first study we focused on the role of ORMDL3 in mast cell activation via the high affinity IgE receptor 1 (FcεRI). We prepared bone marrow-derived mast cells with decreased (ORMDL3-KD) or increased (ORMDL3-OE) ORMDL3 expression. We showed that ORMDL3 is a negative regulator of mast cell activation events like degranulation, cytokine release and migration, without any effect on calcium mobilization. ORMDL3 was previously...

Although glucocorticoids are often used as an adjunct to epinephrine to treat anaphylactic shock, glucocorticoids can also be a rare cause of anaphylactic shock. Only through the administration of a challenge dose of different glucocorticoids and different substrates that glucocorticoids are delivered in can the determination be made about which glucocorticoid or accompanying solvent may be the culprit which caused the anaphylactic reaction. These challenge tests should only be performed in a controlled environment as repeat anaphylaxis is a risk, especially if the patient has a history of glucocorticoid-induced anaphylaxis.

The purpose of this thesis project was to demonstrate the ability to utilize the capabilities of aptamers so that they may act as capture reagents for paper microfluidic devices. Several characterization experiments were conducted as a precursor before the final experimentation was performed. Paper characterization, manufacturing protocols for printing and heating, as well as 3D chip fabrication were all performed and analyzed. The results confirmed that the control of fluid through a 3D microfluidic device based in nitrocellulose is possible. For the biochemistry portion of this thesis report, antibodies and aptamers were chosen to react with IgE, an antibody that is present in high concentrations in the urine of patients diagnosed with respiratory distress. Antibody chips were successfully created as a baseline lateral flow assay for comparison to new aptamer detector reagents. The aptamer experiments were able to demonstrate that it is possible to utilize the capabilities of aptamers so that they may behave as capture reagents in paper microfluidic devices. Overall, the experiments performed were extremely supportive of the ability to develop the field of paper microfluidics with the use of aptamers so that patient populations across the globe can be more accurately and effectively diagnosed.

ZCa is an engineered three-helical affinity protein that through replacement of the loop between helix two and three with an EF-hand motif has a calcium-dependent binding to its target. By using the difference of calcium concentration in the plasma and the cell along with the favorable properties of ZCa, the interest in creating therapeutics toward different diseases based on the ZCa scaffold has come about. In this project novel variants from a phage display library based on the ZCa scaffold are characterized and their ability to bind to their intended target is evaluated. The targets used in this project are IgE for its involvement in allergic reactions and IL-23 for its ability to cause autoimmune diseases. Through several different analysis methods this project shows that novel variants towards different targets are capable of calcium-dependent target binding.