Streptococcus pneumoniae é o agente causador de diversas doenças, tais como meningite e pneumonia. PspC (Pneumococcal surface protein C) foi descrito como um importante candidato vacinal proteico de ampla cobertura e com baixo custo de produção. Trata-se de um fator de virulência, capaz de ligar-se ao Fator H (FH) e a IgA secretório (sIgA). Como PspC é um antígeno polimórfico, é crucial a avaliação da sua variabilidade. Foi determinado o grupo de PspC de treze linhagens de pneumococo isoladas no Hospital Universitário da Universidade de São Paulo. Soros contra diferentes grupos de PspC foram produzidos e PspC do grupo 3 (PspC3) foi capaz de induzir anticorpos que reconhecem diferentes grupos de PspC. Foi observada ainda uma pequena redução na ligação de FH e sIgA por anticorpos anti-PspC3 em ensaios in vitro. No entanto, não foi possível observar proteção contra um modelo de colonização da nasofaringe de camundongos através da imunização com PspC3, possivelmente por deficiências no modelo experimental. / Streptococcus pneumoniae is the causative agent of several diseases, such as meningitis and pneumonia. PspC (Pneumococcal surface protein C) has been described as an important vaccine candidate protein as it could provide wide coverage with low cost of production. PspC is a virulence factor capable of binding to Factor H (FH) and secretory IgA (sIgA). PspC is polymorphic antigen, and therefore it is crucial to evaluate its variability. In the present work we have determined the PspC group of 13 pneumococcal isolates obtained at the University Hospital of the University of São Paulo. Antisera against different PspC groups were produced and PspC group 3 (PspC3) was able to induce antibodies that recognized different groups of PspC. Antibodies to PspC3 reduced binding of FH and sIgA to pneumococcus in in vitro assays. However, no protection was observed against a murine model of nasopharyngeal colonization by immunization with PspC3. This was possibly due to deficiencies in the experimental model.

O rotavírus é a principal causa de diarréia em crianças em todo o mundo. Infecta também adultos, mas não há dados completos sobre a sua incidência nesse grupo nem sobre o papel de anticorpos preexistentes na proteção contra o vírus. O objetivo do trabalho foi avaliar a presença de anticorpos IgA e IgG anti-rotavírus SA-11, por ELISA, em amostras de soro de adultos saudáveis e sua ação neutralizante frente ao vírus, em ensaios de neutralização. Por Immunoblotting foi avaliado o reconhecimento de proteínas virais pelos anticorpos séricos. Observou-se que os títulos das amostras foram muito variáveis, sendo os de IgG superiores aos de IgA. Todas as amostras mostraram-se capazes de neutralizar o vírus em diferentes níveis, porém não foi possível estabelecer uma correlação com os títulos de anticorpos. Foi observado que anticorpos da classe IgG reconhecem mais proteínas virais que os da classe IgA. Este trabalho pode ser considerado mais um passo na elucidação do papel dos anticorpos séricos IgA e IgG anti-rotavírus na infecção em adultos. / Rotavirus has been considered the leading cause of diarrhea in children worldwide. The virus also infects adults but there is no conclusive data neither on the incidence of infection on this group nor on the role of pre-existing antibodies. The aim of the work was to evaluate the presence of anti-rotavirus SA-11 IgA and IgG by ELISA in serum samples of healthy adults and the serum neutralizing ability against the virus by neutralization assays. Immunoblotting was used to evaluate viral proteins recognition by serum antibodies. The antibody titers were extremely variable where IgG titers are greater than IgA ones. All samples were able to neutralize the virus in different levels but it was not possible to establish a correlation between antibody titers and neutralization ones. Immunoblotting assays revealed that IgG antibodies recognize more viral proteins than IgA did. This work can be considered a valuable step for elucidating the role of serum anti-rotavirus IgG and IgA antibodies in adults infection.

Signalling through the EGF receptor is subject to a complex and multilayered regulation. One such mode of regulation is through control of ligand production which plays an important role in fine- tuning EGF receptor activation. In mammals, the production of soluble, biologically active forms of EGF receptor ligands relies on ADAM metalloproteases, predominantly ADAM10 and ADAM17. Recently, a pseudoprotease from the rhomboid-like family of intramembrane proteases, iRhom, emerged as a key positive regulator of ADAM17. However, Drosophila iRhom has also been implicated in the negative regulation of EGF receptor signalling by promoting the degradation of precursors of its ligands. Cell culture based assays suggest that mammalian iRhoms might also be involved in a similar process. In this thesis, the effect of mammalian iRhom overexpression on the levels of EGF receptor ligands has been investigated. Contrary to previous findings, the data presented in this thesis suggest that the observed effect might not be entirely iRhom specific, for the inactive mutants of rhomboid proteases also diminish the levels of EGF receptor ligands. Nor do we find the effect to be specific to EGF receptor ligands, as unrelated transmembrane proteins were also depleted by iRhom overexpression. The coexpression of ADAM17 was...

Signalling through the EGF receptor is subject to a complex and multilayered regulation. One such mode of regulation is through control of ligand production which plays an important role in fine- tuning EGF receptor activation. In mammals, the production of soluble, biologically active forms of EGF receptor ligands relies on ADAM metalloproteases, predominantly ADAM10 and ADAM17. Recently, a pseudoprotease from the rhomboid-like family of intramembrane proteases, iRhom, emerged as a key positive regulator of ADAM17. However, Drosophila iRhom has also been implicated in the negative regulation of EGF receptor signalling by promoting the degradation of precursors of its ligands. Cell culture based assays suggest that mammalian iRhoms might also be involved in a similar process. In this thesis, the effect of mammalian iRhom overexpression on the levels of EGF receptor ligands has been investigated. Contrary to previous findings, the data presented in this thesis suggest that the observed effect might not be entirely iRhom specific, for the inactive mutants of rhomboid proteases also diminish the levels of EGF receptor ligands. Nor do we find the effect to be specific to EGF receptor ligands, as unrelated transmembrane proteins were also depleted by iRhom overexpression. The coexpression of ADAM17 was...

Orientador: Rinaldo Poncio Mendes / Coorientador: James Venturini / Coorientador: Ana Pardini Vicentini / Coorientador: Daniela Vanessa Moris de Oliveira / Banca: Mário León Silva-Vergara / Banca: Anamaria Mello Miranda Peniago / Resumo: O reaparecimento de manifestações clínicas após tratamento eficaz, neste texto identificado como recaída, tem sido pouco avaliado. Assim, foram estudados os casos de recaída observados em 400 pacientes, 93 com a forma aguda/subaguda (FA) e 307 com a crônica (FC), que já tinham apresentado cura aparente, isto é, com cura clínica, normalização da velocidade de hemossedimentação e cura sorológica - caracterizada pela presença de teste negativo à imunodifusão dupla em gel de agar por dois anos. Vinte e um (5,2%) desses pacientes apresentaram recaídas da doença. Três (14,3%) eram do sexo feminino e 18 (85,7%) do masculino, com razão de masculinidade de 6:1. Dos 21 pacientes com recaída, 15 (4,8%) apresentavam a FC e 6 (6,4%) a FA (p>0,05). As reativações ocorreram de 46 a 296 meses após introdução do tratamento (Md=96) e de 4 a 267 meses (Md= 60) após sua suspensão. As formas clínicas não diferiram quanto aos tempos decorridos até a reativação. O diagnóstico sorológico de recaída pela IDD foi feito em apenas 45% dos casos, o que levou à avaliação de outros testes para esse fim. Assim, foi realizado o enzimaimunoensaio (ELISA) e o immunoblotting (IB). A sensibilidade da IDD e do ELISA / 0,710 foi 76,1% em amostras de soro obtidos no pré-tratamento (p=0,25). No diagnóstico de recaída, a sensibilidade da IDD foi menor que no pré-tratamento (80% vs 45%; p=0,017), enquanto o ELISA / 0,710 foi igual (80% vs 65%;p=0,125). A sensibilidade do IB para diagnóstico de recaída foi de 12,5% na identificação da gp70 e 43,8% na da gp43 (p<0,05). A avaliação da acurácia do teste ELISA revelou sensibilidade de 96%, especificidade de 95%, valor preditivo positivo de 95%, valor preditivo negativo de 96% e acurácia de 95,5% quando o cut-off utilizado foi a densidade óptica de 0,710, obtido em função da construção da receiver operator characteristc - ROC, para um intervalo de confiança ... / Abstract: The reappearance of clinical manifestations after efficacious treatment, here identified as relapse, has been rarely evaluated. Thus, the cases of relapse observed in a cohort of 400 patients, 93 with acute/subacute form (AF) and 307 with chronic form (CF) were studied. They had already reached apparent cure, characterized as clinical cure, normalization of the erythrocyte sedimentation rate and serological cure, with a negative double agar gel immunodiffusion test (DID) for two years after antifungal discontinuation. Twenty-one (5.2%) of these patients had relapses. Three (14.3%) were female and 18 (85.7%) were male, with a male:female ratio of 6:1. Of the 21 relapsed patients, 15 (4.8%) presented the CF and 6 (6.4%) the AF (p>0.05). The relapse occurred 46-296 months after introduction of the treatment (Md=8 yrs) and from 4 to 267 months (Md=5 yrs) after withdrawal. Clinical forms did not differ regarding to the time elapsed until relapse. DID was positive in only 45% of the relapsed cases, which led to the evaluation of other tests to diagnose this condition. Thus, the enzyme-linked immunosorbent assay (ELISA) was standardized and the cut off was determined using the curve receiver operator characteristic - ROC and confidence intervals of 95% and 99%, showing optical densities of 0.710 and 0.850, respectively. Then, serological evaluation was performed using ELISA/0.710 and ELISA/0.850, and immunoblotting identifying gp43 (IBgp43) and gp70 (IBgp70). ELISA 0.710 and DID showed the same sensitivity, 76.1%, in serum samples obtained before treatment (p=0.25). DID sensitivity was lower at relapse than before the initial treatment (45% vs 80%; p=0.017), whereas ELISA/0,710 was the same (65% vs 80%; p=0.125). IBgp70 showed a 12.5% and IBgp43 a 43.8% sensitivity for diagnosing relapse (p<0.05). ELISA/0.710 showed a 96% sensitivity, 95% specificity, 95% positive predictive value, 96% negative ... / Mestre

M.Sc. / Heat shock proteins (HSP) are a group of highly conserved proteins induced in pro- and eukaryotes by a wide variety of environmental stresses such as heat shock (HS) and oxidative injury. HSP are classified into families according to their apparent molecular mass and respective inducers. Induction of HSP is primarily regulated on transcriptional level through multiple copies of a conserved cis-acting heat shock element (HSE) in the promoter region of all hsp genes to which the heat shock transcription factor (HSF) binds. Members of the HSP family function collectively as molecular chaperone systems, and fulfil essential roles under normal conditions and provide protection and adaptation during and following stress. The induction of HSP following stress and the subsequent protection confer HSP the potential application in stress therapy and in biomarking of stress. During a previous study in which the effect of Mycobacterium tuberculosis (M.tb) on the stress response of peripheral blood moncytes (PBM) from different donors was investigated, it was observed that different individuals from different South African populations showed differential a HSP synthesis in response to M.tb. This compelled us to investigate the following: Variation in HSP synthesis in peripheral blood monocytes (PBM) from different individuals in response to the classical HSP inducer, HS. The most appropriate technique to study HSP expression on protein level. HSP synthesis was studied in PBM from 36 individuals (European (E): n=22; non-Europeans (nE): n=14) using biometabolic labelling. Three techniques were compared in the determination of HSP expression in six donors in terms of HSP synthesis, which is measured by biometabolic labelling, and accumulation of hsp70 that were measured by Western blot analysis and flow cytometry. Results obtained are : European (E) and non-European (nE) populations differed significantly (p < 0.05) from each other in spite of a prominent variation in HSP synthesis within donors ; Flow cytometry is the technique of choice for the analysis of HSP levels, since it allows fast and safe measurement of HSP levels in single cel populations within a mixed population. Data from flow cytometry correlate with Western blot analysis, but not with biometabolic labelling. The means and ranges for different HSP synthesis in different populations reported in this study, set a standard for the use of HSP as biomarker of pa environmental stress for populations inhabiting southern Africa. Efficient measurement of HSP expression as biomarker of stress can therefore be implemented in routine analysis of environmental stress, as well as investigations concerning the implications of HSP in pathology.

Liu , Huan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 43-45). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.iv / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Gel electrophoresis of proteins --- p.1 / Chapter 1.1.1 --- Principles of gel electrophoresis --- p.1 / Chapter 1.1.2 --- Polyacrylamide gel --- p.3 / Chapter 1.1.3 --- Buffer systems --- p.5 / Chapter 1.1.4 --- Capillary electrophoresis --- p.6 / Chapter 1.2 --- Methods to transfer proteins from gel to membrane --- p.7 / Chapter 1.2.1 --- Simple diffusion --- p.8 / Chapter 1.2.2 --- Ultrasound transfer --- p.8 / Chapter 1.2.3 --- Tank transfer --- p.9 / Chapter 1.2.4 --- Semidry transfer --- p.9 / Chapter 1.2.5 --- Transfer efficiency improvements --- p.10 / Chapter 1.3 --- Visualizing immunoblots --- p.11 / Chapter 1.3.1 --- Membrane staining --- p.11 / Chapter 1.3.2 --- Radiometric detection in blotting --- p.12 / Chapter 1.3.3 --- Bioluminescence-enhanced detection --- p.13 / Chapter 1.3.4 --- Chemiluminescence-enhanced detection --- p.14 / Chapter 1.4 --- Miniaturized electrophoresis and blotting methods --- p.15 / Chapter 1.5 --- Objective of the project --- p.18 / Chapter Chapter 2 --- Dip-pen gel electrophoresis in capillaries --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Experimental section --- p.21 / Chapter 2.2.1 --- Materials and reagents --- p.21 / Chapter 2.2.2 --- PA gel fabrication in capillary --- p.22 / Chapter 2.2.3 --- Setup for electrophoresis in capillary --- p.23 / Chapter 2.3 --- Results and discussion --- p.24 / Chapter 2.3.1 --- PA gel polymerization quality at tip --- p.24 / Chapter 2.3.2 --- Separation efficiency in capillary --- p.25 / Chapter 2.4 --- Conclusions --- p.27 / Chapter Chapter 3 --- Western blotting by dip-pen electrophoresis --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Experimental section --- p.30 / Chapter 3.2.1 --- Materials and reagents --- p.30 / Chapter 3.2.2 --- Protein sample preparation --- p.31 / Chapter 3.2.3 --- Dip-pen electrophoresis based Western blot --- p.31 / Chapter 3.2.4 --- Detection on membrane --- p.32 / Chapter 3.3 --- Results and discussion --- p.33 / Chapter 3.3.1 --- Separation performance on nitrocellulose membrane --- p.33 / Chapter 3.3.2 --- Comparison among different %T PA gel --- p.34 / Chapter 3.3.3 --- SDS-protein complexes capture and immunoblotting --- p.38 / Chapter 3.4 --- Conclusions --- p.39 / Chapter Chapter 4 --- Conclusions --- p.40 / Chapter 4.1 --- Summary --- p.40 / Chapter 4.2 --- Future perspective --- p.41 / References --- p.43

<p>ABSTRACT</p><p>Allergy to mustard has been reported for many years, in some cases as severe anaphylactic reactions. Recent studies imply that this allergy is increasing. Three major allergens have been isolated and characterised; Sin a 1 and Sin a 2 in yellow mustard (Sinapis alba), and Bra j 1 in oriental mustard (Brassica juncea). Yellow mustard and black mustard (Brassica nigra) are the most common species in Europe, whereas oriental mustard is more frequent outside Europe. Mustard plants belong to the Brassicaceae/Cruciferae family. Mustard is present as an ingredient in different foods, sauces and spices, often in small amounts. According to the European labelling directives, mustard and products thereof must always be declared. To monitor this regulation, methods need to be developed to detect mustard. Polyclonal antibodies, produced in rabbits, against yellow and black mustard were characterised with immunodiffusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and immunoblotting. Rocket-immunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA) were developed for the detection and quantification of mustard protein. With indirect competitive ELISA a concentration of 156ng mustard protein per ml food extract was detected, which is more than enough to cover the lowest reported reactive doses.</p>

ABSTRACT Allergy to mustard has been reported for many years, in some cases as severe anaphylactic reactions. Recent studies imply that this allergy is increasing. Three major allergens have been isolated and characterised; Sin a 1 and Sin a 2 in yellow mustard (Sinapis alba), and Bra j 1 in oriental mustard (Brassica juncea). Yellow mustard and black mustard (Brassica nigra) are the most common species in Europe, whereas oriental mustard is more frequent outside Europe. Mustard plants belong to the Brassicaceae/Cruciferae family. Mustard is present as an ingredient in different foods, sauces and spices, often in small amounts. According to the European labelling directives, mustard and products thereof must always be declared. To monitor this regulation, methods need to be developed to detect mustard. Polyclonal antibodies, produced in rabbits, against yellow and black mustard were characterised with immunodiffusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and immunoblotting. Rocket-immunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA) were developed for the detection and quantification of mustard protein. With indirect competitive ELISA a concentration of 156ng mustard protein per ml food extract was detected, which is more than enough to cover the lowest reported reactive doses.

Monoclonal antibodies (MCA) produced against infectious laryngotracheitis virus (ILTV) of chickens reacted in western blotting experiments with several different ILTV protein bands in the absence of tunicamycin which inhibits carbohydrate synthesis. Most of the MCA lost their reactivity in western blotting experiments when extracts of tunicamycin-treated ILTV CELC were used, suggesting their specificity for carbohydrate-based epitopes. In an indirect immunofluorescence test most of the MCA bound primarily to cytoplasmic antigens except some MCA which bound primarily to nuclear antigens. Additivity ELISA was also performed to study whether MCA are against the same epitope or different epitopes. The polymerase chain reaction (PCR) was developed as a diagnostic technique for detection of ILTV using primers made from a portion of the ILTV thymidine kinase gene. The 647-basepair amplified ILTV PCR product was labeled to create a non-radioactive, biotinylated DNA probe. Hybridization was performed using the probe to detect ILTV. Both PCR and hybridization detected ILTV, and neither hybridization nor PCR gave positive results with any other pathogen. Hybridization was specific for ILTV, However, slight hybridization occurred with CELC DNA when relatively relaxed conditions were used. In another experiment, diagnostic tests to detect ILTV in tracheas of experimentally-infected chickens, including the indirect fluorescent antibody test (IFAT), immunoperoxidase (IP), virus isolation (VI), histopathology, PCR, and hybridization, were performed and compared. Using virus isolation as a reference, the sensitivity and specificity of the tests were calculated. The IP test and IFAT performed better than any other test used in this study. / Graduation date: 1995