Submitted by CRISTIANE DE CASTRO PERNET HARA null (cris_hara@hotmail.com) on 2017-01-19T01:22:00Z No. of bitstreams: 1 tese Cristiane Hara (final).pdf: 5145540 bytes, checksum: 622fefbf8e4e6d0b87d3139952cc8f17 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-01-24T13:45:46Z (GMT) No. of bitstreams: 1 hara_ccp_dr_bot.pdf: 5145540 bytes, checksum: 622fefbf8e4e6d0b87d3139952cc8f17 (MD5) / Made available in DSpace on 2017-01-24T13:45:46Z (GMT). No. of bitstreams: 1 hara_ccp_dr_bot.pdf: 5145540 bytes, checksum: 622fefbf8e4e6d0b87d3139952cc8f17 (MD5) Previous issue date: 2016-12-01 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / INTRODUÇÃO - Durante a gravidez, a hiperglicemia materna altera a expressão e a transferência de células imunorregulatórias e de imunoglobulinas e o perfil de citocinas na interface materno-fetal. OBJETIVO – Avaliar, em gestações complicadas por hiperglicemia, a expressão de células NK e o perfil de citocinas no sangue, materno e do cordão umbilical, e na placenta (artigo 1); quantificar a produção de anticorpos e a passagem de IgG total, e respectivas subclasses, via receptor FcRn (artigo 2). MÉTODO - Foram avaliadas 120 gestantes, distribuídas nos grupos: não-diabético (ND; N = 30), hiperglicemia gestacional leve (HGL; N = 30), diabetes mellitus gestacional (DMG; N = 30) e diabetes mellitus tipo 2 (DM2; N = 30). Técnicas de citometria de fluxo foram utilizadas para análise de células e citocinas e, de ELISA, para avaliação das concentrações de IgG total e subclasses. A transferência placentária de anticorpos totais, e respectivas subclasses, foi definida pela relação [(concentrações no sangue de cordão umbilical/sangue materno) x 100]. Na análise estatística foram realizadas análises de variância (ANOVA), seguida pelo teste de Tukey, e de correlação de Pearson, com p < 0,05. RESULTADOS - No sangue materno dos grupos hiperglicêmicos, as células NK CD16+CD56– aumentaram, enquanto que CD16+CD56+ foi menor no grupo DMG. No sangue do cordão do grupo DM2 mostrou uma maior proporção de células CD16+CD56– e CD16–CD56+. As camadas extravilosas da placenta dos grupos DMG e DM2 mostraram maior expressão de CD16+CD56– e independente da camada, a proporção de CD16–CD56+ foi maior em HGL e DMG e menor em DM2. Em relação às citocinas, IL-2 foi menor no sangue materno e IFN-y mais elevados no sangue materno e do cordão do grupo DMG. IL-17 foi mais elevada no sangue materno e do cordão do grupo DM2. A camada extravilosa placentária da HGL mostrou níveis elevados de IL-4, IL-6, IL-10, IL-17, e IFN-γ e baixos níveis de IL-1β e IL-8, enquanto que a camada vilosa placentária continha alta níveis de IL-17 e IFN-γ. O grupo HGL, independentemente da região, apresentaram maiores níveis de IL-8. O grupo DM-2, independentemente da região da placenta mostrou níveis elevados de TNF-α, IL-17, e IFN-γ. O sangue materno de DM-2 e o sangue do cordão umbilical de HGL exibiram uma poporção mais elevada de CD19+ expressas por células B. DM2 mostrou a menor proporção de células CD19+na placenta. A expressão de FcRn aumentou nas células do cordão e placenta de HGL. As células do sangue materno, do sangue do cordão e da placenta de DM2 mostraram menor expressão de FcRn. A maior expressão de FcRn, independente do estado hiperglicêmico foi observada nas células da placenta. Houve menores níveis de IgG no sangue materno em DM2 e maiores no sangue do cordão umbilical de HGL. Os níveis mais elevados de IgG4 foi detectado no sangue de mães hiperglicêmicas. Os níveis mais elevados de IgG3 e IgG4 no sangue do cordão foram detectados em HGL, e os menores de IgG2 e IgG3 níveis em DM2. CONCLUSÃO - A hiperglicemia produz ambiente inflamatório com elevada produção de citocinas, apresentando alterações na expressão de células NK, de FcRn na placenta, na produção e taxa de transferência de IgG. Os níveis celulares que expressam CD16+ e de citocinas no sangue materno, sangue do cordão umbilical e tecido placentário são modificados em gestações complicadas pelo diabetes. A hiperglicemia materna compromete a transferência placentária de IgG1, IgG3 e IgG4. Os resultados sugerem que a hiperglicemia materna, diminui a expressão FcRn em células da placenta e sangue e compromete a produção e transferência de anticorpos maternos para os recém-nascidos. / INTRODUCTION - During pregnancy, the immune response associated with diabetes alters the expression and the transfer of immune cells, including regulatory, immunoglobulins and the profile of cytokines in the maternal-fetal interface. OBJECTIVE - To evaluate the expression of NK cells, and the profile of cytokines in maternal blood, umbilical cord and placenta, quantify the production of antibodies, as well as, the passage of IgG and subclasses, via receptors FcRn in pregnancies complicated by diabetes or hyperglycemia. METHOD - were assessed 120 pregnant women, distributed as non-diabetic (ND; n=30), Mild Gestational Hyperglycemia (MGH; n=30), Gestational Diabetes Mellitus (GDM; n=30) and type 2 Diabetes Mellitus (DM2; N=30). The cells and cytokines were evaluated by flow cytometry. The concentrations of total IgG and subclasses were analyzed by ELISA. Placental transfer of the total and subclasses antibodies were defined in each assay by the ratio [(cord concentrations/maternal concentrations) x 100]. In the statistical analysis we used analysis of variance (ANOVA), followed by Tukey test, and Pearson's linear correlation, with p < 0.05. RESULTS - In the maternal blood from the hyperglycemic groups, the CD16+CD56− NK cells increased, whereas that of CD16+CD56+ decreased in GDM group. Cord blood from DM2 showed a higher proportion of CD16+CD56− and CD16−CD56+. The placental extravillous layer of GDM and DM2 showed an increase of CD16+CD56− cells and, irrespective of region, the proportion of CD16−CD56+ cells was higher in MGH and GDM and lower in DM-2. IL-2 was lower in maternal blood and IFN-����������������������� higher in maternal and cord blood from the GDM group. IL-17 was higher in maternal and cord blood from the DM-2 group. The placental extravillous layer of the MGH showed high levels of IL-4, IL-6, IL-10, IL- 17, and IFN-����������������������� and low levels of IL-1����������������������� and IL-8, whereas the placental villous layer contained high levels of IL-17 and IFN-�����������������������. The GDM group, irrespective of region, showed higher levels of IL-8. The DM-2 group, irrespective of region, placenta showed high levels of TNF-�����������������������, IL-17, and IFN-�����������������������. Maternal blood from DM-2 and cord blood from MGH exhibited a higher proportion of CD19+ expression by B cells. DM-2 showed a lower proportion of CD19+ cells in placenta. FcRn expression increased in cells from cord blood and placenta from MGH. Maternal blood, cord blood and placenta cells from DM-2 showed lower FcRn expression. The highest FcRn expression, irrespective of glycemic status, was observed in placenta cells. Maternal blood IgG levels were lower in DM-2, and cord blood IgG levels were higher in MGH. The highest levels of IgG4 were detected in the blood of hyperglycemic mothers. The highest IgG3 and IgG4 levels in cord blood were detected in MGH, and the lowest IgG2 and IgG3 levels in DM-2. CONCLUSIONS - Hyperglycemia produces inflammatory environment with high production of cytokines, presenting changes in expression of NK cells, FcRn in the placenta, in production and rate of transfer of IgG. The levels of cells expressing CD16+ and cytokines in maternal blood, cord blood, and placental tissue are modified in pregnancies complicated by diabetes. Maternal hyperglycemia compromised placental transfer of IgG1, IgG3 and IgG4. The results suggest that regardless of hyperglycemia degree, it decreases FcRn expression in placenta and blood cells and compromises the production and transfer of antibodies from maternal blood to newborns. / FAPESP: 2012/24212-4 / FAPESP: 2013/13017-9

The bacteria Streptococcus pyogenes produces a multitude of proteins which interact with and alter the functions of the host immune system. Two such proteins, Endoglycosidase S (EndoS) and Immunoglobulin G-degrading enzyme from S. pyogenes (IdeS) are able to specifically alter the effector functions of immunoglobulin G (IgG). EndoS is a glycoside hydrolase which removes the conserved <i>N</i>-linked glycan from IgG Fc whereas IdeS is a cysteine protease that cleaves the exible protein hinge of IgG. The activity of both proteins results in the reduced ability of IgG to elicit immune responses through Fc receptor binding and complement activation. Amongst other applications, both EndoS and IdeS are actively being explored as new therapeutics for IgG-mediated autoimmune diseases. Given the therapeutic potential of EndoS and IdeS, experiments were designed to investigate the structural and functional characteristics of these enzymes in an effort to understand their specficity for and activity against IgG. Here, bioinformatic and biophysical characterisation of EndoS identified subdomains outside of the catalytic domain which contribute to glycoside hydrolase activity. The substrate specificity of EndoS was also explored and showed that EndoS hydrolyses a broad range of glycans from the IgG scaffold. EndoS was also shown to have activity against alternative glycoprotein substrates, however, this non-specific activity was negligible in the context of whole serum. The effect of EndoS-mediated deglycosylation on the structure of the IgG Fc domain was explored using both X-ray crystallography and small-angle X-ray scattering. Small angle X-ray scattering was also used to characterise both EndoS and IdeS in complex with IgG Fc. Solution-state models of each complex were produced providing preliminary data towards how these enzymes interact with IgG. Overall, the results presented here contribute to our understanding of these enzymes which is of importance as they go forward into clinical applications.

This thesis deals with biosensors based on modified semiconductor core/shell quantum dots (QDs) for diagnosis. The work is divided into four main parts. The first one discusses the theory required for the use of QDs in bioaplications, there are described methods of synthesis, modification, application and bioconjugation of QDs. In the experimental part, CdTe/ZnS QDs with core/shell structure were prepared, in which the core was modified by MPA, GSH and TGA. In the next step, these QDs were further modified using CDI, EDC and NHS as mediators in order to increase affinity to BSA (bovine serum albumine) and IgG (imunoglobuline G). Prepared conjugates were characterized by fluorescence spectroscopy (Infinite M200Pro, Tecan) and capillary electrophoresis (Agilent 7100).

Department of Animal Science / MSCAGR (Animal Science)

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21771号 / 工博第4588号 / 新制||工||1715(附属図書館) / 京都大学大学院工学研究科電子工学専攻 / (主査)教授 山田 啓文, 教授 川上 養一, 教授 田畑 修 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Primary sclerosing cholangitis (PSC) is a rare inflammatory chronic liver disease that causes damage to the intra- and or extrahepatic bile ducts leading to cholestasis. As the disease proceeds the development of cirrhosis and eventually liver failure occurs. This study aims to determine the role of IgG subclasses in the prognosis of PSC and its outcome. A retrospective analysis was performed of 183 patients followed at the Department of Upper Abdominal Diseases at the Karolinska University Hospital. Factors that were analysed were sex, age at PSC diagnosis, total IgG values, IgG subclasses values and events of autoimmune hepatitis (AIH), inflammatory bowel disease (IBD), colectomy, cirrhosis, cholangitis, endoscopic retrograde cholangiopancreatography (ERCP), liver transplantation and cholangiocarcinoma. This study showed that high IgG4 levels were associated with a higher incidence of cirrhosis, liver transplantation, cholangitis and ERCP, while low IgG4 levels were associated with a prior IBD diagnosis. In conclusion, elevated IgG4 levels were associated with a higher occurrence of cirrhosis, cholangitis, ERCP and liver transplantation. It seems that IgG4 could be of importance for outcome prediction in PSC.

No description available.

No description available.

Background: Post COVID-19 Condition (PCC, also known as long COVID and post-acute sequelae of COVID-19) is a major public health concern with severe and pervasive impacts on physical and mental health. PCC is highly heterogeneous and may manifest as different clusters of symptoms of varying intensity and duration. The etiology of PCC remains uncertain, though several underlying pathophysiological mechanisms, such as cellular damage, inflammatory cytokines, and a hypercoagulable state, are thought to contribute to PCC inception and trajectory. Examination of potential serological markers of PCC, accounting for clinical covariates, may yield emergent pathophysiological insights. Objectives: Primary objectives of this thesis are to 1) Identify key clinical and potential serological predictors of PCC; 2) Acquire clinical and serological data in a large-scale prospective observational study; 3) Assess relationships between PCC and serological markers, accounting for clinical covariates; 4) Systematically review evidence to date on primary observational studies comparing serological response between people with and without persistent symptoms post COVID-19 recovery; 5) Discuss persisting gaps in knowledge and data quality, and propose strategies for resolve. Methods: This thesis is framed around three core efforts: 1) The design of survey questions and study materials, recruitment of participants, and data collection in a large-scale prospective cohort study launched in 2020; 2) The assessment of relationships between pre-defined serological predictors and PCC, accounting for clinical covariates; and 3) A robust rapid review of PCC onset and phenotype as functions of serological markers. Expert opinion was sought to define serological predictors. Clinical predictors were defined a priori based on systematic reviews meeting AMSTAR 2 guidelines. Conclusions: To address objectives, we described efforts to collect clinical and serological data from a large-scale prospective cohort study; identify PCC-cases and infected-controls; assess associations between pre-defined serological predictors (IgG titres targeting SARS-CoV-2 spike (S), nucleocapsid (N), and receiver binding domain (RBD) antigens, and efficient neutralization) and PCC; and synthesized findings from an extensive rapid review on PCC as a function of serological markers. Our multivariate analysis using Stop the Spread Ottawa data is, to our knowledge, the first Canadian study to report the direction and magnitude of association between selected serological predictors (anti-IgG response to S, N, and RBD SARS-CoV-2 antigens, and neutralizing efficiency) and PCC status and impact on quality of life. Finally, we described five potential strategies which may improve the accessibility, quality, and amalgamation of data pertaining to PCC: 1) Fostering comparability between studies to enable synthesis of multiple datasets; 2) Advancing the characterization and consensus on PCC phenotypes; 3) Employing innovative modelling strategies that could potentially yield novel insights; 4) Promoting robust collaboration and knowledge sharing among research teams; and 5) Engaging people with lived experience at all stages of research.

Intimina é uma adesina de natureza protéica das bactérias diarreiogênicas Escherichia coli enteropatogenica (EPEC) e enterohemorrágica (EHEC), capazes de induzir a lesão \'attaching e effacing\' em enterócitos. Os principais subtipos de intiminas de EPEC e EHEC prevalentes no Brasil são &#945;, &#946; e &#947;. Nosso objetivo foi investigar a transferência de anticorpos maternos anti-intiminas aos recém-nascidos de mães saudáveis de São Paulo, Brasil. Foram pesquisados anticorpos SIgA no colostro e IgG no soro de 50 mulheres saudáveis e no soro de cordão umbilical de seus recém-nascidos, por ELISA utilizando como antígeno proteínas recombinantes purificadas das regiões conservadas e variáveis de intiminas &#945;, &#946; e &#947;. As concentrações de anticorpos no colostro foram superiores quando comparadas com as concentrações do soro para todos os tipos de intiminas. Não se observaram diferenças estatísticas entre as concentrações de anticorpos reativos com as diferentes intiminas nas amostras de colostro. As concentrações de anticorpos reativos com a região conservada da intimina foram significativamente mais elevadas em comparação com as regiões variáveis no soro dos grupos de mães e de recém-nascidos. Houve alta correlação entre todos os anticorpos anti-intiminas nas amostras de colostro. Comparando-se as concentrações de anticorpos séricos, os coeficientes foram maiores entre anti-&#945; e anti-&#946; que entre os outros pares. Nossos resultados confirmam a transferência de anticorpos maternos para o recém-nascido pela placenta e pelo aleitamento materno e reforça o efeito protetor da amamentação contra infecção por EPEC. / Intimin is a proteic adhesin of enteropatogenic (EPEC) and enterohemorragic (EHEC) Escherichia coli, capable of inducing attachment and effacement lesion in enterocytes. The main subtypes of intimins of EPEC and EHEC prevalent in Brazil are &#945;, &#946; and &#947;. Our aim is to investigate the transference of maternal anti-intimin antibodies to the newborns of healthy mothers from Sao Paulo, Brazil. IgG and SIgA antibodies were determined in sera and colostrum from 50 healthy women and cord sera from their newborns, by ELISA using as antigens purified recombinant proteins, conserved and variable regions of &#945;, &#946; and &#947; intimins. The IgA antibody concentrations of colostrum are higher than IgG antibodies in serum for all intimins and there were no statistical differences between them in colostrum samples. The concentrations of antibodies reactive with the conserved region of intimin are significantly higher compared to the variable regions in the sera groups, mothers and newborns. There were high correlation coefficients between all the anti-intimins antibodies in colostrum samples. In the comparison of the seric antibody concentrations, the coefficients were higher between anti-&#945; and anti-&#946; than all the other pairs. Our results confirm the transference of maternal antibodies to the newborns by placenta and breastfeeding and reinforce the high protection effect of breastfeeding against EPEC infection.