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published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Histidine-rich glycoprotein (HRG) is a plasma protein that regulates key cardiovascular processes such as coagulation, angiogenesis and immune response. The protein consists of six distinct functional domains: two N-terminal domains (N1 and N2), two proline-rich regions (PRR1 and PRR2), a central histidine-rich region (HRR) and a C-terminal domain. The HRR binds Zn²⁺, which alters the affinity of HRG towards various ligands including the anticoagulant, heparin. A key aim of this study was to structurally characterise HRG. The 1.93 Å crystal structure of the HRG N2 domain presented here represents the first crystallographic snapshot of the molecule. The N2 domain is cystatin-like and N-glycosylated at Asn184. An S-glutathionyl adduct was observed at Cys185, providing in vivo evidence that release of an anti-angiogenic HRR/PRR fragment is controlled in part by a redox mechanism, representing a novel further role for GSH in regulation of angiogenesis. Since Zn²⁺ regulates some of the functions of HRG, the dynamics of Zn²⁺ in plasma were investigated using a combination of ITC, ELISA and thrombin assay systems. Zn²⁺ is normally associated with albumin in circulation, but its ability to bind Zn²⁺ is allosterically inhibited upon fatty acids binding to albumin. Elevated plasma fatty acid levels are associated with some disease states. It is proposed that this may alter the proportion of Zn²⁺ bound to HRG, which could in turn activate thrombin to promote coagulation. These studies provide evidence to suggest that Zn²⁺-dependent activation of HRG (following fatty acid binding to albumin) may play a role in the development of haemostatic complications in susceptible individuals. Finally, the Zn²⁺ binding ability of albumin was probed in order to locate unidentified sites using recombinant albumin mutants. H9A, H67A, E252A, D256A and H288A mutants all exhibited diminished Zn²⁺ binding ability, indicating that these residues are involved directly or indirectly in Zn²⁺ binding.

A glicosilação é uma das modificações mais comuns ocorridas naturalmente nas cadeias polipeptídicas. As glicoproteínas exercem papéis essenciais para os seres vivos desde o ínicio vida e, por essa razão, qualquer mutação nos resíduos de açúcares a elas ligados causam diversos efeitos não desejados ao indivíduo. O padrão de glicosilação de proteínas é regido tanto por fatores genéticos quanto por fatores externos. Em relação aos defeitos de glicosilação hereditários, diversas mutações em genes específicos causam anormalidades na síntese de glicoproteínas. No grupo de doenças causadas por defeitos de glicosilação hereditários estão incluídas algumas distrofias musculares relacionadas a mutações em proteínas que são glicosiltransferases comprovadas ou putativas. O uso de modelos animais facilita o estudo dessas doenças neuromusculares e, por isso, o presente trabalho foi desenvolvido utilizando tecidos de camundongos controle (C57Black6) e LARGE. O camundongo LARGE possui características fenotípicas semelhantes às de humanos afetados pela distrofia muscular congênita tipo 1D. Diferentes estratégias de análise e de instrumentação foram empregadas para a obtenção de informações relacionadas tanto ao conjunto de glicoproteínas em geral quanto à alfa-distroglicana especificamente. A alfa-distroglicana mostra-se modificada em relação aos resíduos de açúcares nela ligados em animais com mutação no gene LARGE, resultando em diversos problemas de saúde. A técnica de eletroforese bidimensional, aliada à pré-purificação das glicoproteínas por colunas de lectinas e posterior identificação por espectrometria de massas, não garantiu a obtenção de resultados adequados para esta classe de estruturas. Portanto, a comparação glicoproteômica de tecidos musculares de animais controle e LARGE não foi bem sucedida por esta estratégia instrumental. Técnicas imunoanalíticas, em destaque o western blot, por sua vez, garantiram a visualização das diferenças de glicosilação da alfa-distroglicana, e experimentos de cromatografia de imunoafinidade iniciados neste trabalho mostraram o potencial da especificidade de interação anticorpo-antígeno no isolamento desta glicoproteína para estudos futuros de seus resíduos de oligossacarídeos. Finalmente, análises de espectrometria de massas dos resíduos de oligossacarídeos isolados das glicoproteínas foram realizadas. Os resultados obtidos indicaram a necessidade de otimização do preparo e purificação mais eficiente dessas amostras, mas alguns íons puderam ser relacionados a N- e O-glicanas. / Glycosylation is one of the most common modifications that occur naturally in the polypeptide chains. Glycoproteins play key roles in living organisms from the beginning of life and, therefore, any mutation in the sugar residues attached to them may cause many undesirable effects. The glycosylation pattern of proteins is regulated by both genetic and external factors. Regarding hereditary defects of glycosylation, different mutations in specific genes cause abnormalities in the synthesis of glycoproteins. In the group of diseases caused by defective glycosylation are included some hereditary muscular dystrophies, related to mutations in proteins that are proven or putative glycosyltransferase. The use of animal models facilitates the study of neuromuscular diseases and, therefore, this study was conducted using tissues from control mice (C57Black6) and LARGE. The LARGE mouse has phenotypic characteristics similar to those of humans affected by congenital muscular dystrophy type 1D. Different strategies for analysis and instrumentation were employed here to obtain information related both to the set of glycoproteins in general and specifically to alpha-dystroglycan. The alpha-dystroglycan is modified in relation to its linked sugar residues in animals with LARGE gene mutation, resulting in several health problems. Twodimensional electrophoresis technique, coupled with the pre-purification of glycoproteins by lectin column and subsequent identification by mass spectrometry, did not guarantee resultssuited for this class of structures. Therefore, the glycoproteomic comparison of muscle tissues from LARGE and control animals was not effective by this instrumental strategy. Immunoanalytical techniques, highlighting here the western blot, in turn, assured the visualization of the differences in glycosylation of alpha-dystroglycan, and immunoaffinity chromatography experiments undertaken in this work indicate the potential of the specific antibody-antigen interaction in isolation of this glycoprotein for the future study of their attached oligosaccharides. Finally, mass spectrometer analyses of the isolated oligosaccharides residues of the glycoproteins were performed. The results indicated the need for optimization of preparation and purification of these samples more efficiently, but some ions could be related to N-and O-glycans.

To Wah Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 118-142). / Acknowledgements --- p.1 / Summary --- p.3 / List of Abbreviations --- p.7 / Chapter Part I --- Alpha2-HS glycoprotein: Identification and Characterization of the Jacalin Binding Properties --- p.8 / Chapter Chapter 1 --- Introduction --- p.9 / Chapter 1.1 --- Jackfruit and jacalin --- p.10 / Chapter 1.2 --- Biochemical and immunological properties of jacalin --- p.11 / Chapter 1.2.1 --- Molecular Weight of Jacalin --- p.11 / Chapter 1.2.2 --- Molecular structure of jacalin --- p.11 / Chapter 1.2.3 --- Specificity of jacalin to Thomsen-Fredenreich- antigen (T-antigen) --- p.13 / Chapter 1.2.4 --- The internal repeated sequence in the jacalin --- p.13 / Chapter 1.2.5 --- Jacalin-Bound Proteins (JBP) --- p.15 / Chapter 1.2.6 --- Interaction of jacalin to JBP --- p.15 / Chapter 1.2.7 --- Immunological properties of jacalin --- p.16 / Chapter 1.3 --- Application of jacalin in medical research --- p.17 / Chapter 1.4 --- Background knowledge of α2HSG --- p.18 / Chapter Chapter 2 --- Materials and Methods --- p.20 / Chapter 2.1 --- Design of experiment --- p.21 / Chapter 2.2 --- Identification of the Unknown JBP --- p.21 / Chapter 2.2.1 --- Sera --- p.21 / Chapter 2.2.2 --- Isolation of JBP by Affinity Chromatography --- p.22 / Chapter 2.2.3 --- Fast protein liquid chromatography (FPLC) of JBP --- p.22 / Chapter 2.2.4 --- Affinity chromatography with anti-human IgA column --- p.23 / Chapter 2.2.5 --- Preparation of non-IgA JBP fraction --- p.24 / Chapter 2.2.6 --- N-terminal sequencing of the non-IgA JBP fraction --- p.24 / Chapter 2.2.7 --- SDS-PAGE and immunoblot of gel filtration fractions --- p.25 / Chapter 2.2.8 --- ELISA of FPLC fractions of JBP --- p.26 / Chapter 2.2.9 --- Immunochemical analysis --- p.27 / Chapter 2.3 --- α2HSG: the property of jacalin binding --- p.28 / Chapter 2.3.1 --- α2HSG -jacalin binding curve and competitive ELISA --- p.28 / Chapter 2.3.2 --- Purification of jacalin-crude extract (JCE) --- p.28 / Chapter 2.3.3 --- Characterization of JCE and ASJ --- p.29 / Chapter 2.3.4 --- Comparison of jacalin from different sources for binding to α2HSG by competitive ELISA --- p.29 / Chapter Chapter 3 --- Results --- p.31 / Chapter 3.1 --- Identification of the unknown JBP --- p.32 / Chapter 3.1.1 --- Isolation and FPLC of JBP --- p.32 / Chapter 3.1.2 --- Identification of non-IgA JBP by anti-human IgA affinity column --- p.32 / Chapter 3.1.3 --- Identification of the known JBP in the FPLC fractionated JBP --- p.36 / Chapter 3.1.4 --- Characterization and confirmation of non-IgA JBP --- p.36 / Chapter 3.2 --- α2HSG: the property of jacalin binding --- p.42 / Chapter 3.2.1 --- Characterization of α2HSG-jacalin binding --- p.42 / Chapter 3.2.2 --- Characterization of the purified jacalin --- p.45 / Chapter 3.2.3 --- Comparison of different batches of jacalin to interact with α2HSG --- p.45 / Chapter Chapter 4 --- Discussion --- p.53 / Chapter Part II --- Jacalin-α2HSG binding: the Clinical Values --- p.57 / Chapter Chapter 5 --- Introduction --- p.58 / Chapter Chapter 6 --- Materials and Methods --- p.61 / Chapter 6.1 --- Preparation of IgA-specific jacalin (ASJ) by IgA-Sepharose 4B affinity column --- p.62 / Chapter 6.2 --- Preparation of JCE- and ASJ-Sepharose-4B affinity column --- p.62 / Chapter 6.3 --- Factors affecting the yield of α2HSG --- p.62 / Chapter 6.4 --- Miscellaneous methods --- p.63 / Chapter Chapter 7 --- Results and Discussion --- p.65 / Chapter Part III --- Application of Jacalin for Studying the Pathogenesis of IgA Nephropathy --- p.77 / Chapter Chapter 8 --- An Overview of IgA Nephropathy --- p.78 / Chapter 8.1 --- Clinical manifestation of IgA nephropathy --- p.79 / Chapter 8.2 --- Mesangial deposits in IgAN --- p.80 / Chapter 8.3 --- Human IgA system --- p.81 / Chapter 8.4 --- The role of circulating IgA in the pathogenesis of IgA nephropathy --- p.84 / Chapter 8.5 --- Pathogenesis of primary IgA nephropathy --- p.86 / Chapter 8.6 --- Interaction between circulatory IgA and fibronectin (FN) in primary IgAN --- p.87 / Chapter Chapter 9 --- Materials and Methods --- p.90 / Chapter 9.1 --- Design of experiment --- p.91 / Chapter 9.2 --- Sera --- p.91 / Chapter 9.3 --- Analysis of IgAl/IgA ratio in sera and JBP --- p.91 / Chapter 9.4 --- Purification and Fast protein liquid chromatography (FPLC) of jacalin-bound protein (JBP) --- p.92 / Chapter 9.5 --- Analysis of FPLC-fractionated JBP --- p.93 / Chapter 9.5.1 --- ELISA of IgA-containing immune complexes (IgA-IC) --- p.93 / Chapter 9.5.2 --- "Quantitative ELISA of IgA, K-IgAl, and λ-IgAl" --- p.94 / Chapter 9.5.3 --- "Measurement of sIgA,dIgA and IgA containing immune complex (IgA-IC)" --- p.95 / Chapter 9.5.4 --- SDS-PAGE analysis --- p.95 / Chapter 9.6 --- Statistics --- p.95 / Chapter Chapter 10 --- Results --- p.96 / Chapter 10.1 --- IgA1/IgA ratio of serum and JBP --- p.97 / Chapter 10.2 --- Isolation and FPLC of JBP --- p.97 / Chapter 10.3 --- SDS-PAGE analysis of FPLC fractionated JBP --- p.97 / Chapter 10.4 --- ELISA of the FPLC fractionated JBP --- p.99 / Chapter 10.4.1 --- "Distribution of IgA, secretory IgA (sIgA) and dimeric IgA (dIgA) in the FPLC fractions" --- p.99 / Chapter 10.4.2 --- Distribution of IgA containing immune complex (IgA-IC) in the FPLC fractions --- p.99 / Chapter 10.4.3 --- Quantitation of IgA --- p.106 / Chapter 10.4.4 --- "Quantitation of K-IgAl and λ-IgA1, and determination of k/λ ratio of IgAl" --- p.106 / Chapter 10.4.5 --- "Quantitation of sIgA,dIgA, and IgA-containing immune complex (IgA-IC)" --- p.109 / Chapter Chapter 11 --- Discussion --- p.112 / References --- p.118

Yam Hin-Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 138-157. / ABSTRACT --- p.2 / ACKNOWLEDGEMENT --- p.5 / CONTENT --- p.6 / Chapter I. --- INTRODUCTION --- p.8 / Chapter II. --- LITERATURE REVIEWS / Chapter 1. --- Esophagus and Esophageal Carcinoma --- p.11 / Chapter 2. --- Characterization of Cell Line --- p.23 / Chapter 3. --- Membrane Surface --- p.26 / Chapter 4. --- Differentiation and Cancer --- p.36 / Chapter 5. --- Calcium Ion --- p.42 / Chapter III. --- MATERIALS AND METHODS / Chapter 1. --- Characterizations of EC/CUHK2 Cell Line --- p.48 / Chapter 2. --- SQM1 Localization on EC/CUHK2 Cells --- p.57 / Chapter 3. --- SQM1 Localization on Other Cells and Cell Lines --- p.62 / Chapter 4. --- Characterizations of EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.65 / Chapter 5. --- SQM1 Localization on EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.71 / Chapter 6. --- SQM1 Localization on EC/CUHK2 Cells with Changes of Extracellular Calcium Ion Concentrations --- p.73 / Chapter IV. --- RESULTS / Chapter 1. --- Characterizations of EC/CUHK2 Cell Line --- p.74 / Chapter 2. --- SQM1 Localization on EC/CUHK2 Cells --- p.81 / Chapter 3. --- SQM1 Localization on Other Cells and Cell Lines --- p.83 / Chapter 4. --- Characterization of EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.87 / Chapter 5. --- SQM1 Localization on EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.96 / Chapter 6. --- SQM1 Localization on EC/CUHK2 Cells with Changes of Extracellular Calcium Ion Concentrations --- p.105 / Chapter V. --- DISCUSSIONS / Chapter 1. --- Characterizations of Carcinoma Cell Line --- p.107 / Chapter 2. --- SQM1 Distribution on Esophageal Cancer Cells --- p.118 / Chapter 3. --- SQM1 Distribution on Other Cells --- p.122 / Chapter 4. --- Calcium-Induced Differentiation of Esophageal Carcinoma Cells --- p.125 / Chapter 5. --- SQM1 Distribution on Calcium-Induced Esophageal Carcinoma Cells 6 --- p.132 / Chapter VI. --- CONCLUSION --- p.136 / Chapter VII. --- REFERENCES --- p.138 / Chapter VIII. --- ILLUSTRATIONS --- p.158

Orientador: Mirela Tinucci Costa / Banca: Aleksandro Schafer da Silva / Banca: Leucio Câmara Alves / Banca: Aureo Evangelista Santana / Banca: Annelise Carla Camplesi / Resumo: O tratamento de cães com leishmaniose visceral no Brasil tem gerado polêmicas discussões entre os diversos segmentos de profissionais da saúde. De um lado os Médicos Veterinários pressionados pelos proprietários dos cães que querem ter o direito de decidir por um tratamento ou pela eutanásia de seus animais; de outro os Ministérios da Saúde e da Agricultura, Pecuária e Abastecimento (MAPA) que se mostram contrários a qualquer tratamento, motivados pelo risco de se criar cepas do parasita resistentes aos fármacos, os quais também podem ser empregados em pacientes humanos. Argumentos científicos que possam embasar essa polêmica devem ser explorados. Uma das possibilidades de se avaliar o desenvolvimento de resistência a fármacos pode ser conseguida por meio da pesquisa da expressão da glicoproteína-P (gp-P) e proteína de resistência a múltiplas drogas (MRP1), que atuam como bombas de efluxo expulsando xenobióticos de células, responsáveis pelo fenótipo MDR (resistência a múltiplas drogas). Avaliamos, pela técnica de imuno-histoquímica, a reatividade da gp-P e MRP1, clone C494 e ABCC1, respectivamente, na pele de 11 cães naturalmente infectados por Leishmania (L.) infantum chagasi antes e após serem submetidos a dois protocolos de tratamento: Alopurinol e vacina inativada (n=4) e Alopurinol, vacina inativada e domperidona (n=7) e quantificamos formas amastigotas na pele pela mesma técnica. Todos os animais após 3 e 6 meses de tratamento tiveram melhora clínica evidente e tiveram contagem negativa de formas amastigotas do parasita. Pele de cães com LV expressaram gp-P e MRP1, antes e após o tratamento, no entanto, este experimento permitiu concluir que a gp-P e MRP1 não foram bons marcadores para avaliar a eficácia terapêutica da LVC e para prever possíveis estados de resistência a fármacos. Todavia, o entendimento sobre a participação de marcadores de resistência a múltiplas drogas na pele de ... / Abstract: The treatment of dogs with visceral leishmaniasis in Brazil has generated controversial debate among various segments of health professionals. On one side, veterinarians pressured by dog owners want to have the right to decide between treatment and euthanasia of their animals. On the other hand, the Ministry of Health and the Ministry of Agriculture, Livestock, and Supply (MAPA) are opposed to treatment because of the risk of creating strains of the parasite resistant to drugs that are also used for human patients. Scientific arguments that can support this controversy should be explored. One possibility for assessing the development of drug resistance can be achieved through research of the expression of Pglycoprotein (P-gp) and multidrug resistance-associated protein (MRP1). These proteins, which are responsible for the MDR phenotype (multiple drug resistance), act as efflux pumps which expel xenobiotics from cells. Using immunohistochemistry, the reactivity of P-gp (clone C494) and MRP1 (clone ABCC1) in the skin of 11 dogs naturally infected by Leishmania (L.) infantum chagasi was analyzed before and after undergoing two treatment protocols: Allopurinol and inactivated vaccine (n=4) and Allopurinol, inactivated vaccine, and domperidone (n=7). Quantification of Leishmania amastigotes in the skin was also achieved via immunohistochemistry. All of the animals after 3 and 6 months of treatment had clinical improvement and tested negative for amastigote forms of the parasite. Yet, the skin of dogs positive for CVL (canine visceral leishmaniasis) expressed P-gp and MRP1 before and after treatment, illustrating that P-gp and MRP1 were neither good markers for evaluating the therapeutic efficacy of CVL nor for predicting the possible states of drug resistance. However, an understanding of the participation of markers for multiple drug resistance in the skin of dogs under treatment for CVL can be of great importance for the development of an ... / Doutor

A glicosilação é uma das modificações mais comuns ocorridas naturalmente nas cadeias polipeptídicas. As glicoproteínas exercem papéis essenciais para os seres vivos desde o ínicio vida e, por essa razão, qualquer mutação nos resíduos de açúcares a elas ligados causam diversos efeitos não desejados ao indivíduo. O padrão de glicosilação de proteínas é regido tanto por fatores genéticos quanto por fatores externos. Em relação aos defeitos de glicosilação hereditários, diversas mutações em genes específicos causam anormalidades na síntese de glicoproteínas. No grupo de doenças causadas por defeitos de glicosilação hereditários estão incluídas algumas distrofias musculares relacionadas a mutações em proteínas que são glicosiltransferases comprovadas ou putativas. O uso de modelos animais facilita o estudo dessas doenças neuromusculares e, por isso, o presente trabalho foi desenvolvido utilizando tecidos de camundongos controle (C57Black6) e LARGE. O camundongo LARGE possui características fenotípicas semelhantes às de humanos afetados pela distrofia muscular congênita tipo 1D. Diferentes estratégias de análise e de instrumentação foram empregadas para a obtenção de informações relacionadas tanto ao conjunto de glicoproteínas em geral quanto à alfa-distroglicana especificamente. A alfa-distroglicana mostra-se modificada em relação aos resíduos de açúcares nela ligados em animais com mutação no gene LARGE, resultando em diversos problemas de saúde. A técnica de eletroforese bidimensional, aliada à pré-purificação das glicoproteínas por colunas de lectinas e posterior identificação por espectrometria de massas, não garantiu a obtenção de resultados adequados para esta classe de estruturas. Portanto, a comparação glicoproteômica de tecidos musculares de animais controle e LARGE não foi bem sucedida por esta estratégia instrumental. Técnicas imunoanalíticas, em destaque o western blot, por sua vez, garantiram a visualização das diferenças de glicosilação da alfa-distroglicana, e experimentos de cromatografia de imunoafinidade iniciados neste trabalho mostraram o potencial da especificidade de interação anticorpo-antígeno no isolamento desta glicoproteína para estudos futuros de seus resíduos de oligossacarídeos. Finalmente, análises de espectrometria de massas dos resíduos de oligossacarídeos isolados das glicoproteínas foram realizadas. Os resultados obtidos indicaram a necessidade de otimização do preparo e purificação mais eficiente dessas amostras, mas alguns íons puderam ser relacionados a N- e O-glicanas. / Glycosylation is one of the most common modifications that occur naturally in the polypeptide chains. Glycoproteins play key roles in living organisms from the beginning of life and, therefore, any mutation in the sugar residues attached to them may cause many undesirable effects. The glycosylation pattern of proteins is regulated by both genetic and external factors. Regarding hereditary defects of glycosylation, different mutations in specific genes cause abnormalities in the synthesis of glycoproteins. In the group of diseases caused by defective glycosylation are included some hereditary muscular dystrophies, related to mutations in proteins that are proven or putative glycosyltransferase. The use of animal models facilitates the study of neuromuscular diseases and, therefore, this study was conducted using tissues from control mice (C57Black6) and LARGE. The LARGE mouse has phenotypic characteristics similar to those of humans affected by congenital muscular dystrophy type 1D. Different strategies for analysis and instrumentation were employed here to obtain information related both to the set of glycoproteins in general and specifically to alpha-dystroglycan. The alpha-dystroglycan is modified in relation to its linked sugar residues in animals with LARGE gene mutation, resulting in several health problems. Twodimensional electrophoresis technique, coupled with the pre-purification of glycoproteins by lectin column and subsequent identification by mass spectrometry, did not guarantee resultssuited for this class of structures. Therefore, the glycoproteomic comparison of muscle tissues from LARGE and control animals was not effective by this instrumental strategy. Immunoanalytical techniques, highlighting here the western blot, in turn, assured the visualization of the differences in glycosylation of alpha-dystroglycan, and immunoaffinity chromatography experiments undertaken in this work indicate the potential of the specific antibody-antigen interaction in isolation of this glycoprotein for the future study of their attached oligosaccharides. Finally, mass spectrometer analyses of the isolated oligosaccharides residues of the glycoproteins were performed. The results indicated the need for optimization of preparation and purification of these samples more efficiently, but some ions could be related to N-and O-glycans.