An outline of the work contained in this thesis is presented. The first chapter is a critical review of the literature pertaining to the pathophysiological mechanisms operational with regards to the antiphospholipid syndrome (APS). The syndrome is characterised by venous and arterial thrombosis, and recurrent fetal loss, in association with the persistent presence of antibodies targeting the main autoantigen beta 2-glycoprotein I (β2GPI). The second chapter reviews the literature delineating the diverse physiological functions of β2GPI, and then relates them to its role in our current understanding of the pathophysiology of APS. The third chapter presents a critical review of the evidence base for the diagnosis and management of APS. The fourth chapter describes the interaction between β2GPI and the glycoprotein Ib alpha (GPIbα) subunit of the platelet receptor GPIb-IX-V. GPIbα is an important platelet adhesion receptor, which mediates multiple additional functions on the platelet surface, including binding coagulation factor XI (FXI). The implication of the interaction between β2GPI and GPIbα on platelet activation and the release of thromboxane in the presence of anti-β2GPI antibodies is explored, as well as the intracellular pathways via which this activation occurs. The relevance of these findings to understanding APS pathogenesis, in particular thrombosis, is discussed. The fifth chapter delineates the interaction between the fifth domain of β2GPI and FXI and its activated form factor XIa (FXIa). The ability of FXIa to cleave β2GPI between lysine (Lys) 317 and threonine (Thr) 318, and modulate its function is reported. The sixth chapter describes the ability of β2GPI to inhibit FXIa autoproteolytic hydrolysis at the specific FXIa residues arginine (Arg) 507, Arg532 and Lys539. This interaction with β2GPI stabilizes FXIa activity over time, and leads to enhanced FXIa mediated fibrin formation. This is a novel physiological function of β2GPI with important implications. Recent epidemiological studies by others have emphasized the critical role of FXIa in pathological thrombus propagation. The seventh chapter defines the relevance of the FXIa residues Arg507, Arg532 and Lys539 to FXIa mediated inactivation by the main FXIa inhibitor Protease Nexin 2 (PN2), and by Antithrombin III (ATIII). Insights into future directions for research are presented and discussed within each individual chapter.

Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS. Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 200-242). Also available on the World Wide Web.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Antiphospolipidsyndrome (APS) is one of the most common autoimmune diseases characterized bythrombosis, fetal loss and presence of antiphospholipid antibodies. In APS research the antibodies of biggestinterest are anti-β2-glycoprotein I antibodies (Aβ2GPIA). β2-glycoprotein I (β2GPI)is a plasma protein which becomes activated and obtains a open structure incontact with negative charged surface molecules such as phospholipids. Inactiveβ2GPI has a closed, circular shape which can’t bind autoantibodies. Thereis no golden standard for APS diagnosing and the methods used often giveinconsistent results. The purpose of this examination project work was toconvert β2GPI into the open and closed forms, respectively, by dialyzing againsthigh ionic strength, low and high pH and determine if there is any differencein binding capacity between the two forms and Aβ2GPIAon a microtiter plate.                                                The binding capacity was tested inan ELISA (enzyme-linkedimmunosorbent assay) using purified IgG from patient sera and thedifferent conformational forms of β2GPI. An ELISA for measuring of Aβ2GPIAon several patient samples was also performed.               No difference in binding capacitycould be detected which might be explained by that the conversion of β2GPI was unsuccessful.Perhaps no difference can be measured between the structures because the closedform is expected to open on microtiter plates. An unexpected result was thepresence of immune complexes of β2GPI-Aβ2GPIA found in the serum of one of the patients. In theory an ELISA based on theopen form of β2GPI would provide more reliable diagnoses and furtherresearch is needed in this area.

Made available in DSpace on 2014-06-11T19:23:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-30Bitstream added on 2014-06-13T18:50:11Z : No. of bitstreams: 1 neo_ta_me_arafcf.pdf: 1125295 bytes, checksum: 23377b72364333a1ac1bf48473dfab17 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O vírus da Hepatite C (VHC) é o principal agente etiológico das hepatites não-A e não-B, infectando aproximadamente 170 milhões de pessoas no mundo (3% da população mundial). O vírus da hepatite C (HCV; hepatitis C-virus) é envelopado tem de 50 a 70nm de diâmetro, possui uma única fita positiva de RNA e pertence ao gênero do Hepacivirus e à família Flaviridae. Seu genoma é constituído por cerca de 9.500 nucleotídeos com regiões curtas não codificadoras e hiperconservadas nas extremidades 5’ e 3’UTR, flanqueando uma única ORF. A região estrutural do vírus é constituída por 3 genes: core, E1 e E2. As proteínas do envelope, E1 e E2 do VHC, são altamente glicosiladas e apresentam 30 e 70 kDa, respectivamente. Estudos demonstram que ambas apresentam funções específicas em diferentes etapas do ciclo de replicação do vírus, atuando de forma essencial para entrada, ligação ao receptor e fusão com a membrana da célula hospedeira. A glicoproteína E2 do VHC liga-se com alta afinidade a uma alça do receptor CD81, também denominado de TAPA-1, uma tetraespanina encontrada na superfície de muitas células, incluindo hepatócitos. No entanto, o CD81 isoladamente não é suficiente para mediar a entrada celular do vírus, e vários outros co-fatores podem atuar nessa interação. Os receptores de lipoproteína de baixa densidade (LDL-r) e receptor scavenger tipo B classe I apresentam grande importância nessa relação com o VHC. Estudos sobre as glicoproteínas E1 e E2 têm mostrado que estas se associam com os LDL-r, sugerindo que o VHC use estes receptores para invadir a célula hospedeira. Além disso, estudos anteriores relatam o fato de que, as lipoproteínas poderiam proporcionar acréscimos da infectividade ao VHC. Desta forma, neste trabalho foram desenvolvidas estratégias de clonagem e expressão heteróloga da proteína E2, e avaliou-se sua imunogenicidade... / Hepatitis C is currently recognized as the primary cause of hepatitis non A - non B associated to the blood transfusion. The hepatitis C virus (HCV) is enveloped in about 50 to 70nm in diameter, presenting a positive single strand RNA and belongs to the genus Hepacivirus and the family Flaviridae. Its genome consists of 9,500 nucleotides with short non-coding regions and hiperconservadas ends 5´ and 3'UTR flanking a single ORF. The virus structural region is based on three core genes, E1 and E2. HCV E1 and E2 are highly glycosylated and have 30 and 70 kDa, respectively. Studies show that both have key role in different stages of the cycle of virus replication, acting as essential for entry, receptor binding and fusion with host cell membrane. Glycoprotein E2 of HCV binds with high affinity to a loop of CD81, a tetraspanin, also named TAPA-1, found on the surface of many cells, including hepatocytes. However, the CD81 alone is not sufficient to mediate the cellular entry of the virus, and several other co-factors may be operating in this interaction. Recipients of low density lipoprotein (LDL-r) and scavenger receptor class B type I (SR-BI) would present great importance in relation to HCV. The LDL-r plays an important role in infection for virus of the hepatitis C. Studies on the glycoproteins E1 and E2 have shown that these are associated with the LDL-r suggesting that HCV uses the LDL-r to invade the host cell. Besides, previous studies showed that lipoprotein could improve HCV infectivity. Thus, in this work the capacity of recognition of the antibodies present anti-HCV was evaluated in the positive human serum for HCV of recognizing the protein E2 recombinant produced in bacteria of the lineage Rosetta and also the capacity of connection of the protein E2 of HCV in bind LDL-r present in the surface of human cells with characteristics endoteliais (ECV 304), and such capacity was... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:23:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-21Bitstream added on 2014-06-13T18:51:20Z : No. of bitstreams: 1 calado_amc_me_jabo.pdf: 1098325 bytes, checksum: e69e94e5c8654a5cdbae32a66649fbe3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A leishmaniose visceral canina (LVC) é uma doença sistêmica e imunomediada. É provocada pelo protozoário da espécie Leishmania (L.) chagasi, que é transmitido ao homem e aos animais através da picada do vetor infectado, o flebotomíneo Lutzomyia longipalpis. Até o momento não há tratamento que leve a cura parasitológica dos cães e o arsenal de fármacos leishmanicidas é limitado. A resistência aos medicamentos constitui um impedimento importante para o controle de enfermidades e a manifestação de resistência a vários medicamentos é conhecida como MDR (“multidrug resistance”). Um desses mecanismos de resistência a múltiplas drogas é o aumento da expressão da glicoproteína-P (gp-P), um sistema de efluxo de xenobióticos codificadas pelo gene MDR1. Na presente pesquisa, buscou-se avaliar a possibilidade do fenômeno de resistência a múltiplas drogas em cães naturalmente parasitados por Leishmania (L.) chagasi . Foram objetivos do estudo avaliar a expressão da glicoproteína-P (gp-P) e da proteína de resistência a múltiplas drogas (MRP) por meio da imuno-histoquímica, em amostras fígado, adrenais, rins, baço e pele de cães portadores da LV. Os resultados mostraram que o fígado, rins e adrenais expressam significativamente (p<0,001) mais gp-P do que a pele e o baço; também que as adrenais expressam significativamente (p<0,001) mais MRP do que a pele e o baço e que este ultimo expressa significativamente (p<0,001) mais MRP do que a pele. Quando se comparou os dois anticorpos verificou-se que o fígado, rins e adrenais expressam porcentagens semelhantes de células imunomarcadas e que o MRP é significativamente (p<0,01) mais reativo na pele que a gp-P. Esses resultados poderiam justificar a redução da carga parasitária na pele que se acompanha durante o tratamento. Todavia, a não obtenção da cura parasitológica poderia ser devido a alta expressão... / Canine visceral leishmaniasis (LVC) is a systemic immunomediated disease. It is caused by the protozoary from the Leishmania (L.) chagasi species, wich is transmitted to man and animals through the infected vector bite, the phlebotomine sand fly Lutzomyia longipalpis. Until present, there is no treatment leading to a parasitologic cure for dogs and the leishmanicide medicine arsenal is limited. Drug resistance is one of the important impediments for infirmity control and the resistance manifestation to multiple drugs is known as MDR. One of those multiple drugs resistance mechanisms is the increase of the glycoprotein-P (gp-P) expression, a xenobiotic efflux system encoded by the MDR1 gene. At the present research, it aimed to evaluate the possibility of resistance phenomenon the multiple drugs in dogs naturally parasited by Leishmania (L.) chagasi. The objectives of this study was to evaluate glycoprotein-P (gp-P) and multiple drugs resistance protein (MRP) immunohistochemical expressions in LVC infected dogs’ liver, adrenals, kidneys, spleen and skin samples. Results showed that liver, kidneys and adrenals express significantly (p<0,001) more gp-P than skin and spleen; also, adrenals express significantly (p<0,001) more MRP than skin and spleen and that spleen express significantly (p<0,001) more MRP than skin. When the two antibodies were compared, it was verified that liver, kidneys and adrenals express similar percentages of immunomarked cells and that MRP is significantly more reactive in skin than gp-P. These results could justify the parasitary load reduction in skin during treatment. Nevertheless, the lack of parasitologic cure could be due to the high pg-P and MRP1 expressions in liver, adrenals and kidneys, which could increase the pharmac efflux at these organs

Orientador: Paulo Inácio da Costa / Banca: Márcia Aparecida Silva Graminha / Banca: Fernanda de Freitas Aníbal / Resumo: O vírus da Hepatite C (VHC) é o principal agente etiológico das hepatites não-A e não-B, infectando aproximadamente 170 milhões de pessoas no mundo (3% da população mundial). O vírus da hepatite C (HCV; hepatitis C-virus) é envelopado tem de 50 a 70nm de diâmetro, possui uma única fita positiva de RNA e pertence ao gênero do Hepacivirus e à família Flaviridae. Seu genoma é constituído por cerca de 9.500 nucleotídeos com regiões curtas não codificadoras e hiperconservadas nas extremidades 5' e 3'UTR, flanqueando uma única ORF. A região estrutural do vírus é constituída por 3 genes: core, E1 e E2. As proteínas do envelope, E1 e E2 do VHC, são altamente glicosiladas e apresentam 30 e 70 kDa, respectivamente. Estudos demonstram que ambas apresentam funções específicas em diferentes etapas do ciclo de replicação do vírus, atuando de forma essencial para entrada, ligação ao receptor e fusão com a membrana da célula hospedeira. A glicoproteína E2 do VHC liga-se com alta afinidade a uma alça do receptor CD81, também denominado de TAPA-1, uma tetraespanina encontrada na superfície de muitas células, incluindo hepatócitos. No entanto, o CD81 isoladamente não é suficiente para mediar a entrada celular do vírus, e vários outros co-fatores podem atuar nessa interação. Os receptores de lipoproteína de baixa densidade (LDL-r) e receptor scavenger tipo B classe I apresentam grande importância nessa relação com o VHC. Estudos sobre as glicoproteínas E1 e E2 têm mostrado que estas se associam com os LDL-r, sugerindo que o VHC use estes receptores para invadir a célula hospedeira. Além disso, estudos anteriores relatam o fato de que, as lipoproteínas poderiam proporcionar acréscimos da infectividade ao VHC. Desta forma, neste trabalho foram desenvolvidas estratégias de clonagem e expressão heteróloga da proteína E2, e avaliou-se sua imunogenicidade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hepatitis C is currently recognized as the primary cause of hepatitis " non A - non B " associated to the blood transfusion. The hepatitis C virus (HCV) is enveloped in about 50 to 70nm in diameter, presenting a positive single strand RNA and belongs to the genus Hepacivirus and the family Flaviridae. Its genome consists of 9,500 nucleotides with short non-coding regions and hiperconservadas ends 5' and 3'UTR flanking a single ORF. The virus structural region is based on three core genes, E1 and E2. HCV E1 and E2 are highly glycosylated and have 30 and 70 kDa, respectively. Studies show that both have key role in different stages of the cycle of virus replication, acting as essential for entry, receptor binding and fusion with host cell membrane. Glycoprotein E2 of HCV binds with high affinity to a loop of CD81, a tetraspanin, also named TAPA-1, found on the surface of many cells, including hepatocytes. However, the CD81 alone is not sufficient to mediate the cellular entry of the virus, and several other co-factors may be operating in this interaction. Recipients of low density lipoprotein (LDL-r) and scavenger receptor class B type I (SR-BI) would present great importance in relation to HCV. The LDL-r plays an important role in infection for virus of the hepatitis C. Studies on the glycoproteins E1 and E2 have shown that these are associated with the LDL-r suggesting that HCV uses the LDL-r to invade the host cell. Besides, previous studies showed that lipoprotein could improve HCV infectivity. Thus, in this work the capacity of recognition of the antibodies present anti-HCV was evaluated in the positive human serum for HCV of recognizing the protein E2 recombinant produced in bacteria of the lineage Rosetta and also the capacity of connection of the protein E2 of HCV in bind LDL-r present in the surface of human cells with characteristics endoteliais (ECV 304), and such capacity was... (Complete abstract click electronic access below) / Mestre

A epilepsia, doença descrita pela primeira vez em 2000 a.C., tem a crise convulsiva ou epiléptica como fenômeno paroxístico, e a International League Against Epilepsy define a crise epiléptica como \"manifestação excessiva e/ou hipersincrônica, normalmente autolimitada, da atividade dos neurônios no cérebro\". Há 40 anos surgiram os medicamentos antiepilépticos, mas a resistência múltipla a fármacos antiepilépticos (FAEs) é um problema significante que afeta pelo menos 30% dos pacientes portadores dessa doença devastadora. O mecanismo exato da fármaco-resistência desenvolvida em pacientes epilépticos ainda é desconhecido, porém uma possível causa seria a inadequada acumulação intraparenquimal do fármaco antiepiléptico relacionada a expressão aumentada da glicoproteína-P (PgP). Neste contexto, nosso objetivo foi investigar a correlação da expressão da PgP, codificada pelo gene ABCB1, no córtex temporal de 12 pacientes fármaco-resistentes frente aos FAEs fenobarbital, carbamazepina, fenitoína e lamotrigina; comparamos também a expressão da PgP nesse mesmo grupo de pacientes, selecionados no Centro de Cirurgia de Epilepsia (CIREP) frente a um grupo controle composto por indivíduos não epilépticos que evoluíram para óbito examinados no Serviço de Verificação de Óbito do interior (SVOi). Utilizamos a Cromatografia Líquida de Alta Eficiência para determinar as concentrações dos FAEs no plasma e no cérebro, sendo que a metodologia utilizada no tecido cerebral foi desenvolvida e validada especialmente para esse fim. Essas concentrações foram então determinadas e a razão entre as duas medidas foi comparada com a expressão de PgP no tecido cerebral. Analisando os resultados concluímos que não há correlação linear entre a razão dos fármacos estudados e a expressão de PgP no córtex temporal de pacientes com epilepsia refratária. / Epilepsy, desease described for the first time in 2000 B.C., presents the convulsion or epileptic seizure as its paroxysmal event, and the International League Against Epilepsy defines the epileptic seizure as \"excessive and/or hypersynchronous manifestation, usually self-limiting, from brain neurons activity\". Forty years ago the antiepileptic drugs (AEDs) emerged, but multiple resistance to AEDs is a significant problem which affects at least 30% of epileptic patients. The mechanism underlaying pharmacoresistance is still unknown, but a possible cause is the inadequate accumulation of AEDs in the brain tissue related to P-glycoprotein over expression. In this context, our aim was to investigate the correlation between PgP expression, codified by ABCB1 gene, in temporal cortex of 12 pharmacoresistant patients towards AEDs phenobarbital, carbamazepine, phenytoin and lamotrigine; we also compared PgP expression between the same group of patients, selected inside Epilepsy Surgery Center (CIREP), and a control group composed by non-epileptic deceased individuals examined at the Death Verification Service from our Medicine School (SVOi). We used High Performance Liquid Chromatography to determine AEDs concentrations in plasma and brain, and the methodology applied to brain quantification was specially developed for this purpose. These two concentrations were then determined and the ratio between them was compared with PgP expression in brain tissue. Analyzing the results we concluded that there is no linear correlation between the ratio of the AEDs studied and PgP expression in temporal cortex of patients with pharmacoresistant epilepsy.