Dissertations / Theses on the topic 'ICA512'

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75.
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department Cell and Developmental Biology." Discipline: Cell and Developmental Biology; Department/School: Medicine.

Les carcinomes évoluent dans un microenvironnement inflammatoire composé de cellules stromales (fibroblastes, cellules endothéliales et immunitaires) immergées dans une matrice extracellulaire (MEC). Les fibroblastes associés aux carcinomes (FACs) déposent et remodèlent la MEC dans le but de la rendre permissive à la croissance et l’invasion tumorale. Parmi les facteurs pro-inflammatoires responsables de l’activation des fibroblastes résidents, la cytokine Leukemia Inhibitory Factor (LIF) détient un rôle capital. En régulant l’activité de la chaîne légère de la myosine II (MLC-II), LIF induit la contractilité du cytosquelette d’actomyosine, générant des forces de tension et le remodelage de la MEC par les FACs. En revanche, les gènes régulés par LIF impliqués dans le phénotype pro-invasif des FACs ne sont pas connus. A l’aide d’un criblage phénotypique en trois dimensions, nous avons identifiés ICAM-1 comme régulateur majeur du remodelage de la MEC par les FACs. Nous démontrons qu’ICAM-1 est nécessaire et suffisant pour induire la réorganisation de la MEC indispensable à l’invasion collective des cellules de carcinome squameux. En effet, ICAM-1 est un régulateur de la contractilité cellulaire dépendante de la voie de signalisation RhoA-ROCK et de la kinase Src. De plus, la contractilité cellulaire régule l’expression d’ICAM-1, menant ainsi à une boucle de régulation positive. Nous proposons alors qu’ICAM-1 représente une cible thérapeutique afin de lutter contre l’invasion tumorale et la dissémination métastatique
Acto-myosin contractility in carcinoma-associated fibroblasts leads to the assembly of the tumor extracellular matrix. The pro-inflammatory cytokine LIF governs fibroblast activation in cancer by regulating the myosin light chain 2 activity. So far, however, how LIF mediates cytoskeleton contractility remains unknown. Using phenotypic screening assays based on knock down of LIF-dependent genes in fibroblasts, we identified ICAM1 as a crucial regulator of stroma fibroblast proinvasive matrix remodeling. We demonstrate that ICAM1 is necessary and sufficient to promote inflammation-dependent extracellular matrix organization, which leads to cancer cell invasion. Indeed, ICAM1 mediates generation of acto-myosin contractility downstream of the Src kinases in stromal fibroblasts. Moreover, acto-myosin contractility regulates ICAM1 expression, establishing a positive feedback signaling. Thus, targeting stromal ICAM1 might constitute a possible therapeutic mean to counteract tumor cell invasion and dissemination

Orientador: Fernando Ferreira Costa
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Anemia falciforme (AF) é uma doença monogênica, mas com uma apresentação clínica variável, devido a possível associação com outros genes. Especula-se que nesta doença genes envolvidos com a resposta inflamatória tenham efeito epistático. A mieloperoxidase (MPO) é uma enzima com atividade antimicrobicida, sendo encontrada nos grânulos azurofilicos dos neutrófilos. O polimorfismo A-463MPO diminui a expressão desta enzima, podendo este alelo ser possível marcador para eventos infecciosos em pacientes com defesa imune já comprometida. Neste estudo 97 pacientes com AF acompanhado pelo ambulatório de hematologia-Unicamp foram inicialmente divididos em dois grupos, conforme o número de internamentos hospitalares para antibioticoterapia: pacientes que não apresentaram infecção com necessidade de intemação (64 pacientes) e um segundo grupo onde ocorreu pelo menos 1 episódio de hospitalização devido a infecção (33 pacientes). O grupo controle foi composto por 48 indivíduos sadios negros da Babia-BrasiL Pacientes e controles foram genotipados para o polimorfismo G/A-463MPO, através da técnica de "Conformation Sensitive Gel Eletrophoresis" (CSGE) e sequenciamento automatizado. Observou-se que a presença do genótipo AA ou AG está associado a maior número de eventos infecciosos (P=O.005 OR=3.8). Esta correlação também foi observada na análise quanto à presença do alelo A-463MPO (P=0.004 OR=2.7). Este achado sugere que deficiência de MPO em pacientes com AF provavelmente favorece a ocorrência de eventos infecciosos. Vaso oclusão é o principal evento nas crises dolorosas dos pacientes com anemia falciforme (AF) e marcadores genéticos de risco para eventos vaso oclusivos ainda são desconhecidos. Polimorfismos em genes envolvidos na adesão celular PSGL-l (VNTR no exon 2), ICAM-1 G241R e K461E) e CD18 (V441V) foram genotipados em 103 pacientes com AF. Os pacientes foram inicialmente divididos em dois grupos: pacientes em que ocorre acidente vascular cerebral (AF+A VC,16 pacientes) e pacientes sem ocorrência de A VC (AF-A VC, 87 pacientes). Posteriormente, os pacientes foram divididos em outros dois grupos: pacientes em que foi detectado um dos seguintes eventos: A VC, síndrome torácica aguda (STA), necrose asséptica de cólo de fêmur (NACF) e priapismo (AF+FVO, 35 pacientes) e pacientes em que não ocorrem essas complicações (AF-FVO, 70 pacientes). PSGL-l é um receptor nas células mielóides e linfócitos T estimulados de alta afinidade para P-selectinas, tendo fundamental papel na adesão dos leucócitos às plaquetas e ao endotélio. O gene contém 3 variantes alélicas (A, B, C) de um número variável de repetição "in tandem" (VNTR). A análise das freqüências dos genótipos e alelos revelou associação da variante B com o grupo AF+FVO [p=O.04, IC 95% OR=2 (1-4)] e quase significante com os casos de A VC [p=0.09, IC 95% OR=2 (0.8-4.9)], enquanto que o alelo A revelou nível significativo de associação com o grupo AF+FVO [p=O.Ol IC 95% OR=O.5 (0.2 0.9)]. O gene da molécula de adesão leucócitário CD-I8 é a beta-2 subunidade das integrinas a_heterodímeros. O polimorfismo C1323T foi recentemente implicado como fator de risco para fenômenos vaso oc1usivos. O estudo deste polimorfismo em pacientes portadores de anemia falciforme não revelou associação significativa com FVO ou A VC. ICAM-l é uma molécula da superfamília das imunoglobulinas com importante papel na adesão das células endotelial-leucócitos durante a resposta inflamatória. No mínimo dois sítios polimórficos são conhecidos R241E e K469E. Ambos sítios estão envolvidos na ligação aos contrareceptores Mac-I e LF A-I. A análise destes polimorfismos através da técnica de CSGE e seqüenciamento, revelou uma associação significativa do heterozigoto K469E para risco a A VC [P=0.02 IC 95% OR=3.6 (1.1-12.3)]. No entanto, o estudo falhou em demonstrar associação alélica com os casos de A VC ou FVO. Como não se conhece bem a relação funcional deste polimorfismo, maiores estudos são necessários incluindo análise funcional, para se esclarecer o real papel deste polimorfismo ao risco a AVC
Abstract: Not informed
Mestrado
Genetica Medica
Mestre em Genética e Biologia Molecular

Pharmacology
Ph.D.
Background: Epidemiology, clinical trials and meta-analysis studies have established that Hyperhomocysteinemia (HHcy) is an independent risk factor for stroke. However, the exact molecular mechanism underlying the HHcy-induced risk of stroke is unclear. Our study aims to investigate the role of HHcy in stroke. Methods and results: We established a mice mode of focal ischemic stroke, termed transient Middle Cerebral Artery Occlusion (tMCAO) and conducted surgery on a mice model of HHcy (plasma homocysteine level ~150μM), in which a Zn2+ inducible human cystathionine β-synthase (CBS) transgene was introduced to circumvent the neonatal lethality of the CBS gene deficiency (Tg-hCBS Cbs-/- mice). Fourteen-week-old male mice were used in the experiment. A student’s t-test was used for the evaluation of the statistical significance between the two groups. For the comparison across multiple groups, one-way ANOVA was used. We found that HHcy 1) increased the infarction volume from 42.3 ± 4.9 mm
Temple University--Theses

INTRODUÇÃO: A síndrome coronariana aguda (SCA) constitui uma síndrome clínica geralmente causada por doença arterial coronariana (DAC) aterosclerótica e está associada ao infarto agudo do miocárdio (IAM) que pode muitas vezes levar a óbito. Aterosclerose é uma doença progressiva, sistêmica e de inicio precoce caracterizada pelo acúmulo de lipides e elementos fibrosos nas grandes artérias e recentemente foi considerada como uma afecção de origem inflamatória. OBJETIVO: Avaliar a frequência genotípica de E-SELECTINA, MMP9, LIGHT, LTα, VCAM1, ICAM1, LGALS2 e NFκB em pacientes com infarto agudo do miocárdio (IAM), angina instável (AI) e indivíduos que foram submetidos á angiocoronariografia e que apresentaram ausência de processo ateromatoso significativo. Assim como analisar a associação dos polimorfismos com a concentração sérica das formas solúveis das proteínas com a expressão gênica em leucócitos do sangue periférico, procurando estabelecer modelo de analise menos invasiva da aterosclerose. CASUÍSTICA E MÉTODOS: O estudo foi realizado em um grupo de pacientes recrutados no Instituto Dante Pazzanese de Cardiologia (IDPC) com infarto agudo do miocárdio, angina instável e indivíduos que foram submetidos á angiocoronariografia e que apresentaram ausência de processo ateromatoso significativo. Foram incluídos no estudo 93 indivíduos sendo 47 com IAM com e sem supra ST compondo o grupo IAM e 46 com AI ou que foram submetidos á angiocoronariografia e que apresentaram ausência de processo ateromatoso significativo compondo o grupo SIAM, de ambos os sexos com idades entre 45 e 90 anos. Foi realizado o estudo dos polimorfismos dos genes LIGHT (rs344560 e rs2291668), MMP9 (rs17576), LTα (rs909253 e rs1041981), LGALS2 (rs7291467), VCAM1 (rs3176878), ICAM1 (rs281432), E-SELECTINA (rs5368) e NFκB (rs17032705) por pirosequenciamento, a análise da expressão dos genes LIGHT, MMP9, LTα, VCAM1, ICAM1 e NFκB por PCR em tempo real e a dosagem das formas solúveis de VCAM1, ICAM1, E-SELECTINA e MMP9 utilizando o sistema LUMINEX. RESULTADOS: A frequência alélica e genotípica dos polimorfismos estudados (LIGHT [rs344560 e rs2291668], MMP9 [rs17576)] LTα [rs909253 e rs1041981], LGALS2 [rs7291467], VCAM1 [rs3176878], ICAM1 [rs281432], E-SELECTINA [rs5368] e NFκB [rs17032705]) não apresentou relação com doença arterial coronariana. Foi encontrada associação da expressão de LTα com síndrome coronariana aguda (p<0,05) e relação de alguns dos polimorfismos estudados (6+3279C>T de LGALS2, 8+10029G>A de NFκB, 332-3499C>G de ICAM1, Lys178Glu de LIGHT e Asp693Asp de VCAM1) com alterações na expressão gênica, formas solúveis e parâmetros bioquímicos. CONCLUSÕES: Não houve associação dos polimorfismos estudados com síndrome coronariana aguda assim como não houve associação das formas solúveis. Em relação á expressão gênica, foi encontrada associação da expressão de LTα com síndrome coronariana aguda (p<0,05) e alguns dos polimorfismos estudados (6+3279C>T de LGALS2, 8+10029G>A de NFκB, 332-3499C>G de ICAM1, Lys178Glu de LIGHT e Asp693Asp de VCAM1)ocasionaram alterações da expressão gênica, formas solúveis e parâmetros bioquímicos.
BACKGROUND: : Acute coronary syndrome (ACS) is a clinical syndrome usually caused by atherosclerotic coronary artery disease (CAD) and is associates with acute myocardial infarction (AMI) and sometimes can take to death. Atherosclerosis is a progressive disease characterized by the accumulation of lipides and fibrous elements in arteries and recently was considered a disease of inflammatory origin. OBJECTIVE: : Evaluated the genotypic frequency of E-SELECTIN, MMP9, LIGHT, LTα, VCAM1, ICAM1, LGALS2 e NFκB in patients with acute myocardial infarction, unstable angina and individuals who were submitted to coronary angiography and had absence of significant atheromathous process. As well as analyze the polymorphism association with serum concentration of protein soluble forms with gene expression in peripheral blood leukocytes trying to establish a model less invasive to analyze atherosclerosis MATERIALS AND METHODS: : Study in a group of patients recruited in Dante Pazzanese of Cardiology Institute with acute myocardial infarction, unstable angina and in individuals who showed no significant atheromatous process. The study included 93 patients (47 with acute myocardial infarction and 46 with unstable angina or individuals who showed no significant atheromatous process) of both sexes with ages between 45 and 90 years. The genes polymorphisms study was by pyrosequencing, the gene expression was by PCR real time and soluble forms was dosage by LUMINEX. RESULTS: : The allelic and genotypic polymorphisms frequency studied (LIGHT [rs344560 e rs2291668], MMP9 [rs17576)] LTα [rs909253 e rs1041981], LGALS2 [rs7291467], VCAM1 [rs3176878], ICAM1 [rs281432], E-SELECTINA [rs5368] e NFκB [rs17032705]) don\'t presented relation with coronary arterial disease. Was found association with LTα gene expression and coronary acute syndrome (p<0,05) and relation of some polymorphism studied (6+3279C>T de LGALS2, 8+10029G>A de NFκB, 332-3499C>G de ICAM1, Lys178Glu de LIGHT e Asp693Asp de VCAM1) with changes in gene expression, serum soluble forms and biochemical parameters. CONCLUSION: : Do not have association between polymorphisms studied and serum soluble forms with acute coronary syndrome. Was found association with LTα gene expression and coronary acute syndrome (p<0,05) and relation of some polymorphism studied (6+3279C>T de LGALS2, 8+10029G>A de NFκB, 332-3499C>G de ICAM1, Lys178Glu de LIGHT e Asp693Asp de VCAM1) with changes in gene expression, serum soluble forms and biochemical parameters.

Nesta tese estudamos os repertórios das famílias multigênicas var (domínio DBLa), rif e stevor, importantes fatores de virulência, de Plasmodium falciparum em isolados brasileiros. Mostramos que estas famílias multigênicas podem ser conservadas entre diferentes isolados no Brasil, sugerindo que o repertório de genes variantes pode ser limitado em algumas localidades. Identificamos menos polimorfismos nos genes stevor que os genes var ou rif. Constatamos que genes var podem ser conservados ao longo do tempo em isolados brasileiros. Identificamos também os domínios DBLa e DBLb de genes var associados a citoaderência a CHOICAM1 em isolados de Rondônia. Mediante ensaios de ELISA, mostramos que domínios DBLbICAM foram reconhecidos de modo heterogêneo. Por citometria de fluxo, demonstramos que o parasita aderente em ICAM1 foi reconhecido majoritariamente pelo plasma de pacientes com infecção sintomática e menos de infecção assintomática. Além disso, identificamos por PCR em tempo real os genes var transcritos no clone 3D7 aderente em diferentes linhagens de células CHO.
Herein, we studied the repertoire of multigenic families of var (DBLa domain), rif and stevor from brazilian isolates of Plasmodium falciparum. We showed that these multigenic families could be conserved among different isolates from Brazil, suggesting that the repertoire of variant genes can be limited in certain areas. We identified less polymorphism in stevor genes than in var or rif genes. We also demonstrated that var genes can be conserved over time in Brazilian isolates and identified the DBLa and DBLb domains from var genes associated with cytoadherence to CHOICAM1 in Rondonian isolates. ELISA assays showed that these DBLbICAM domains were recognized in a heterogeneous fashion. By flow cytometry, we demonstrated that ICAM1-adherent parasites were stronger recognized by plasma from symptomatic infections than from non-symptomatic infections. Finally, we profiled the var gene transcripts in 3D7 parasites adherent to different CHO cells by real time PCR.

La diapedese leucocytaire est basee sur une cascade d'interactions adhesives etablies entre leucocytes et cellules endotheliales. L'adherence et la migration trans-endotheliale font intervenir des molecules adhesives de la superfamille des immunoglobulines, en particulier icam-1 dans l'adherence et pecam-1 dans la migration. La participation specifique de la proteine icam-1 a l'extravasation des leucocytes n'a jamais ete clairement demontree, du fait de son implication dans la phase precedente d'adherence, ainsi que de la multiplicite des recepteurs adhesifs presents sur l'endothelium. J'ai donc etabli un modele d'etude de l'adherence et de l'extravasation des neutrophiles, base sur l'utilisation de cellules cho recombinantes exprimant les proteines icam-1 et pecam-1 humaines, seules ou en combinaison. Ce modele m'a permis de demontrer le role specifique d'icam-1 dans la migration intercellulaire des neutrophiles et plus particulierement, l'implication de l'interaction fibriogene/icam-1 dans ce phenomene. La partie cytoplasmique d'icam-1 est indispensable pour cette fonction et met en jeu une voie de signalisation impliquant la proteine rho. Enfin, la mutation de l'acide amine d26 sur icam-1, qui est situe sur le site de liaison du fibrinogene, aboutit a l'inhibition de l'extravasation des neutrophiles.

碩士
中國醫藥大學
環境醫學研究所碩士班
97
Background: Asthma is a chronic respiratory disease. It is still uncertain how genes, environmental factors, and their interaction are related to asthma occurrence, although several mechanisms have been proposed. Intercellular adhesion molecule-1(ICAM1) plays an important role in the inflammatory pathway, and is an asthma susceptibility gene. We investigated the role of two ICAM1 functional single nucleotide polymorphisms (SNPs) at codon 56 (K56M, rs5491, A/T) and codon 469 (K469E, rs5498, A/G) in childhood asthma, and examined the effect modification associated with children’s exposure status of environmental tobacco smoke (ETS). Methods: We conducted a population-based cross-sectional study of elementary schools in Yilan, Taichung, and Chiayi during April 2006 and April 2008. We used structure questionnaire to definite case and control group in this study. After excluding individuals with missing genotypes on ICAM1 polymorphisms, 771 participants were included in the analyses. The associations between ICAM1 SNPs, ETS exposure, and asthma were estimated using logistic regression model with adjustments for confounders including gender, age, and school locations. Results: After adjusting for confounders, the T allele of rs5491 (56M) was associated with a higher risk of asthma (OR = 1.42, 95% CI = 0.70-2.88); an individual carrying an AA or AG genotype at codon 469 was also at an increased risk of asthma (OR = 1.30, 95% CI = 0.56-3.03) compared to carrying an GG genotype. Our results revealed that there was no interaction between rs5491 and rs5498 (p-value for the interaction > 0.05) and no haplotype effect on asthma (p-value for global test = 0.612). The associations between rs5491, rs5498, and asthma were not statistically significantly modified by ETS exposure (p-value for interaction > 0.05 for both SNPs). However, our results suggested that ICAM1 rs5791 genotypes might have different effects on asthma by ETS exposure. Conclusions: The present study was the first one to investigate the associations between ICAM1 gene and asthma in Taiwanese children. These findings suggest that the genetic variations in ICAM1 gene and ETS exposure may play important roles in the development of asthma in Taiwanese children. Our results also provide a direction for further studies on the association between ICAM1 gene polymorphisms, exposure of ETS, and childhood asthma.

博士
國立中正大學
心理學研究所
103
Episodic memory is the ability to consciously recollect specific events from one’s past personal experience including what happened in where and when. In animals, it focuses on the capability for combining these three main elements: “what”, “where” and “when”, to create “episodic-like memory”. Previous studies have shown that the medial prefrontal cortex and hippocampus may play an important role in the formation of episodic-like memory. Furthermore, anatomical evidence indicated that a reciprocal circuit connects the hippocampus iCA1 with mPFC via the anterior thalamus, while there is no direct neural connection between these two brain areas cross hemispheres. However, there is no clear conclusion about how the two areas interact in the processing of episodic-like memory. Thus, the present study intends to address this issue.We applied the O’Brien’s task to test episodic-like memory of a fear conditioning event in rats. The task comprises three stages: a when and where pair-association is formed in the pre-exposure stage; the combination of when, where, and the foot shock event is formed in the conditioning stage; the episodic-like memory is recalled through a spatial clue in the test stage. The experimental design of the present study is to temporarily deactivate mPFC and hippocampus iCA1 using lidocaine, and attempt to understand the roles of the two brain areas in episodic-like memory process.Results of experiment one showed that rats with temporary inhibition of bilateral hippocampus iCA1 in the pre-exposure and test stages failed in the task. We inferred that the processes of forming when-where connection and recalling episodic-like memory require involvement of hippocampus iCA1. In experiment two, we found that rats with temporary inhibition of bilateral mPFCin the conditioning and test stages failed in the task. Thus we inferred that the processes of integrating what-when-where connection and recalling episodic-like memory need involvement of mPFC. In experiment three, results showed that rats with temporary ipsilateral deactivationof mPFC and hippocampus iCA1 performed episodic-like memory task at the same level as the normal rats. It was interpreted that the processing of episodic-like memory requires the cooperation of intact mPFC and hippocampus iCA1 on at least one hemisphere of the brain. Results of experiment four showed that rats with contralateral deactivation of mPFC and hippocampus iCA1 in the conditioning and test stages showed deficits in processing episodic-like memory. Therefore, we inferred that the integrating and recalling processes of episodic-like memory requires the cooperation of mPFC and hippocampus iCA1. As a conclusion, this study found that mPFC and hippocampus iCA1 involves in different process of episodic-like memory respectively; and further, it was found that performing of episodic-like memory requires the cooperation of mPFC and hippocampus iCA1.