FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Apesar de muito se discutir sobre os efeitos citotÃxicos dos venenos ofÃdicos, pouco ainda à conhecido sobre os mecanismos de aÃÃo sobre as diversas cÃlulas, e em especial, sobre as cÃlulas renais. No caso particular da citotoxicidade dos venenos crotÃlicos, tem-se postulado a participaÃÃo de diversos metabÃlitos da hidrÃlise de lipÃdios de membrana, e, mais recentemente, da disfunÃÃo mitocondrial. O presente trabalho teve como objetivo estudar o efeito da fosfolipase A2 (FLA2) isolada do veneno da Crotalus durissus terrificus sobre rim isolado de rato assim como estudar a toxicidade e as alteraÃÃes da funÃÃo mitocondrial induzidas pelas FLA2s de pÃncreas de porco (PFLA2) e de veneno da C. d. terrificus (VSFLA2) em suspensÃes de tÃbulos proximais (TP). No rim isolado foi observado aumento no fluxo urinÃrio, no ritmo de filtraÃÃo glomerular (RFG) e na pressÃo de perfusÃo (PP) enquanto ocorreram decrÃscimos nos percentuais de transporte total de sÃdio (%TNa+), de potÃssio (%TK+) e de cloreto (%TCl-). Na anÃlise histopatolÃgica foi observada a deposiÃÃo de material proteinÃceo nos tÃbulos de rins perfundidos com FLA2. No estudo de suspensÃes de TP tratadas com PFLA2 e com VSFLA2 foi observado que estas induziram injÃria celular, sugerida pelo aumento na liberaÃÃo de lactato desidrogenase (LDH), promoveram aumentos nos nÃveis de Ãcidos graxos nÃo esterificados (AGNE) e diminuÃram o potencial de membrana mitocondrial (ΔΨm), sem, no entanto, alterar os nÃveis de ATP. Em relaÃÃo ao ΔΨm, a PPFLA2 nÃo produziu efeitos nas concentraÃÃes mais elevadas, apesar de ter aumentado, significativamente, na menor concentraÃÃo. As concentraÃÃes mais elevadas da FLA2 crotÃlica, entretanto, induziram um decrÃscimo significativo no ΔΨm. A adiÃÃo de BSA reverteu completamente os efeitos das FLA2s sobre o ΔΨm. No estudo da permeabilidade mitocondrial de transiÃÃo (PMT) foi observado que a PFLA2 e a VSFLA2 promoveram a liberaÃÃo da safranina O e, por entanto, induziu a formaÃÃo de PMT, apesar da leve edema mitocondrial produzido. Conclui-se que as fosfolipases A2 de pÃncreas de porco e do veneno da C. d. terrificus produziram um efeito citotÃxico em preparaÃÃes de tÃbulos proximais evidenciado pelo aumento na liberaÃÃo de LDH, alÃm de promoverem alteraÃÃes no potencial mitocondrial de membrana, o que sugere alteraÃÃo da funÃÃo mitocondrial por essas enzimas. Em rim isolado, foi observado que a FLA2 crotÃlica promoveu alteraÃÃes da funÃÃo renal / Although the increasing interest on biological effects of snake venoms, their cytotoxic as well as their nephrotoxic mechanisms are still unknown. In the particular case of crotalic venoms, it has been postulated the participation of several metabolites from membrane phospholipids hydrolysis and more recently, mitochondrial dysfunction. The present work investigated the renal effects promoted by the phospholipase A2 (PLA2) isolated from Crotalus durissus terrificus venom in the isolated rat kidney. Addition of PLA2 increased UF, GFR and PP, while reducing %TNa+, %TK+ and %TCl-. The histological analysis showed a mild amount of a proteinaceous substance in the renal tubules of kidneys perfused with PLA2. In the present study also showed that the phospholipase A2 isolated from porcine pancreas (PPLA2) and from C d terrificus snake venom (SVPLA2) produced cellular injury suggested by the increase in LDH release and increased nonesterified fatty acid (NEFA) levels from rabbit proximal tubules in suspension. Furthermore, the SVPLA2 induced a decrease in mitochondrial membrane potential (ΔΨm) assessed by both JC-1 uptake and safranin O uptake. PPLA2 produced no effects on ΔΨm with the highest concentrations, and an unexpected increase in the group treated with the lowest concentration. Addition of BSA completely reversed the effects induced by phospholipases on ΔΨm. It was observed no changes in cell ATP levels. Finally, to determine whether mitochondrial membrane permeability was affected by PPLA2 and SVPLA2, we measured the change safranin O uptake to assess both changes in mitochondrial volume and in ΔΨm. The latter was affected by both PLA2s although small alterations in the mitochondrial volume were observed. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic PLA2s disturbed the membrane integrity as well as the mitochondrial function. Furthermore, crotalic PLA2 altered renal function in the isolated rat kidney preparation.

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva que causa comprometimento cognitivo e demência. O diagnóstico é baseado em parâmetros clínicos, mas sua confirmação é post-mortem, após avaliação patológica durante a autópsia. Os tratamentos disponíveis para a DA são os inibidores da colinesterase (IChEs) e os antagonistas de receptores de N-metil-D-aspartato (NMDA), sendo que os IChEs compõe o principal grupo. Diversos estudos tem mostrado um efeito neuroprotetor dos IChEs, levando a alterações na patogênese da DA. Avaliar e mensurar essas alterações são papeis atribuídos aos biomarcadores. Neste sentido podemos destacar a fosfolipase A2 (PLA2), a principal responsável pelo metabolismo de fosfolípides de membrana, e que tem sido achada diminuída na DA, assim como a glicogênio sintase-quinase (GSK), responsável pela fosforilação da proteína Tau, que é um dos processos alterados na DA. O objetivo deste trabalho foi avaliar o efeito do tratamento com IChE sobre a atividade da PLA2 e expressão da GSK3B em plaquetas de 30 pacientes com DA após 3 e 6 meses de tratamento. Como grupo controle foram investigados 42 individuos idosos sem doença neurodegenerativa. Encontramos nos pacientes com DA antes do tratamento uma diminuição da atividade da iPLA2 quando comparada ao grupo controle. Após três e seis meses de tratamento a PLA2 aumentou, voltando ao nível dos controles. Os pacientes que apresentaram um aumento maior da iPLA2 apos 3 meses de tratamento apresentaram melhora cognitiva mais marcante após seis meses de tratamento, avaliado pelo CAMCOG. Apos 6 meses de tratamento encontramos um inativação da GSK3B, medida por um aumento em sua forma fosforilada. Nossos resultados sugerem que o donepezil apresenta propriedades modificadoras na doença de Alzheimer, e ainda que a medida da atividade da iPLA2 poderia ser usada como marcador de resposta terapêutica ao donepezil e, possivelmente, a outros IChEs, na doença de Alzheimer / Alzheimer\'s disease (AD) is a progressive neurodegenerative disorder that causes dementia and cognitive impairment. The Diagnosis is based on clinical parameters, but confirmation is post-mortem after pathologic evaluation during autopsy. The treatments available for AD are cholinesterase inhibitors (IChEs) and N-methyl-D-aspartate (NMDA) antagonists. The main group comprises the IChEs. Several studies have shown a neuroprotective effect of IChEs, leading to alterations in the pathogenesis of AD. Evaluate and measure these changes are assigned to biomarkers. In this regard we can highlight the phospholipase A2 (PLA2) the main enzyme in membrane phospholipids metabolism and that has been found decreased in AD as well as Glycogen Synthase kinase (GSK), a major responsible for tau phosphorylation which is one processes altered in AD. The objective of this study was to evaluate the effect of treatment with IChE on PLA2 activity and GSK3B expression in platelet of 30 AD patients after 3 and 6 months of treatment. The control group comprised 42 elderly individuals without neurodegenerative disease The results obtained were a decreased iPLA2 activity in patients with AD before treatment as compared to controls. After 3 and 6 months of treatment, we observed a significant increase in iPLA2 activity, restoring enzymatic activity similar to that observed among control. The patients who showed higher iPLA2 activity in the first three months were those showing cognitive improvement after six months of treatment, measured by CAMCOG. After 6 months of treatment a GSK3B inactivation were found, measured by an increase in its phosphorylated form. Our results suggest that donepezil present modifying properties in Alzheimer disease and that iPLA2 activity measurement could be used as a marker of therapeutic response to donepezil and possibly other IChEs in Alzheimer\'s disease

Os envenenamentos ofídicos constituem um problema relevante de saúde pública em diversas regiões do mundo, particularmente em países da zona tropical e neotropical. A fisiopatologia do acidente ofídico é constituída por uma série de eventos complexos tanto a nível local quanto sistêmico, e o soro antiofídico é o único tratamento utilizado. No entanto, os efeitos tóxicos locais induzidos durante o envenenamento por serpentes, principalmente do gênero Bothrops, não são eficientemente neutralizados pela soroterapia tradicional. Por esta razão, procuram-se alternativas complementares, como as plantas medicinais antiofídicas que são usadas por comunidades que não têm acesso a soroterapia. A flora brasileira possui uma ampla variedade de plantas medicinais com potencial antiofídico, as quais têm sido pouco estudadas cientificamente. Neste estudo foram realizados ensaios in vitro e in vivo de neutralização de peçonhas ofídicas com o extrato aquoso das entrecascas de Anacardium humile (EAAh), e, o isolamento e a caracterização fitoquímica de um inibidor de miotoxinas, o ácido gálico (AG). Para os ensaios de inibição, foram utilizadas soluções contendo peçonha bruta ou toxina isolada misturadas com diferentes quantidades de extrato vegetal que foram previamente incubadas por 30 min a 37°C. Também foi realizada administração do extrato após o envenenamento em diferentes intervalos de tempo para os testes de inibição da miotoxicidade. Observou-se que EAAh tem atividade inibitória sobre os efeitos tóxicos (letalidade, miotoxicidade, e hemorragia) e farmacológicos/enzimáticos (edema, atividade fosfolipásica e coagulante) induzidos pelas peçonhas de serpentes dos gêneros Bothrops, Crotalus, Lachesis e das toxinas isoladas. O extrato vegetal inibiu 100% a letalidade induzida pela peçonha de C. d. terrificus e sua principal neurotoxina, a crotoxina. O EAAh foi submetido a fracionamento cromatográfico analítico, e em condições polares foi possível identificar e isolar o ácido gálico, o qual demonstrou tempo de retenção e espectros de ressonância magnética nucleares similares ao padrão comercial e a dados de literatura deste mesmo composto, respectivamente. O ácido gálico isolado foi capaz de inibir a atividade miotóxica induzida pela peçonha bruta de B. jararacussu e sua principal miotoxina, a bothropstoxina-I, uma PLA2-símile Lys49. A análise dos espectros de dicroísmo circular e os estudos de interação por modelagem molecular sugerem que o ácido gálico forma um complexo com a BthTX-I de B. jararacussu em seu sítio ativo, inibindo sua atividade tóxica. A ligação do ácido gálico com as miotoxinas não modificou nem a forma e nem a intensidade dos espectros de dicroísmo circular, não induzindo alterações significativas na porcentagem dos diversos domínios que constituem a estrutura secundária destas proteínas. O ácido gálico assim como outros taninos, tem revelado-se um bom inibidor das ações tóxicas de peçonhas de serpentes e está relacionado com a ação inibitória do extrato de Anacardium humile. / Ophidian envenomations are a significant problem of public health in several regions of the world, particularly in tropical and neotropical countries. The pathophysiology of snakebite accidents is constituted by a complex series of events both locally and systemically, and the antivenom serum is the only treatment used. However, local toxic effects induced during envenomation by snakes, especially from the genus Bothrops, are not effectively neutralized by the traditional serum therapy. For this reason, additional alternatives are made necessary, such as the use of medicinal plants that are used by communities with no access to serum therapy. The Brazilian flora possesses a wide variety of medicinal plants with antiophidian potential, which have been little-studied scientifically. In the present study, we performed in vitro and in vivo neutralization of snake venoms with the aqueous extract of inner bark of Anacardium humile (EAAh), and the isolation and phytochemical characterization of an inhibitor of myotoxins, the gallic acid (GA). For the inhibition assays, we used solutions containing crude venom or isolated toxin mixed with different amounts of plant extracts that were previously incubated for 30 min at 37°C. Administration of the extract after envenomation was also performed at different time intervals for myotoxicity inhibition assays. It was observed that EAAh has inhibitory activity against the toxic (lethality, myotoxicity and hemorrhage) and pharmacological/enzymatic effects (edema-inducing, coagulant and phospholipase activities) induced by snake venoms of the genera Bothrops, Crotalus, Lachesis and isolated toxins. The plant extract inhibited 100% of the lethality induced by C. d. terrificus venom and its major neurotoxin, crotoxin. The EAAh was subjected to analytical chromatographic separation, and in polar conditions, it was possible to identify and isolate the gallic acid, which showed retention time and nuclear magnetic resonance spectra similar to the commercial standard and to literature data of this same compound, respectively. Gallic acid alone was able to inhibit the myotoxic activity induced by crude venom of B. jararacussu and its main myotoxin, BthTX-I, a Lys49 PLA2-like enzyme. The analysis of circular dichroism spectra and interaction studies by molecular modeling suggest that gallic acid forms a complex with BthTX-I in its active site, which inhibits its toxic activity. The binding of gallic acid to myotoxins did not change neither the form nor the intensity of circular dichroism spectra, not inducing significant changes in the percentage of the various domains that form the secondary structure of these proteins. The gallic acid and other tannins have been showed to be good inhibitors of the toxic effects of snake venoms, and our study showed that this acid is related to the inhibitory action of the Anacardium humile extract.

Os envenenamentos causados pelas serpentes do gênero Bothrops são os mais importantes do ponto de vista médico e econômico na América Central. Dentre estas, a serpente Bothrops asper é responsável por 90% dos envenenamentos ofídicos registrados no Panamá anualmente. Apesar da relevância médica e econômica, só a peçonha de populações de B. asper da Costa Rica e Guatemala tem sido estudadas em detalhes. Neste trabalho apresentamos a caracterização da peçonha da B. asper do Panamá e o isolamento, a caracterização funcional e estrutural de quatro fosfolipases A2 básicas denominadas MTX-I, MXT-II, MTX-III e MXT-IV e de uma fosfolipase A2acídica denominada Basp-I-PLA2. As fosfolipases A2 foram isoladas da peçonha em duas etapas usando cromatografia de troca iônica em CM-Sepharose (0,05 M NH4HCO3 pH 8,1), e cromatografia hidrofóbica em Fenil-Sepharose (0,05 M Tris-HCl pH 7,4) seguida de gradiente de concentração de 4 a 0 M NaCl no mesmo tampão a temperatura ambiente (25°C). A isoforma acídica demonstrou maior atividade catalítica que as isoformas básicas, quando atuou sobre fosfatidilcolina e fosfatidilglicerol. A focalização isoelétrica evidencia pIs de 8,1 a 8,3 para as MTXs e 4,6 para isoforma Basp-I-PLA2. A determinação da massa molecular por espectrometria de massa mostrou que MTX-1 14,156.5; MTX-2 14,249.5 e MTX-3 14,253.0 e Basp-I-PLA2 14,246.0 Da. As PLA2s (MTX-I, II, III e IV) induziram atividade miotóxica, reação inflamatória com migração de leucócitos ao músculo e ativação de macrófagos para exercer fagocitose e produção de superóxido. MTX-II exerceu um forte efeito citotóxico contra células tumorais JURKAT, C. albicans e E. coli. A fosfolipase A2 acídica, testada no plasma enriquecido com plaquetas, mostrou potente efeito inibitório na agregação plaquetária induzida pela ADP e colágeno. A análise de seqüência N- terminal demonstrou que as proteínas MTX-I, MTX-III e Basp-I-PLA2 pertencem à subclasse de fosfolipases A2 Asp49 cataliticamente ativas, enquanto que, MTX-II e MTX-IV pertencem à subclasse de fosfolipases A2 Lys49-homólogas cataliticamente inativas. Além disso, a sequência N-terminal das fosfolipases A2 básicas isoladas, demonstrou claramente que as miotoxinas isoladas neste trabalho são similares às miotoxinas previamente isoladas da peçonha da B. asper da Costa Rica. A Basp-I-PLA2 é uma nova fosfolipase A2 acídica isolada da peçonha de B. asper do Panamá e sua seqüência N-terminal revelou uma alta homologia com outras PLA2s acídicas Asp49 isoladas de peçonhas de serpentes. / Envenoming by Bothrops snakes is the most serious kind of envenoming from the medical and economical point of view in Central America. Bothrops asper snake is responsible for 90% of the snakebites registered in Panama every year. Despite its medical and economical relevance, only the venom of Costa Rica and Guatemala populations of this species has been studied to some detail, and there is very little information on intraspecies variability in venom composition and toxicity. In this study the crude venom of B. asper from Panama was characterized and its pharmacological and biochemistry activities were investigated with standard laboratory assays. Furthermore, we describe the isolation, functional and structural characterization of four basic phospholipases A2, named MTX-I, MTX-II, MTX-III, MTX-IV and a new acid phospholipase A2 named Basp-I-PLA2. The proteins were isolated from the crude venom by a combination of two chromatographic steps, using ion-exchange chromatography on CM-Sepharose (0.05 M NH4HCO3 pH 8.1 buffer), and hydrophobic chromatography on Phenyl-Sepharose (0.05 M Tris-HCl pH 7.4), followed by concentration gradient from 4 to 0 M NaCl at 25°C in the same buffer. Analyses of phospholipids hydrolyzed by these enzymes have shown that all phospholipases belong to type A2. The acidic isoform demostraded more catalytic activity than the basic PLA2s. This enzyme was more active on substrates as phosphotidylcholine and phosphatidylglycerol. The isoelectric focusing evidenced pIs beetwen 8.1 to 8.3 for the MTXs and 4.6 for the isoform Basp-I-PLA2. Its mol. Wt was estimated by Mass spectrometry to be MTX-1 14,156.5; MTX-2 14,249.5 and MTX-3 14,253.0 and Basp-I-PLA2 14,246.0 Da. The PLA2s (MTX-I, II, III and IV) induced myotoxic activity, inflammatory reaction mainly leukocyte migration to the muscle and activation of macrophages to exert phagocytic activity and production of superoxide. MTX-II, the most abundant one showed to be cytotoxic against JURKAT tumor cell line, C. albicans and E. coli. The acidic phospholipases A2 when tested in platelet rich plasma, showed a potent inhibitory effect on aggregation induced by ADP and collagen. The analysis of the sequence N-terminal demonstrated that the MTX-I, MTX-III and BASP-I-PLA2 belong to the subclass of Asp49 phospholipases A2 catalytically active, whereas, MTX-II and MTX-IV belong to proteins of the subclass of the enzymatically inactive Lys49 PLA2 s-like. In addition, a sequence of the region N-terminal of the PLA2s basic isolated, demonstrated clearly, that the isolated myotoxins in this work are similar of the previously isolated myotoxins of the snake venom Bothrops asper from Costa Rica. The Basp-I-PLA2 is a new acidic PLA2 and his sequence N-terminal revealed a high homology with other Asp49 acidic PLA2 s from snake venoms.

Les maladies cardiovasculaires (MCV) sont les principales causes de mortalité et de morbidité à travers le monde. En Amérique du Nord, on estime à 90 millions le nombre d’individus ayant une ou plusieurs MCV, à près de 1 million le nombre de décès reliés par année et à 525 milliards de dollars les coûts directs et indirects en 2010. En collaboration avec l’équipe du Dre. Boileau, notre laboratoire a récemment identifié, le troisième locus impliqué dans l’hypercholestérolémie familiale. Une étude publiée dans le New Engl J Med a révélé que l’absence de la convertase PCSK9 réduit de 88% le risque de MCV, corrélé à une forte réduction du taux de cholestérol plasmatique (LDL-C). Il fut démontré que PCSK9 lie directement le récepteur aux lipoprotéines de faible densité (LDLR) et, par un mécanisme méconnu, favorise sa dégradation dans les endosomes/lysosomes provoquant ainsi une accumulation des particules LDL-C dans le plasma. Dans cet ouvrage, nous nous sommes intéressés à trois aspects bien distincts : [1] Quels sont les cibles de PCSK9 ? [2] Quelle voie du trafic cellulaire est impliquée dans la dégradation du LDLR par PCSK9 ? [3] Comment peut-on inhiber la fonction de PCSK9 ? [1] Nous avons démontré que PCSK9 induit la dégradation du LDLR de même que les récepteurs ApoER2 et VLDLR. Ces deux membres de la famille du LDLR (fortes homologies) sont impliqués notamment dans le métabolisme des lipides et de la mise en place de structures neuronales. De plus, nous avons remarqué que la présence de ces récepteurs favorise l’attachement cellulaire de PCSK9 et ce, indépendamment de la présence du LDLR. Cette étude a ouvert pour la première fois le spectre d’action de PCSK9 sur d’autres protéines membranaires. [2] PCSK9 étant une protéine de la voie sécrétoire, nous avons ensuite évalué l’apport des différentes voies du trafic cellulaire, soit extra- ou intracellulaire, impliquées dans la dégradation du LDLR. À l’aide de milieux conditionnées dérivés d’hépatocytes primaires, nous avons d’abord démontré que le niveau extracellulaire de PCSK9 endogène n’a pas une grande influence sur la dégradation intracellulaire du LDLR, lorsqu’incubés sur des hépatocytes provenant de souris déficientes en PCSK9 (Pcsk9-/-). Par analyses de tri cellulaire (FACS), nous avons ensuite remarqué que la surexpression de PCSK9 diminue localement les niveaux de LDLR avec peu d’effet sur les cellules voisines. Lorsque nous avons bloqué l’endocytose du LDLR dans les cellules HepG2 (lignée de cellules hépatiques pour l’étude endogène de PCSK9), nous n’avons dénoté aucun changement des niveaux protéiques du récepteur. Par contre, nous avons pu démontrer que PCSK9 favorise la dégradation du LDLR par l’intermédiaire d’une voie intracellulaire. En effet l’interruption du trafic vésiculaire entre le réseau trans-Golgien (RTG) et les endosomes (interférence à l’ARN contre les chaînes légères de clathrine ; siCLCs) prévient la dégradation du LDLR de manière PCSK9-dépendante. [3] Par immunobuvardage d’affinité, nous avons identifié que la protéine Annexine A2 (AnxA2) interagit spécifiquement avec le domaine C-terminal de PCSK9, important pour son action sur le LDLR. Plus spécifiquement, nous avons cartographié le domaine R1 (acides aminés 34 à 108) comme étant responsable de l’interaction PCSK9AnxA2 qui, jusqu’à présent, n’avait aucune fonction propre. Finalement, nous avons démontré que l’ajout d’AnxA2 prévient la dégradation du LDLR induite par PCSK9. En somme, nos travaux ont pu identifier que d’autres membres de la famille du LDLR, soit ApoER2 et VLDLR, sont sensibles à la présence de PCSK9. De plus, nous avons mis en évidence que l’intégrité du trafic intracellulaire est critique à l’action de PCSK9 sur le LDLR et ce, de manière endogène. Finalement, nous avons identifié l’Annexine A2 comme unique inhibiteur naturel pouvant interférer avec la dégradation du LDLR par PCSK9. Il est indéniable que PCSK9 soit une cible de premier choix pour contrer l’hypercholestérolémie afin de prévenir le développement de MCV. Cet ouvrage apporte donc des apports considérables dans notre compréhension des voies cellulaires impliquées, des cibles affectées et ouvre directement la porte à une approche thérapeutique à fort potentiel. / Cardiovascular disease (CVD) is the primary cause of death and morbidity worldwide, claiming about 900 000 lives yearly in North America alone. A high level of circulating LDL-cholesterol is a major risk factor positively correlated with premature development of complex CVD mainly due to a rapid buildup of lipid deposition in the arteries. In collaboration with Dre Boileau, we recently discovered that the convertase PCSK9 is the third locus of familial hypercholesterolemia. A study published in the New Eng J Med revealed that the absence of PCSK9 reduces the risk of CVD by ~88%, resulting from a strong reduction of cholesterol in the bloodstream (LDL-C). It has been shown that PCSK9 directly binds the low-density lipoprotein receptor (LDLR) and by an unknown mechanism, reroutes it towards degradation in late endosomes/lysosomes, resulting in the accumulation of LDL-C particles in plasma. In this thesis, we addressed three different aspects of PCSK9 biology: [1] What are the targets of PCSK9? [2] Which cellular trafficking components are involved in PCSK9-induced LDLR degradation? [3] How can we inhibit the function of PCSK9? [1] We first demonstrated that PCSK9 induces the degradation of the LDLR and two of its closest family members. These include the very-low-density-lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. In addition, we demonstrated that these receptors enhance the cellular association of PCSK9 independently of the presence of the LDLR. This study represents the first evidence that PCSK9 could target other proteins for degradation, reinforcing its role as a key regulator of some members of the LDLR family. [2] Since PCSK9 is a secreted protein, we decided to investigate the contributions of both the intra- and extracellular trafficking pathways in LDLR degradation. Using conditioned media derived from mice primary hepatocytes, we showed that endogenously secreted PCSK9 was not able to influence LDLR levels of PCSK9-deficient primary hepatocytes (Pcsk9-/-). By flow cytometry (FACS), we observed that overexpression of the gain-of-function PCSK9-D374Y, but not wild type PCSK9, decreases cell surface LDLR on adjacent cells suggesting that its spectrum of action is local. We also noticed that blockade of endocytosis in HepG2 cells (commonly used to study endogenous LDLR degradation by PCSK9) does not affect total LDLR protein levels. In contrast, disruption of the intracellular trafficking between the trans-Golgi network (TGN) and endosomes (siRNAs against clathrin light chains; CLCs) prevented LDLR degradation in a PCSK9-specific manner. [3] By Far Western blotting, we identified that Annexin A2 (AnxA2) specifically interacts with the C-terminal domain of PCSK9, which is crucial for its function in LDLR degradation. Moreover, we determined that the R1 domain (amino acids 34 to 108) is responsible for the PCSK9AnxA2 interaction, which confers a new function for this protein. Finally, we showed that addition of AnxA2 prevents PCSK9-induced LDLR degradation. In summary, this work allowed us to identify that PCSK9 induces the degradation of the LDLR and its closest family members, ApoER2 and VLDLR. We also highlighted that the integrity of the intracellular trafficking pathway is crucial for endogenous PCSK9-induced LDLR degradation. Furthermore, we discovered that AnxA2 is a unique, natural inhibitor capable of interfering with the action of PCSK9 in LDLR degradation. It is undeniable that PCSK9 is a genetically validated target to reduce circulating LDL-cholesterol and prevent CVD. This thesis brings forth important contributions in our understanding of the cellular pathways involved and opens the door for novel therapeutic approaches.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Uma linhagem celular de CHO, previamente modificada geneticamente pela introdução de cDNA da 2,6 sialiltransferase de rato, gerou, pela primeira vez, um hTSH recombinante com sialilação humanizada (hlsr-hTSH), mais similar ao hormônio nativo, com 61% de ligação de ácido siálico na conformação 2,3 e 39%, na conformação 2,6. O clone mais produtivo, quando submetido à amplificação gênica com 8 M de metotrexato, apresentou um nível de secreção de aproximadamente 2 g de hTSH/106 células/dia, nível este útil para a purificação e caracterização do produto. A massa molecular relativa do heterodímero e das subunidades e do hlsr-hTSH purificado, determinada por espectrometria de massa MALDI-TOF, e a hidrofobicidade relativa, determinada por RP-HPLC, não apresentaram diferenças significativas com relação às preparações de hTSH recombinante derivadas de células CHO sem a modificação devida ao gene da 2,6 sialiltransferase. Entretanto, algumas diferenças foram observadas na composição dos N-glicanos, com mais estruturas tri- e tetra-sialiladas no hlsr-hTSH. O hlsr-hTSH mostrou-se eqüipotente (p>0,05) à preparação comercial de r-hTSH (Thyrogen) e 1,5 vezes mais potente do que a preparação nativa de hTSH (p<0,001), quando analisado por um bioensaio in vivo baseado na capacidade do TSH de estimular a liberação de T4. / A CHO cell line, previously genetically modified by the introduction of rat 2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of 2,3- and 39% of 2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 M methotrexate, presented a secretion level of ~2 g hTSH/106 cells/day, useful for product purification and characterization. The relative molecular masses (Mr) of the heterodimer and of the - and -subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T4 release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.5-fold more potent than native hTSH (p < 0.001).

Orientador: Marcos Roberto de Mattos Fontes / Resumo: As fosfolipases A2 (PLA2s) são um dos maiores constituintes protéicos do veneno botrópico e um dos responsáveis pela necrose muscular, consequência esta não eficazmente neutralizada pela administração do soro antiofídico. Estas proteínas são tóxicas através do rompimento ou perturbação da membrana celular em um mecanismo catalítico dependente de cálcio e outro independente, sendo este último não totalmente elucidado. Usualmente, estas toxinas são obtidas diretamente do veneno das serpentes, sendo sua purificação um desafio pela co-existência de diferentes isoformas. O objetivo desta tese foi compreender o mecanismo miotóxico independente de cálcio através de estudos estruturais e propor nova metodologia que trate de dados cristalográficos de toxinas provenientes de amostras impuras, chamada SEQUENCE SLIDER. Para tanto, cristalografia e outras técnicas biofísicas, como espalhamento de raios X a baixo ângulo, serão utilizados para estudar três miotoxinas ofídicas em estado nativo e complexado com produtos naturais e inibidores. Nós propusemos medidas locais e globais para caracterizar e relacionar a estrutura dessas toxinas a função. Com o SEQUENCE SLIDER, pudemos elucidar as estruturas de toxinas inéditas cuja sequência era parcialmente conhecida. Esta nova metodologia proposta consiste em avaliar diferentes cadeias laterais contra o coeficiente de correlação em espaço real calculado a partir dos dados cristalográficos. Em paralelo, desenvolvemos o SEQUENCE SLIDER no âmbito do... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

La cycline A2 est un régulateur essentiel du cycle cellulaire qui, en association avec des kinases dépendantes des cyclines (CDK) régule la réplication de l'ADN et l’entré des cellules en mitose. Dans de nombreux types de cancers humains, la cycline A2 a été considérée comme un facteur de prolifération contribuant à la cancérogenèse de par ses fonctions dans la régulation du cycle cellulaire. Récemment, la complexité des fonctions de cycline A2 a été révélée. Certaines études in vitro ont démontré que l'inactivation de la cycline A2 induit une augmentation de la motilité cellulaire et de l'invasion dans les fibroblastes consécutive à une activation défective de RhoA. De plus, il a été montré que l'inactivation de la cycline A2 induit la EMT par l’intermédiaire d’une augmentation de l'activité transcriptionnelle de la β-caténine, mais aussi via la voie TGFβ/Prefoldin. Ces études suggèrent que des niveaux réduits de cycline A2 sont liés à une invasion accrue et à l’apparition de métastases dans certains types de cancer. A l’aide d’un modèle de souris mutante pour la cycline A2, une étude récente a établi une fonction de cette dernière, indépendante des CDK, dans la réparation des cassures double brin de l'ADN et a montré que la perte de la cycline A2 favorise l’apparition de cancers de la peau et du poumon. L’ensemble de ces études met en évidence l’existence de multiples fonctions pour la cycline A2. Mon travail de thèse a consisté à explorer le rôle de la cycline A2 dans l'homéostasie du colon et le développement du Le cancer colorectal.Pour évaluer la valeur pronostique de la cycline A2 dans le CCR, nous avons analysé l'expression de la cycline A2 par IHC sur un grand nombre d'échantillons tumoraux dérivés de patients atteints de CCR de différents stades. Nous avons trouvé que les niveaux élevés de la cycline A2 sont corrélés avec un mauvais pronostic et une survie plus faible chez les patients atteints de CCR de stade I et II. Cependant, une diminution de l'expression de la cycline A2 a été détectée aux stades III et IV par comparaison aux biopsies de CCR de stade I et II. Il est tentant de proposer que le profil d'expression de la cycline A2 reflète ses rôles distincts au cours de la cancérogenèse du côlon, comme la prolifération cellulaire au stade précoce, lorsqu'elle est fortement exprimée, mais favorise l'invasion et l'agressivité à des stades plus avancés, lorsque ses niveaux d’expression sont réduits.En complément de cette étude clinique, nous avons généré des modèles murins porteurs d’une mutation constitutive ou inductible de la cycline A2 dans l’épithélium intestinal. Nous avons montré que la déplétion de la cycline A2 dans l'épithélium intestinal de la souris provoque une rupture de la crypte colique, une inflammation, une augmentation de la prolifération des cellules épithéliales et l’apparition de dysplasies de bas et haut grade, reconnues comme lésions précancéreuses dans le CCR. Ces observations suggèrent un rôle majeur pour la cycline A2 dans la régulation de l'homéostasie du côlon et l'initiation de la tumorigénèse. Une analyse plus poussée a révélé une proportion accrue de lésions au niveau de l'ADN et une activation aberrante de la β-caténine, anomalies couramment détectées chez les patients humains atteints de CCR et considérées comme les premières altérations de cette pathologie. En outre, nous avons détecté une expression élevée de NFkB et YAP1 chez les souris mutantes pour la cycline A2, voies qui jouent un rôle critique dans la régénération tissulaire et peuvent conduire la dédifférenciation des cellules épithéliales du côlon contribuant ainsi à la tumorigenèse. Finalement, les souris mutantes cycline A2 ont été soumises à un protocole de colite associé au cancer et ont développé une proportion accrue d'inflammation, mais aussi de dysplasies et d'adénocarcinomes, en taille et en nombre, suggérant que la perte de la cycline A2 participe à la carcinogenèse colorectale chez la souris. / Cyclin A2 is an essential cell cycle regulator required for accurate DNA replication and mitotic entry in association with cyclin-dependent kinases (CDKs). In multiple types of human cancers, cyclin A2 was considered as a proliferation driver contributing to carcinogenesis based on its function to promote cell cycle.Recently, the complexity of cyclin A2 functions has been revealed. Some in vitro studies demonstrated that cyclin A2 inactivation induces increased cell motility and invasiveness of mouse fibroblasts due to defective RhoA activation. Moreover, cyclin A2 inactivation has been shown to induce EMT through the upregulation of β-catenin transcriptional activity, but also via the TGFβ/Prefoldin pathway. These studies suggest that reduced levels of cyclin A2 are linked to increased invasiveness and metastasis in some cancer types. Using a cyclin A2 mutant mouse model, a recent study established the CDK-independent function of cyclin A2 in the repair of double-stranded DNA breaks and showed that loss of cyclin A2 promotes tumorigenesis in skin and lung due to deficient DSBs repair.Altogether, these studies highlight the multiple functions cyclin A2 can execute. The aim of my thesis was to explore the role of cyclin A2 in colon homeostasis and colorectal cancer development.To evaluate the prognostic value of cyclin A2 in CRC, we analyzed cyclin A2 expression by IHC on tumor samples derived from CRC patients of different stages. We found that high levels of cyclin A2 correlate with bad prognosis and lower survival in patients with stage I and II CRC. However, decreased cyclin A2 expression was detected in stage III and IV by comparison to stage I and II CRC biopsies. Complementary to the clinical study, we generated tissue-specific mutant mouse models bearing either a constitutive or inducible cyclin A2 deletion in the intestinal epithelium. We showed that depletion of cyclin A2 in mouse intestinal epithelium causes colonic crypt disruption, inflammation, increased proliferation of epithelial cells and occurrence of low- and high-grade dysplasia, recognized as precancerous lesions of CRC. These observations suggest a major role for cyclin A2 in the regulation of normal colon homeostasis and tumor initiation. Further analysis revealed an increased proportion of DNA damage and aberrant activation of β-catenin, commonly detected in human patients with CRC and which are considered as the first occurring alterations in this pathology. Furthermore, we detected elevated expression of NFkB and YAP1 in the colons of cyclin A2 mutant mice, pathways that have been previously shown to play critical roles for tissue regeneration after tissue damage and to drive dedifferentiation of colonic epithelial cells thus contributing to tumorigenesis. Finally, cyclin A2 mutant mice were subjected to a modified colitis-associated CRC model and developed increased proportion of inflammation, but also dysplasia and adenocarcinomas, in size and numbers, suggesting that loss of cyclin A2 contributes to inflammation-associated colorectal carcinogenesis in mice.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / O Aspergillus fumigatus é o principal agente etiológico da aspergilose invasiva, uma infecção fúngica oportunista que acomete, principalmente, pacientes de Unidades Hematológicas, como aqueles com neutropenia profunda e prolongada. Após a filamentação este fungo angioinvasivo é capaz de ativar e causar danos em células endoteliais de veia umbilical humana (HUVEC) que passam a expressar um fenótipo pró-trombótico. A ativação destas células, dependente de contato célulacélula, é mediada por TNF-&#945; e caracterizada pela expressão de moléculas próinflamatórias, como citocinas, quimiocinas e moléculas de adesão. Recentemente, nosso grupo comparou a ativação endotelial de HUVECs desafiadas com cepas selvagens e uma cepa mutante para o gene UGM1. Nestes experimentos a cepa mutante &#916;ugm1, que apresenta um fenótipo de maior produção de galactosaminogalactana (GAG) na parede celular, mostrou um fenótipo hiperadesivo e uma capacidade maior de ativar células endoteliais. Entretanto, os receptores e as vias de sinalização envolvidos nesta ativação permanecem desconhecidos. Assim, o objetivo deste trabalho foi verificar as proteínas envolvidas nestes processos através do estudo das proteínas diferencialmente expressas nas HUVECs após a interação com A. fumigatus, usando a técnica proteômica 2D-DIGE. Brevemente, as HUVECs foram infectadas com tubos germinativos da cepa selvagem (AF293) e da cepa &#916;ugm1 de A. fumigatus. Em seguida, as proteínas foram marcadas com diferentes fluorocromos e separadas por eletroforese bidimensional. A análise quantitativa foi realizada utilizando o software DeCyder. Foram identificadas por MS/MS cinco proteínas diferencialmente expressas, incluindo a galectina-1 e a anexina A2, ambas mais expressas após a interação, sendo a primeira ~25% mais expressa após a interação com a mutante &#916;ugm1. Este trabalho propõe que a galectina-1 poderia ser o receptor endotelial para polímeros de galactose presentes na parede celular do A. fumigatus, e que a Anexina A2 poderia estar envolvida na sinalização intracelular em resposta a este patógeno. No entanto, experimentos complementares, em curso, são necessários para comprovar esta hipótese. / Aspergillus fumigatus is the main etiological agent of invasive aspergillosis, the main opportunistic fungal infection of Hematologial Unitys patients, especially those with long-term neutropenia. Upon filamentation, this angioinvasive fungus can activate and damage the human umbilical vein endothelial cells (HUVEC), which in response switch to a pro-thrombotic phenotype. HUVEC activation is mediated by TNF-&#945; once cell-cell contact occurs. This activation is characterized by the expression of pro-inflammatory molecules such cytokines, chemokines and adhesion molecules. Recently, our group performed the comparison of HUVEC activation upon interaction with a wild type and the UGM1 mutant strains of A. fumigatus. The &#916;ugm1 strain, which presents an increased production of the cell wall galactosaminogalactan, showed a hyper adherent phenotype and an increased capability to cause endothelial cell stimulation and activation, when compared with the wild type strain. The receptors involved in the pathogen-host interaction or the signaling pathways after endothelial activation by A. fumigatus remain unknown. Thus, the aim of this study was to investigate the differentially expressed proteins in HUVECs upon interaction with A. fumigatus, using the 2D-DIGE proteomic approach. Briefly, HUVECs were challenged with germlings of A. fumigatus wild type Af293 and &#916;ugm1 strains and then submitted to protein extraction. The total HUVEC protein extracts were labeled with different CyDyes and fractionated by 2D electrophoresis. Quantitative analysis to determine the differences in protein abundance amongst interacted cells vs. control endothelial cells was performed using the software DeCyder. Five differentially expressed proteins were identified by MS/MS including galectin-1 and annexin A2, both overexpressed after the interaction. These two proteins are described elsewhere to be associated with host-pathogen interaction. Besides, galectin-1 showed an ~25% increase after interaction with the &#916;ugm1 strain and it is plausible that this particular protein could be a putative receptor for galactose-containing polymers of the A. fumigatus cell wall and annexin A2 could be involved in signalizing pathways upon interaction. However, other experimental evidences, under development, are necessary to confirm this hypothesis.