La synthèse totale de la N1999-A2 représente un défi synthétique qui a intéressé plusieurs grandes équipes spécialisées dans ce domaine. L’approche synthétique envisagée repose sur le savoir-faire du laboratoire. Elle permet de former le cœur diènediyne dans les dernières étapes de la synthèse. Nous avons réussi à réaliser le premier couplage des deux synthons clefs, une avancée majeure dans le cadre de notre approche synthétique. Ce travail a aussi permis le développement de nouvelles méthodologies. Notamment les couplages croisés au palladium avec un nouveau type de partenaire électrophile stable : les nosylates d’aryle ou vinyle. Cette découverte permettra de compenser l’instabilité de plusieurs intermédiaires synthétiques clefs (triflates d’énol). / The total synthesis of N1999-A2 is a synthetic challenge which mobilized several renown groups specialized in this field. Our group is also interested in the construction of this complex molecule. Our strategy is based on the know-how of our laboratory. The considered synthetic approach permits the formation of the dienediyne core at the lasts steps of the synthesis. We managed to achieve the first coupling of the two key building blocks. This is a major advance in our synthetic approach. This project also allowed the development of new synthetic methodologies. In particular the palladium catalyzed cross-coupling reactions with aryl and vinyl nosylates, novel stable and reactive electrophilic partners. This discovery will allow us to bypass the extreme instability of several key intermediates (enol triflates).

As fosfolipases A2 (PLA2s) são enzimas que induzem vários efeitos farmacológicos e geralmente, correspondem a maior porcentagem do conteúdo protéico dos venenos de serpentes. Desta forma, o isolamento e a caracterização bioquímica, funcional e estrutural de PLA2s poderão gerar informações importantes para o melhor entendimento dos efeitos farmacológicos e de efeitos tóxicos ocasionados por estas proteínas. Através de dois métodos cromatográficos (troca-iônica em CM-Sepharose e hidrofóbica em Phenyl-Sepharose) foi isolada uma isoforma de fosfolipase A2 ácida presente na peçonha da serpente Bothrops moojeni, denominada de BmooPLA2. Quando submetida à eletroforese em gel de poliacrilamida com agente desnaturante, BmooPLA2 apresentou massa molar relativa de aproximadamente 14.000. A proteína isolada, BmooPLA2, possui uma única cadeia polipeptídica, pI~5,2, é rica em aminoácidos hidrofóbicos, ácidos e possui 14 resíduos de cisteína. Esta isoforma pH-termoestável apresentou alta atividade fosfolipásica. A enzima induziu edema moderado in vivo, na concentração de 25 g. Além disso, a BmooPLA2 foi capaz de inibir a agregação plaquetária de modo dose dependente e induzir efeito hipotensor nas concentrações de 15 e 30 g . Foi realizada também a construção da biblioteca de cDNA da glândula de peçonha da serpente Bothrops moojeni, onde o cDNA que codifica a proteína BmooPLA2 foi clonado e a proteína recombinante expressa em E. coli. Todos os ESTs (Expressed Sequence Tags) foram classificados de acordo com a homologia de sua estrutura primária com sequências conhecidas. As sequencias codificando para toxinas representaram cerca de 30% do total de sequências identificadas. De acordo com o transcriptoma, as toxinas mais expressas pela serpente Bothrops moojeni são as metaloproteases (SVMP), as quais correspondem a aproximadamente 77% das toxinas encontradas. A proteína recombinante apresentou a mesma sequência de aminoácidos, atividade fosfolipásica e efeito inibitório sobre plaquetas, observados para a proteína nativa BmooPLA2, sugerindo que a recBmooPLA2 foi expressa, purificada e reenovelada em sua forma ativa. Como nenhum estudo abordou a participação das PLA2s ácidas de Bothrops nos processos inflamatórios e os mecanismos envolvidos na liberação desses mediadores, particularmente os prostanóides, as toxinas nativa e recombinante foram avaliadas quanto ao seu efeito sobre a resposta inflamatória (ensaios sobre leucócitos in vitro), avaliando a expressão da enzima COX-2 e liberação do prostanóide PGE2, além de outros mediadores como TXB4 e LTB2, após a incubação com macrófagos isolados, in vitro. Com estes resultados foi possível uma melhor compreensão da composição da peçonha desta serpente, bem como um melhor entendimento da participação das PLA2s ácidas envenenamento ofídico, abrindo novas perspectivas para sua aplicação biotecnológica. / The phospholipase A2 (PLA2s) are enzymes that induce various pharmacological effects and usually correspond to a higher percentage of the protein content of snake venoms. Thus, isolation, biochemical, functional and structural characterization of PLA2s may generate important information for a better understanding of the pharmacological effects and toxicity induced by these proteins. Through two chromatographic steps (ion exchange on CMSepharose and hydrophobic in Phenyl-Sepharose) an acidic phospholipase A2 isoform was isolated from the venom of the snake Bothrops moojeni and named BmooPLA2. Its biochemical and partial functional characterization were also performed. When submitted to electrophoresis on polyacrylamide gel with denaturing agent (SDS-PAGE), BmooPLA2 presented relative molar mass of approximately 14,000. The isolated protein, BmooPLA2, has a single polypeptidic chain, pI ~ 5.2, is rich in hydrophobic amino acids and has 14 cysteine residues. This pH-thermostable isoform showed high phospholipasic activity. The enzyme induced moderate edema in vivo, at the concentration of 25 g. In addition, BmooPLA2 was able to inhibit platelet aggregation in a dose dependent manner and showed hypotensive effect at different concentrations (15 and 30 g). It was also carried out the construction of the cDNA library from the venom gland of the snake Bothrops moojeni, where the cDNA encoding the protein BmooPLA2 was cloned and a recombinant protein expressed in E. coli. All ESTs (Expressed Sequence Tags) were classified according to their primary structure homology with known sequences. The sequences coding for toxins accounted for approximately 30% of all identified sequences. According to the transcriptome, the majority of the toxins expressed by the snake Bothrops moojeni are metalloproteases (SVMP), which correspond to approximately 77% of the toxins found. The recombinant protein presented the same amino acid sequence, phospholipase activity and inhibitory effect on platelets, observed for the native BmooPLA2, suggesting that recBmooPLA2 was expressed, purified and refolded in its active form. Since no study has addressed the involvement of acidic PLA2s from Bothrops genus upon inflammatory processes and the mechanisms involved in the release of such mediators, particularly prostanoids, native and recombinant toxins were evaluated for their effects on the inflammatory response (essays on leukocytes in vitro), evaluating the expression of COX-2 and prostanoid release of PGE2, as well as other mediators as TXB4 and LTB2, after incubation with isolated macrophages, in vitro. With these results it was possible to better understand the composition of the venom of this snake, and a better understanding of the role of acidic PLA2s on the snake envenomation, opening new perspectives for its biotechnological application.

Le paludisme est une maladie tropicale causée par un parasite du genre Plasmodium. Chez l’Homme, un niveau élevé de phospholipase A2 sécrétée de groupe IIA humaine (hGIIA) est mesuré dans le plasma des patients impaludés. Cette enzyme est connue pour son rôle antibactérien et pro-inflammatoire. Cependant, son rôle dans le paludisme n’a jamais été exploré. Pour comprendre le rôle in vivo de la hGIIA dans cette pathologie, nous avons entrepris la caractérisation hématologique, histopathologique et immunohistochimique de l’infection de souris C57BL/6, transgéniques (Tg+) pour l’enzyme humaine, par l’espèce murine Plasmodium chabaudi chabaudi 864VD. Ce modèle reproduit un paludisme non létal. Nos résultats ont permis d’établir que les souris Tg+ ont un meilleur contrôle de l’infection au moment du pic de crise parasitaire (J14 post-inoculation), avec une diminution de 27% de la parasitémie, comparé aux souris « littermates » non transgéniques (Tg-). L’injection de hGIIA recombinante aux jours 12, 13 et 14 p.i. (0,125 mg/kg deux fois par jour) à des souris C57BL/6 wild-type (WT) infectées par P. c. chabaudi 864VD provoque une diminution d’environ 19% de la parasitémie à J14 p.i., démontrant un rôle direct de la hGIIA dans la diminution de la population parasitaire. Les données hématologiques montrent que l’infection chez la souris Tg+ provoque une anémie plus durable que chez la souris Tg- et une élévation nettement plus importante du nombre de leucocytes, en particulier des polynucléaires neutrophiles. Chez la souris Tg+ parasitée, on observe aussi l’activation d’un nombre important de lymphocytes et une activation spécifique des monocytes avant le pic de crise. Chez la souris Tg- infectée, les données histologiques mettent en avant une meilleure récupération des lésions histopathologiques du foie et une hyperplasie des lymphocytes B dans la rate, tandis que les souris Tg+ infectées présentent des lésions hépatiques tardives et une hématopoïèse extramédullaire splénique. Les résultats des analyses par RT-qPCR suggèrent que l’ARNm de la hGIIA augmente au pic parasitaire dans le foie des souris Tg+ infectées, mais diminue dans la rate et les cellules sanguines. L’injection de hGIIA recombinante au début de la phase patente est sans effet sur la parasitémie, ce qui laisse supposer que des événements plus tardifs dans l’infection sont nécessaires à l’activité antiparasitaire de l’enzyme. L’étude du rôle des lipoprotéines oxydées comme substrat potentiel de l’activité antiparasitaire de l’enzyme, basée sur des résultats in vitro, est abordée. En conclusion, nos études ont permis de dresser un tableau large de l’infection à Plasmodium chez la souris exprimant la hGIIA, et ouvrent de nouvelles perspectives dans l’analyse du rôle de l’enzyme dans la physiopathologie du paludisme. / Malaria is a tropical disease caused by a parasite of the Plasmodium genus. High levels of circulating human group IIA secreted phospholipase A2 (hGIIA) have been reported in malaria patients. The enzyme is well known for its bactericidal and pro-inflammatory actions. However, so far its role in malaria is unknown. In order to address the in vivo role of hGIIA in malaria, we performed a hematological, histopathological and immunohistochemical characterization of C57BL/6 hGIIA transgenic mice (Tg+ mice) infected with P. chabaudi chabaudi (864VD strain), a murine Plasmodium species and strain which causes non-lethal chronic malaria. Infected Tg+ mice present a 27% reduction of parasitaemia at the peak of infection (D14 post-inoculation, p.i.) compared to infected non-transgenic littermates (Tg- mice). Intraperitoneal injection of recombinant hGIIA at D12, D13 and D14 p.i. (0.125 mg/kg twice a day) into P. chabaudi 864VD-infected WT C57BL/6 mice leads to a 19% reduction of the parasitaemia at D14 p.i., demonstrating the direct and acute role of hGIIA in lowering parasite population and presumably ruling out a potential effect linked to chronic overexpression of hGIIA in Tg+ mice. Hematological data show a durable anemia in Tg+ mice compared to Tg- mice during the infection and an important increase of leucocytes, especially of polynuclear neutrophils. The parasitized Tg+ mouse also presents a higher activation of lymphocytes and a specific activation of monocyte cells at the pic of crisis. In the infected Tg- mouse, histological data show a better histopathological recovery in the liver and B cells hyperplasia in the spleen, whereas the infected Tg+ mouse presents late hepatic injuries and splenic extra-medullar hematopoiesis. RT-qPCR analyses suggest that hGIIA mRNA increases at the pic of infection in the liver of infected Tg+ mice, but decreases in spleen and blood. Intraperitoneal injection of recombinant hGIIA at the patent phase is without effect on parasitaemia, which suggests that later infection events are needed for the enzyme antiparasitic activity. Involvement of oxidized-lipoproteins as potential hGIIA substrates, based on in vitro studies, is discussed. In conclusion, our studies allowed us to elaborate a larger picture of the infection of Plasmodium in the mice expressing hGIIA and open new perspectives in the analysis of the role of the enzyme in malaria pathophysiology.

Les biotechnologies de la reproduction sont aujourd’hui largement utilisées dans le contrôle de la fertilité animale et humaine. Ces techniques présentent cependant des rendements faibles et font actuellement l’objet de nombreuses recherches. Ce travail de thèse s’inscrit dans ce contexte et se focalise sur la recherche de nouvelles molécules pro-fertilité dans l’espèce bovine, et s’articule autour de deux axes : le premier consiste à tester les propriétés pro-fertilité d’une enzyme du métabolisme lipidique sur la maturation ovocytaire, la fécondation et le développement embryonnaire préimplantatoire in vitro, et le deuxième à découvrir des molécules permettant d’améliorer la fécondance des spermatozoïdes. Nous démontrons ici que l’application de l’enzyme améliore de manière significative le nombre et la qualité des embryons au stade blastocyste. Un savoir-faire quant à l’utilisation de cette enzyme (fenêtre de traitement, concentration,…) a été développé. Cette thèse a également permit de caractériser différents composés avec des propriétés différentes dont une molécule originale permettant d’augmenter la vitesse des spermatozoïdes. Ce composé est prometteur car il est aussi actif sur des spermatozoïdes issus du testicule, de l’épididyme ou de l’éjaculat, avant ou après congélation. / The reproductive biotechnologies are now widely used in control of animal and human fertility. However, these technics have low yields and are currently the subject of much research. In this context, the present thesis focuses on the search for new pro-fertility molecules in cattle, organized around two axis : the first one is to test the pro-fertility properties of an enzyme from lipid metabolism on maturation, fertilization and preimplantation embryo development in vitro, and the second one is to discover molecules to improve sperm fertilizing ability. Here, we show that application of the enzyme significantly improve the number and quality of embryos at the blastocyst stage. Expertise in the use of this enzyme (time of treatment, concentration, etc.) was developed. This thesis also allowed the characterization of different compounds with different properties. Among them, one original molecule increase sperm velocity. This compound is promising because it works on sperm from the testis, epididymis or ejaculate before or after freezing.

L’immunologie anti-tumorale et l’immunothérapie ont connu dernièrement de grandes avancées avec la mise en évidence du processus d’immuno-surveillance et le développement de plusieurs approches vaccinales. Il n’en demeure pas moins que l’induction d’une réponse immunitaire anti-tumorale se traduit peu par l’éradication de la tumeur. Comme phénomène dynamique et interactif, la réponse cytotoxique anti-tumorale implique les effecteurs cytotoxiques et les cibles tumorales; pourtant, le microenvironnement tumoral et sa plasticité influent largement sur l’efficacité de celle-là. Avec l’appui de récentes données expérimentales, il apparaît crucial de prendre en compte la susceptibilité tumorale à la lyse par les effecteurs cytotoxiques anti-tumoraux, notamment les lymphocytes T cytotoxiques (CTLs), et plus particulièrement dans un contexte de plasticité cellulaire. Ainsi, le principal objectif de mes travaux de thèse est de saisir le rôle de la Transition Épithélio-Mésenchymateuse (EMT) dans la susceptibilité des cellules tumorales à la lyse par les CTLs dans des modèles cellulaires de cancer du sein. Nos résultats montrent que l’EMT est capable d’induire une diminution de la susceptibilité des cellules mésenchymateuses à la lyse spécifique. Elle engage de ce fait de multiples acteurs. Tout d’abord, dans les deux modèles d’étude, il s’avère que l’EMT est capable de réguler négativement l’expression de la molécule HLA-A2. Ensuite, dans le premier modèle expérimental, nous avons établi que l’EMT induit une altération de la signalisation au niveau de la synapse immunologique. De plus, le régulateur de l’autophagie, Becline 1, joue un rôle crucial dans l’induction de la diminution de la sensibilité à la lyse par les lymphocytes T-CD8+ suite à l’induction de l’EMT. Dans le deuxième modèle d’étude, le mécanisme mis en jeu par l’EMT pour réguler la susceptibilité des cellules mésenchymateuses à la lyse par les CTLs se manifeste dans l’induction du facteur de transcription inducteur des propriétés de cellules souches cancéreuses, le KLF4 ainsi que via la régulation négative de l’expression du miR-7. Ensemble, ces résultats élucident de nouveaux mécanismes d’échappement des cellules tumorales malignes à la lyse par les lymphocytes T-CD8+ suite à l’induction de l’EMT. Cette étude soutient ainsi l’importance du ciblage des facteurs de transcription inducteurs de l’EMT et responsables de la plasticité cellulaire afin de neutraliser leur fonction. Cela pourrait aider à construire une nouvelle stratégie pour mieux contrôler l’échappement des cellules tumorales invasives à la lyse spécifique et in fine pour garantir une immunothérapie plus efficace contre le cancer. / The anti-tumor immunology and immunotherapy have recently undergone major breakthroughs, with the identification of immune surveillance process and the development of several vaccine approaches. However, the fact remains that the induction of an antitumor immune response is still not effective enough. Certainly, the antitumor cytotoxic response is a dynamic and interactive phenomenon, involving cytotoxic effectors and tumor targets, but its effectiveness is considerably influenced by the tumor microenvironment and its plasticity. Recent studies support the importance of taking into account the tumor susceptibility to lysis by anti-tumor cytotoxic effectors, notably Cytotoxic T Lymphocytes (CTLs), especially in a context of cellular plasticity. On the grounds of these studies, this research aims at understanding the role of Epithelial to Mesenchymal Transition (EMT) in the susceptibility of tumor cells to CTLs mediated lysis in different models of breast cell carcinoma. Our results reveal that EMT is able to induce a decrease in the susceptibility of mesenchymal cells to specific lysis. It calls therefore multiple actors. First, in both study models, it turns out that the EMT is able to downregulate the expression of HLA-A2 molecule. Then, in the first experimental model, we show that EMT induces an alteration of signalling at the immunological synapse. Moreover, the regulator of autophagy, Beclin 1, plays a crucial role in the induction of reduced susceptibility to lysis by T-CD8+ lymphocytes following induction of EMT. In the second experimental model, we show that the mechanisms used by EMT to regulate the susceptibility of mesenchymal cells to lysis by CTLs involve the induction of the transcription factor inducing cancer stem cells properties, KLF4, as well as the downregulation of miR-7 expression. Together, these results shed light on new mechanisms used by malignant tumor cells to escape to lysis by T-CD8+ lymphocytes following the induction of EMT. Thus, this study advocates the importance of targeting transcription factors, which are inducers of EMT and responsible for cellular plasticity, in order to neutralize their function. These insights may prove useful for the development of new strategies aimed at better controlling the escape of invasive tumor cells to specific lysis, and ultimately ensuring a more effective immunotherapy against cancer.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / This study objective to determine the cardiorrespiratory effects and viability of the association between various alpha2-agonists and lidocaine via epidural in bitches submitted to ovaryhysterectomy. Fourty two bitches were castrated under lumbo-sacral epidural anesthesia with lidocaine 1% with vasoconstrictor (CON) or associated with 0,25 mg/kg of xylazine (XYL), 2 mg/kg of dexmedetomidine (DEX), 5 mg/kg of clonidine (CLO), 10 mg/kg of romifidine (ROM), or 30 mg/kg of detomidine (DET), with a total volume of 1ml/4kg. During the procedure, cardiac (HR) and respiratory (RR) rates, and systolic, mean and dyastolic arterial pressures were measured before epidural and every 10 minutes after the epidural injection. Also pH and blood gases were measured before, 30 and 60 minutes after epidural injection. If the animals presented disconfort signals, they were submitted to inhalatory anesthesia with isoflurane 5%, intubated and manteined with 1,5% ETiso. The duration of epidural block and pos-operatory analgesia were also measured. All animals in CON and DEX groups, five animals in ROM and CLO, four animals in XYL and three animals in DET needed complementation with inhalatory anesthesia. All groups, except CLO, presented decrease in HR. There was increase in blood pressures, and DET presented marked hypertesion.Values of RR, blood gases and duration of epidural block were similar between groups. Post-operatory analgesia was prolonged in XYL, persisting for up to four hours. None of the protocols tested was totally efficient, but xylazine prolonged post-operatory analgesia. The second chapter of this dissertation aimed to evaluate the correlation between visual analog scale (VAS), Melbourne pain scale and Von Frey filaments in the evaluation of postoperatory pain in bitches submitted to ovaryhysterectomy. The same animals were evaluated by two observers unaware of the protocol used, in one-hour intervals, using VAS, Melbourne pain scale and Von Frey filaments, applied around the surgical wound. A score of 50 mm in VAS or 13 points in the Melbourne pain scale were considered as the criterion for analgesic administration. VAS and Melbourne pain scale showed a good correlation, with r = 0,74. Correlation between EVA and Von Frey filaments was weak (r = -0,40). Correlation between Melbourne pain scale and Von Frey filaments was -0,37. VAS revealed to be the most sensitive scale. VAS and Melbourne pain scale determined a good correlation degree. We sugest a lower score in the Melbourne pain scale to be considered as criterion for analgesic administration. / Objetivou-se determinar a viabilidade e efeitos cardiorrespiratórios da associação entre diferentes agonistas a2-adrenérgicos e lidocaína via epidural para realização de ovariossalpingohisterectomia (OSH) em cadelas. Foram castradas 42 cadelas, as quais eram mantidas sob anestesia epidural lombo-sacral com lidocaína 1% com vasoconstrictor (CON) ou em associação com 0,25 mg/kg de xilazina (XIL), 2 mg/kg de dexmedetomidina (DEX), 5 mg/kg de clonidina (CLO), 10 mg/kg de romifidina (ROM), ou 30 mg/kg de detomidina (DET), perfazendo um volume final de 1ml/4kg. Avaliaram-se as frequências cardíaca (FC) e respiratória (f) e as pressões arteriais sistólica, média e diastólica antes da epidural e a cada 10 min pós-epidural e pH e gases sanguíneos antes da epidural e 30 e 60 min após. Ao menor sinal de desconforto frente ao procedimento, os animais eram submetidos à anestesia inalatória com 5% de isofluorano, intubados e mantidos com ETiso de 1,5%. Avaliou-se o tempo de bloqueio anestésico e analgesia pós-operatória. Todos os animais do CON e DEX, cinco animais do ROM e CLO, quatro animais do XIL e três do DET necessitaram de complementação com isofluorano. Todos os grupos, exceto CLO, apresentaram diminuição da FC após a epidural. Houve aumento das pressões arteriais em todos os grupos, observando-se hipertensão acentuada no DET. Os valores de ¦, gases sanguíneos e tempo de bloqueio anestésico não diferiram entre grupos. A analgesia pós-operatória foi mais duradoura no XIL, persistindo por até quatro horas. Nenhum dos protocolos foi totalmente eficiente para a realização do procedimento, porém a xilazina produziu analgesia pós-operatória mais duradoura. A segunda parte desta dissertação objetivou avaliar a correlação entre a escala visual analógica (EVA), escala de Melbourne e os filamentos de Von Frey, na avaliação da dor pós-operatória em cadelas submetidas à OSH. Para isso, as mesmas cadelas foram avaliadas em relação à dor pós-operatória por dois observadores cegos, em intervalos de uma hora, utilizando a EVA, a escala de Melbourne e os filamentos de Von Frey, aplicados ao redor da incisão cirúrgica. Foram considerados como critérios para administração da analgesia resgate uma pontuação de 50 mm na EVA ou de 13 pontos na escala de Melbourne. Os valores obtidos na EVA e na escala de Melbourne determinaram boa correlação, com r = 0,74. A correlação de EVA com os filamentos de Von Frey foi fraca (r = -0,40). Já a correlação entre a escala de Melbourne e os filamentos de Von Frey foi de -0.37. A EVA revelou-se a escala mais sensível. A EVA e a escala de Melbourne determinaram boa correlação, sugerindo que se considere uma pontuação menor na escala de Melbourne como critério para administração de analgesia resgate.

Orientador: Marcos Roberto de Mattos Fontes / Resumo: Os acidentes ofídicos constituem um problema de saúde pública, afetando regiões de clima tropical e subtropical e áreas rurais e pobres de países da América Latina, África, Ásia e Oceania. No Brasil, o gênero Bothrops é responsável por cerca de 90% dos acidentes ofídicos notificados, cujo envenenamento é caracterizado por intensa mionecrose local ineficientemente neutralizada pela soroterapia. O veneno botrópico possui uma classe de proteínas miotóxicas estruturalmente semelhantes às fosfolipases A2 (PLA2), responsáveis por induzir lesões musculares por um mecanismo não-catalítico parcialmente explicado por diferentes hipóteses. Contudo, há evidências que os efeitos miotóxico e paralisante in vitro são decorrentes de sua atividade desestabilizadora de membranas e que atuam em sinergia com as PLA2 catalíticas no envenenamento. Neste estudo, foi desenvolvido um novo protocolo de purificação da miotoxina não-catalítica (PLA2 homólogas ou proteínas PLA2-like) botrópica BthTX-I, a qual foi avaliada em testes cristalográficos, calorimétricos, miográficos e morfológicos. Potenciais inibidores vegetais da classe dos cinamatos foram co-cristalizados com a BthTX-I e testados em inibir as lesões e paralisia musculares in vitro promovida pela toxina a fim de evoluir no conhecimento da relação estrutura/atividade das PLA2 homólogas miotóxicas. Dentre todos os compostos testados, os ácidos chicórico e caftárico apresentaram-se como excelentes inibidores da BthTX-I. Contudo, foi possível ap... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Snakebites are a public health problem, concerning tropical and subtropical regions, rural and poor areas of Latin America, Africa, Asia and Oceania countries. In Brazil, Bothrops genus accounts for about 90% of reported snakebites, whose envenomation is characterized by intense local myonecrosis inefficiently neutralized by antivenom. A class of myotoxic proteins found in Bothrops venoms which is structurally similar to phospholipases A2 (PLA2), is responsible for inducing muscle injuries by a non-catalytic mechanism, partially explained by different hypotheses. However, there are evidences that myotoxic and in vitro paralyzing effects are due to their destabilizing-membrane activity and they act in synergy with the catalytic PLA2 myotoxins in envenomation. In this study, it was developed a new protocol for purification of a non-catalytic botropic myotoxin (PLA2 homologues or PLA2-like proteins) BthTX-I, which was evaluated by crystallographic, calorimetric, myographic and morphologic assays. Potential plant inhibitors of cinnamates class were co-crystallized with BthTX-I and tested to inhibit in vitro paralysis and muscle injuries promoted by the toxin. Among all the compounds tested, the chicoric and caftaric acids presented excellent BthTX-I inhibition characteristiscs. However, only chicoric acid (CA) we were able to perform crystallographic experiments, which presented different structural characteristics compared to other ligands and bothropic toxins. According to the ... (Complete abstract click electronic access below) / Doutor

Orientador: Marcos Hikari Toyama / Resumo: Os acidentes ofídicos foram recentemente incorporados na lista de doenças tropicais negligenciadas pela Organização Mundial de Saúde (OMS). No Brasil, uma parcela significativa destes acidentes (10%) é ocasionada pela serpente Crotalus durissus terrificus (Cdt), popularmente conhecida como cascavel. Dentre os componentes do veneno, a fosfolipase A2 secretória (sPLA2) representa aproximadamente 35% da massa do veneno seco. A Cdt sPLA2 responde pela atividade mais importante e clinicamente significativa que inicia com o processo inflamatório logo após o contato com a peçonha. Quando não tratada, a peçonha pode fazer com que o indivíduo sofra com insuficiência renal aguda, a qual não é neutralizada em curto espaço de tempo, nem mesmo pelo antiveneno específico. O processo inflamatório em diversas doenças humanas, está altamente relacionado com a formação de altas quantidades de espécies reativas de oxigênio e nitrogênio (EROs e ERNs), conhecido como explosão oxidativa/nitrosativa. Este processo é combatido por moléculas de baixo peso molecular como (vitaminas D e E, e glutationa), como também por enzimas antioxidantes, como a catalase (Cat), superóxido dismutase (Sod) e peroxirredoxinas (Prx). Já foi demonstrado que as sPLA2 humanas compartilham grande similaridades bioquímicas e farmacológicas com seus homólogos de serpentes e são capazes de induzir a formação de EROs e ERNs em células de mamíferos, mas nenhum estudo até o presente momento abordou de forma sistemática danos em... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Snake envenomation have recently been added to the list of tropical neglected diseases by the World Health Organization (WHO). In Brazil, a significant portion of ophidian accidents (~ 10%) is caused by the Crotalus durissus terrificus (Cdt) snake, popularly known as rattlesnake. Among the components of the venom, secretory phospholipase A2 (sPLA2) accounts for approximately 35% of the mass of the dry venom. Cdt sPLA2 accounts for the most important clinical damages and to the ingflammmatory proccess. When untreated, venomation can cause acute renal failure, which is not neutralized in a short time, not even by the specific antivenom. The inflammatory process, which is very well characterized in several human diseases, is highly related to the formation of high amounts of reactive oxygen and nitrogen species (ROS and RNS), known as oxidative / nitrosative burst. This process is counteracted by low molecular weight molecules such as vitamins D and E and glutathione, as well as antioxidant enzymes such as peroxiredoxins (Prx). human sPLA2 share high biochemical and pharmacological similarities with their snake homologs and are able to induce the formation of ROS and RNS in mammalian cells, but no study to date has systematically addressed damage to biomolecules carried out by ROS and RNS and the expression of peroxiredoxins in response to sPLA2 administration of snakes. We have recently shown that the administration of polyphenolic extracts of Laguncularia racemosais able to in... (Complete abstract click electronic access below) / Mestre

La dégradation des fonctions de reproduction masculine dans les dernières décennies, et par suite, la baisse du taux de fertilité chez l'homme a renforcé les démarches de recherche des causes d'infertilité masculine, qu'elles soient génétiques, ou environnementales, mettant en œuvre les perturbateurs endocriniens ou d'autres produits reprotoxiques. Par ailleurs, les traitements palliatifs tels que l'aide médicale à la procréation ne permettent qu'à 1 couple sur deux d'obtenir une grossesse. La recherche vise donc également à définir de nouvelles pistes d'amélioration de ces techniques. Les phospholipases A2 (PLA2) sont des enzymes abondamment exprimées dans les organes reproducteurs mâles, le sperme éjaculé et dans le tractus génital femelle. Elles jouent des rôles importants dans la capacitation, la réaction acrosomique (RA) et la fécondation. Nous avons montré au laboratoire que la PLA2 murine secrétée de groupe X (mGX) est présente dans l'acrosome des spermatozoïdes de souris. Elle est libérée au cours de la RA et s'avère un inducteur puissant de la RA. Les mécanismes permettant ces effets sont encore mal connus. Par l'utilisation d'inducteurs et d'inhibiteurs des PLA2, sur des modèles murins wild type ou KO pour certaines PLA2, ce travail montre que durant la capacitation, l'activation des iPLA2 β permet de déclencher une RA spontanée dans une sous-population spécifique de spermatozoïdes. Au cours de la RA induite par la progestérone (P4), On a pu aussi valoriser le rôle des iPLA2 β qui initie la cascade de la RA et permet que les sPLA2 soient activées pour amplifier le déroulement de la réaction acrosomique. Par ailleurs, ce travail montre que l'effet de mGX sur le taux de fécondation et de développement embryonnaire ne dépend pas du taux de la RA. Cet effet obtenu grâce à mGX n'est pas observé avec d'autres sPLA2 (murine ou humaine), ni avec la P4 ce qui lui confère une propriété espèce dépendante. / For the last ten years, the impairment of the male reproductive functions has highly increased, while the use of assisted reproductive techniques has also increased. Despite this evolution, the pregnancy rates obtained in in vitro fertilization (IVF) remain low, as only one in two couples will obtain a pregnancy after 4 attempts. Research has to discover new ways to gain in reproductive impact. Hence, many studies have recently been developed to test molecules that could improve the IVF results. Phospholipases A2 (PLA2s) are part of these molecules. They play an important role, because of their abundant expression in: male reproductive organs, in ejaculated sperm and in the female tract. Several studies have suggested a role for members of the secreted phospholipase A2 family in capacitation, acrosome reaction (AR), and fertilization. We demonstrated previously that sperm from mGX knock-out mice had a severely impaired fertilization potential in vitro, but the molecular nature of these enzymes and their specific functions have remained elusive. Our aims were to study the mechanism of the acrosome reaction by focusing on different kinds of PLA2 using inhibitors and knockout mice for each type of PLA2. We demonstrate the importance of iPLA2β in spontaneous AR occurring during capacitation. We also show that iPLA2β and sPLA2 of group X are both involved in progesterone (P4)-induced AR in mouse sperm. In addition we show that in the mouse neither P4 nor any of the other sPLA2s tested are able to mimic the IVF improvement obtained with mGX-treatment. We also demonstrate that this improvement obtained with phospholipase A2 murine group X is not dependent on the rate of AR. These results demonstrate that sPLA2s are not commutable in the context of mouse sperm fertility, indicating that group X sPLA2 is unique to improve fertility outcome.

SILVA, Katiane Queiroz da. Efeito da adição de plasma seminal e de sua composição bioquimica sobre os espermatozóides epididimários de caprinos. 2009. 43 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Zootecnia, Fortaleza-CE, 2009 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-08-02T14:51:18Z No. of bitstreams: 1 2009_dis_kqsilva.pdf: 313426 bytes, checksum: 9fae2725277c1fe6e2b27d9bb34c0908 (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-08-02T14:51:56Z (GMT) No. of bitstreams: 1 2009_dis_kqsilva.pdf: 313426 bytes, checksum: 9fae2725277c1fe6e2b27d9bb34c0908 (MD5) / Made available in DSpace on 2016-08-02T14:51:57Z (GMT). No. of bitstreams: 1 2009_dis_kqsilva.pdf: 313426 bytes, checksum: 9fae2725277c1fe6e2b27d9bb34c0908 (MD5) Previous issue date: 2009 / The objective of this study was to verify the effect of the addition and of the composition of the seminal plasma (SP) on the characteristics of the epididymal spermatozoa (ES) of goat. Eight goat males were used for obtaining of the biological material so that, the SP it was obtained in previous collections and stored at -18 oC until that proceeded to the addition to ES and the biochemical analyses. ES were obtained of the tail of the epididymal by the method of surgical castration. Four sample of 100 μL were removed from the "pool" of ES, and in two of them 120 μL were added of SP. Two sample, one with and other without SP, were diluted in a concentration of 200 x 106 sptz /mL, in the extender citrate-yolk (CY) and in the extender tris-yolk (TY). The samples were evaluated the vigor, motility and motility degradation rate (TDM) in fresh state and for the test of slow thermoresistence, after two and 24 hours of conservation. For the determination of fructose levels and total proteins were used specific kits of In Vitro Diagnóstico S/A®. The determination of activity of the phospholipase A2 was realized by the technique of the pH-stat. For the analysis of the data was used the statistical program SAS@. The results demonstrated that the addition of SP to ES reduced seminal parameters significantly, except in the extender TY when the animals were to grouped in function of concentration of total proteins of SP. Besides, the TY, when used to conserve the ES added of SP with low fructose concentration (540 mg/dL) preserved the seminal characteristics better. It follows that the initial concentration of fructose in the seminal plasma influenced the quality of conservation of the epididymal spermatozoa; as for the total proteins just affected the conservation in the citrate-yolk and the intensity of activity of the phospholipase A2 did not interfere in the conservation at 5 ºC of the epididymal spermatozoa of goat / O objetivo deste estudo foi verificar o efeito da adição e da composição do plasma seminal (PS) sobre as características dos espermatozóides epididimários (EEP) de caprinos. Utilizaram-se oito machos caprinos para obtenção do PS, que foi obtido em coletas prévias e armazenado a -18o C até que se procedessem a sua adição aos EEP e as análises bioquímicas. Os EEP foram obtidos da cauda do epidídimo pelo método de castração cirúrgica. Do “pool” de EEP foram retiradas quatro alíquotas de 100 μL, sendo que em duas delas foram adicionados 120 μL de PS e nas outras duas não. Duas alíquotas, uma com e outra sem PS, foram diluídas a uma concentração de 200 x 106 sptz/ mL, no diluidor citrato-gema (CG) e no diluidor tris-gema (TG). As amostras foram avaliadas quanto ao vigor, à motilidade e à taxa de degradação da motilidade (TDM) no seu estado fresco e pelo teste de termorresistência lento, após duas e 24 horas de conservação. Para a determinação dos níveis de frutose e de proteínas totais foram utilizados os kits específicos da In Vitro Diagnóstico S/A®. A determinação da atividade da fosfolipase A2 foi realizada pela técnica do pH-stat. Para a análise dos dados foi utilizado o programa estatístico SAS@. Os resultados demonstraram que a adição de PS aos EEP diminuiu significativamente os parâmetros seminais avaliados, exceto no diluidor TG quando os animais foram agrupados em função da concentração de proteínas totais do PS. Além disso, o TG, quando utilizado para conservar os EEP adicionados de PS de baixa concentração de frutose (540 mg/dL) preservou melhor as características seminais avaliadas. Concluiu-se que a concentração inicial de frutose no PS influenciou positivamente a qualidade de conservação dos espermatozóides epididimários; já a de proteínas totais afetou apenas a conservação no CG e a intensidade de atividade da fosfolipase A2 não interferiu na conservação a 5 ºC dos espermatozóides epididimários de caprinos