Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-08-24T20:25:23Z No. of bitstreams: 1 viniciusjosepilate.pdf: 4576162 bytes, checksum: b62d296b1537238b9c96d99b034cb665 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-25T12:05:45Z (GMT) No. of bitstreams: 1 viniciusjosepilate.pdf: 4576162 bytes, checksum: b62d296b1537238b9c96d99b034cb665 (MD5) / Made available in DSpace on 2017-08-25T12:05:45Z (GMT). No. of bitstreams: 1 viniciusjosepilate.pdf: 4576162 bytes, checksum: b62d296b1537238b9c96d99b034cb665 (MD5) Previous issue date: 2017-07-05 / Sibynomorphus neuwiedi e Sibynomorphus mikanii são serpentes de pequeno porte, conhecidas como cobras dormideiras, noturnas, não peçonhentas e malacófagas. Ambas ocorrem em áreas florestais de Mata Atlântica e ambientes alterados, porém S. mikanii ocorre mais frequentemente no Cerrado. Estudos parasitológicos, biológicos, ecológicos e morfológicos envolvendo essas espécies ainda são escassos. Os objetivos do presente estudo foram analisar a ecologia de helmintos, a dieta e a ecomorfologia das serpentes S. neuwiedi e S. mikanii de Minas Gerais, Brasil, registrar a helmintofauna de suas presas, verificar os efeitos da sazonalidade e do sexo, estado reprodutivo, tamanho e massa corporal destas serpentes, assim como do tamanho dos órgãos parasitados, sobre seus helmintos, e verificar os efeitos ecológico, filogenético e sexual na variabilidade morfométrica. As 39 serpentes analisadas de S. neuwiedi e as 49 de S. mikanii da Coleção Herpetológica da UFJF – Répteis tiveram aferidos comprimento e largura da cabeça, largura interocular, comprimentos rostro-cloacal, da cauda e total, e massa corporal. Posteriormente foram necropsiadas, sexadas e analisadas quanto à maturidade, tendo a cavidade corporal, órgãos e conteúdos digestórios analisados em busca de helmintos e presas. Órgãos parasitados foram medidos. Foram coletados 30 espécimes de moluscos da família Veronicellidae, eutanasiados, fixados, conservados e, somados aos espécimes encontrados nos estômagos das serpentes, foram dissecados e examinados para a identificação e em busca de helmintos. Os helmintos encontrados nas serpentes e nos moluscos foram lavados, fixados, conservados, quantificados, identificados e fotografados. Foi realizada a morfometria de trematódeos e nematoides. As comunidades de metazoários parasitos das serpentes foram formadas apenas por endoparasitos dos grupos dos helmintos e apresentaram riqueza e diversidade baixas, padrão de distribuição agregado, caráter isolacionista, especificidade quanto aos sítios de infecção e predominância de apenas um táxon parasitário por infracomunidade. As prevalências foram medianas. Ambas espécies de serpentes foram parasitadas por dois táxons de trematódeos, enquanto a serpente S. neuwiedi também apresentou três táxons de nematoides e S. mikanii, quatro (dois em comum entre estas serpentes). Os táxons parasitários predominantes foram Rhabdias sp. e Strongyluris sp. em ambas espécies de serpentes, e Mesocoelium sibynomorphi em S. neuwiedi, não apresentando o tamanho deste último helminto nesta serpente relação de intensidade-dependência. As serpentes apresentaram dietas semelhantes: moluscos de espécies da família Veronicellidae, sendo registradas as espécies-presas Sarasinula linguaeformis na serpente S. neuwiedi e Latipes erinaceus e Sarasinula sp. na serpente S. mikanii. Larvas de nematoides Strongyluris sp. foram os helmintos em comum entre essas serpentes e suas presas. A variação na sazonalidade, o sexo, o estado reprodutivo, o tamanho e a massa corporal de S. neuwiedi e S. mikanii, e o tamanho dos órgãos parasitados, não exerceram influência sobre as comunidades de helmintos destas serpentes. Essas espécies não apresentaram dimorfismo sexual. O habitat não influenciou a morfologia dessas serpentes, enquanto a filogenia influenciou a morfologia de fêmeas adultas, o que não ocorreu em fêmeas jovens e machos. / Sibynomorphus neuwiedi and Sibynomorphus mikanii are small sized snakes, known as sleepy snakes, nocturnal, non-venomous and snail eaters. Both occurs in Mata Atlânica forests and impacted environments, however S. mikanii is more frequently found in Cerrado. Studies about parasitology, biology, ecology and morphology of both species are scarce. The goals of the present study where to analize ecology of helminths, the diet and ecomorphology of the snakes S. neuwiedi and S. mikanii from Minas Gerais, Brazil, register the helminths presentes in their preys, verify the effects of seazonality and gender, reproductive state, size and body mass of these snakes, so as the size of parasitized organs, on their helminths, and verify the ecological, phylogenetic and sexual effects at morphometric variation. The 39 analyzed individuals of S. neuwiedi and the 49 specimens of S. mikanii from the Herpetological Collection of the UFJF – Reptiles have had measured their length and width of head, length between eyes, rostrum-cloacal length, tail and total, and body mass. Subsequently where necropsied, sexed and analyzed the maturity, the body cavity, organs and digestive contents searched in search of helminths and preys. Parasitized organs were measured. It were colected 30 specimens of snails from Veronicellidae family, euthanasia was realized and the animal fixed, conserved and, added to the specimens found in snakes stomach, these were dissected and examined to identification and pursuit of helminths. The helminths found in the snakes and in molluscs were washed, fixed, conserved, quantified, identified and photographed. It was realized morphometry of trematodes and nematodes. The metazoans parasites communities of the snakes where composed only by endoparasites from helminths group and presents low richness and diversity, distribution patttern assembled, isolacionist profile, specificity to infection sites and predominance of only one parasite taxon by infracommunity. The prevalences were average. Both species of snakes were parasitized by two taxa of trematodes, while S. neuwiedi presented also three taxa of nematodes and S. mikanii, four (two in common amongst these snakes). The prevailing parasites taxa were Rhabdias sp. and Strongyluris sp. in both species of snakes, and Mesocoelium sibynomorphi in S. neuwiedi, do not presenting the size of this last helminth in this snake relation of intensity-dependency. The snakes presented similar diet: molluscs from species of Veronicellidae family, being registered the prey-species Sarasinula linguaeformis in S. neuwiedi and Latipes erinaceus and Sarasinula sp. in S. mikanii. Larval forms from the nematode Strongyluris sp. was the common helmint between these snakes and their prey. The seazonality variation, gender, reproductive state, size and body mass of S. neuwiedi and S. mikanii, and the size of parasitized organs, did not influenced the helminth communities of these snakes. These species did not present sexual dimorphism. The habitat did not influence the morphology these snakes, while the phylogeny influenced the morphology of adult females, what did not occurred in young females and male.

The aim of this project is to develop and perform a first evaluation of a software, based on the active contour, which automatically computes the cross-section area of the internal jugular veins through a sequence of 90 ultrasound images. The software is intended to be useful in future research in the field of intra cranial pressure and its associated diseases. The biomechanics of the internal jugular veins and its relationship to the intra cranial pressure is studied with ultrasound. It generates data in the form of ultrasound sequences shot in seven different body positions, supine to upright. Vein movements in cross section over the cardiac cycle are recorded for all body positions. From these films, it is interesting to know how the cross-section area varies over the cardiac cycle and between body positions, in order to estimate the pressure. The software created was semi-automatic, where the operator loads each individual sequence and sets the initial contour on the first frame. It was evaluated in a test by comparing its computed areas with manually estimated areas.  The test showed that the software was able to track and compute the area with a satisfactory accuracy for a variety of sequences. It is also faster and more consistent than manual measurements. The most difficult sequences to track were small vessels with narrow geometries, fast moving walls, and blurry edges. Further development is required to correct a few bugs in the algorithm. Also, the improved algorithm should be evaluated on a larger sample of sequences before using it in research.

Ce travail avait pour objectif de mieux évaluer les critères intrinsèques d’un antivenin et de son utilisation afin d’être le plus efficace possible dans le traitement des envenimations ophidiennes survenant sur le territoire de France métropolitaine. L’immunothérapie est à ce jour le seul traitement étiologique efficace dans la prise en charge des victimes d’envenimation ophidienne. La qualité des antivenins est un élément clé dans l’efficacité et la tolérance de ces traitements. Pour une efficacité optimale, l’immunothérapie doit être administrée le plus rapidement possible. Il ressort enfin que les caractéristiques des envenimations sont en évolution constante sur le territoire métropolitain : émergence de signes neurotoxiques dans les envenimations vipérines ; développement de l’importation de serpents exotiques. Tous ces éléments incitent à une surveillance spécifique par des structures expertes et spécialisées dans le domaine. / This work aimed to assess antivenom criteria in order to be most effective in the treatment of snake bites occurring in metropolitan France. Immunotherapy is the only effective etiological treatment in snake envenomation, considered as a gold standard. Quality of antivenoms is a key element for effectiveness and safety. Immunotherapy should be administered as soon as possible. Finally, it appears that the characteristics of envenomation are constantly changing on the mainland : emergence of neurotoxic signs in viper envenomation; importation of exotic snakes. All these emerging elements need a specific monitoring by expert and specialized structures in France.

This study presents a probabilistic lava flow hazard assessment for the Idaho National Laboratory (INL) and the cities of Idaho Falls and Pocatello, Idaho. The impetus of this work is to estimate the conditional probability that a lava flow on the eastern Snake River Plain (ESRP) will impact the areas of interest given the formation of a new volcanic vent in the region. A list of 288 eruptive events, derived from a previously published inventory of 506 surface and 32 buried vents, was created to reduce the biasing of spatial density maps towards eruptions with multiple dependent vents. Conditional probabilities of new vents and events occurring on the ESRP were modeled using the the Sum of Asymptotic Mean Squared Error (SAMSE) optimal pilot bandwidth estimator with a bivariate Gaussian kernel function. Monte Carlo analyses of potential eruption scenarios were performed using MOLASSES, a cellular automata fluid flow simulator. Results show that Idaho Falls is impacted <1% of the time for both the vent and event simulations; Pocatello is not impacted by any simulated flows. 25.45% of vent flows and 33.74% of event flows breach the boundaries of INL. 18.27%of vent and 25.85% of event simulations initiate on the INL property. Annual inundation probabilities of 1.06 x 10-4 for vent-based flows and 7.12 x 10-5 for event-based flows are reported for INL; annual probabilities of an eruptive center initiating on INL property are 7.60 x 10-5 for vents and 5.45 x 10-5 for events. All of these values exceed the International Atomic Energy Agency’s acceptable risk probability of 10-7 by several orders of magnitude.

Lectins, multivalent carbohydrate-binding proteins of non-immune origin, have the unique ability to decode the information contained in complex carbohydrate structures of glycoproteins and glycolipids by stereo-specifically recognizing and binding to carbohydrates and carbohydrate linkages. The ubiquitous distribution of lectins in all forms of life and viruses along with their involvement in various biological processes such as cell-cell communication, host-pathogen interaction, cancer metastasis, embryogenesis, tissue development and mitogenic stimulation further emphasizes the importance of lectins in biological systems. Although not much is known about the endogenous roles of plant lectins, they constitute the most thoroughly studied class of lectins. On the basis of their subunit folds plant lectins have been divided in six major classes. They include jelly roll fold lectins (or legume lectins), hevein domain lectins (or cereal lectins), β-trefoil fold lectins, β-prism II fold lectins (or bulb lectins), β-prism I fold lectins and the most recently discovered lectin homologous to cyanovirin-N (http://www.cermav.cnrs.fr/lectines). Interestingly, of these, lectin subunits harbor an approximate three-fold symmetry in three cases and each subunit is believed to have evolved through successive gene duplication, fusion and divergent evolution. One of the major research activities in this laboratory involves structural studies on plant lectins. Decades of extensive studies in the laboratory have shed light on various structural and functional aspects of lectins such as variability in quaternary association, lectin-carbohydrate interactions, strategies for generating ligand specificity and multivalency. Furthermore, the β-prism I fold was first identified as a lectin fold in this laboratory through the X-ray analysis of the methyl-α-galactose complex of jacalin, one of the two lectins from the seeds of Artocarpus integrifolia. Subsequently, many other lectins with the same fold have been structurally characterized here and else where (http://www.cermav.cnrs.fr/lectines). They include mannose specific tetrameric artocarpin and dimeric banana lectin studied in this laboratory. Also investigated here is the structure of first dimeric β-prism II fold lectin, namely, garlic lectin. The subsequent work, carried out by the author, on the structure and dynamics of three-fold symmetric lectins form the subject matter of this thesis. Different web-servers available at NCBI and EXPASY web sites were used for sequence annotation studies. MRBAYES and MEGA were used for phylogenetic analysis. Molecular dynamics (MD) simulations were carried out using the simulation package GROMACS v.3.3.1. OPLS-AA/L and GLYCAM-06 force fields were used for proteins and carbohydrates respectively. Simulations were performed in explicit water system with TIP4P water model under NPT conditions with unit dielectric constant. The hanging drop method was used for crystallizing banana lectin and its complexes. Intensity data were collected on a MAR 345 image plate mounted on a Rigaku RU200 rotating-anode X-ray generator. The Oxford cryosystem was used when collecting data at low temperature. The data were processed using DENZO and SCALEPACK of HKL suite of programs. The structure factors from the processed data were calculated using TRUNCATE of CCP4 suite of programs. The molecular replacement program MOLREP was used for structure solution. Structure refinements were carried out using the CNS software package and REFMAC of CCP4. Model building was done using the molecular graphics program COOT. INSIGHT II, ALIGN, CONTACT, MUSTANG and SC of CCP4 were used for analysis of structural features. PROCHECK and web-server MOLPROBITY were used for the validation of the refined structures. The β-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit / domain. Until recently, β-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit / domain. However, the recently determined structure of the β-prism I fold lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for the two lectin folds and which carry one or more relevant carbohydrate-binding motifs. The recent observation of a β-prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The detailed sequence and phylogenetic analysis of all the β-prism I fold lectin or lectin-like sequences, available then, with particular attention to their carbohydrate-binding sites in them, in conjunction with the analysis of available three-dimensional structures demonstrate substantial diversity in the number of binding sites, unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of β-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the prepondence of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the β-prism II fold, is related to the role of plant lectins in defence. Jacalin is the most thoroughly studied β-prism I fold lectin. A wealth of structural and thermodynamic data, mostly from this laboratory, led to a thorough characterization of carbohydrate-recognition in the case of jacalin. One aspect of jacalin that has not been investigated so far was its dynamics. The issue was addressed through reasonably long MD simulations, in explicit solvent system using all atom force field, of all the jacalin-carbohydrate complexes of known structure, models of unliganded molecules derived from the complexes and also models of relevant complexes where X-ray structures are not available. Results of the simulations and the available crystal structures involving jacalin permit delineation of the relatively rigid and flexible regions of the molecule and the dynamical variability of the hydrogen bonds involved in stabilizing the structure. Local flexibility appears to be related to solvent accessibility. Hydrogen bonds involving side chains and water bridges involving buried water molecules appear to be important in the stabilization of loop structures. The lectin-carbohydrate interactions observed in crystal structures, the average parameters pertaining to them derived from simulations, energetic contribution of the stacking residue estimated from quantum mechanical calculations and the scatter of the locations of carbohydrate and carbohydrate-binding residues, are consistent with the known thermodynamic parameters of jacalin-carbohydrate interactions. The simulations, along with X-ray results, provide a fuller picture of carbohydrate binding by jacalin than provided by crystallographic analysis alone. The simulations confirm that in the unliganded structures water molecules tend to occupy the positions occupied by carbohydrate oxygens in the lectin-carbohydrate complexes. Population distributions in simulations of the free lectin, the ligands and the complexes indicate a combination of conformational selection and induced fit. Mannose-specific β-prism I fold lectins, like lectins belonging to other plant families, exhibit interesting variability in their quaternary association. Mannose specific artocarpin and MornigaM are tetrameric, heltuba is octameric in the crystal structure and banana lectin and calsepa are dimeric. The modes of the dimerization in the last two are however, entirely different. This variability was explored through modelling and molecular dynamics simulations based on the known three-dimensional structures. This study, which combines computational approaches and results of X-ray analyses, provides valuable insights into the origin of the variability in quaternary association. MD simulations on individual subunits and the oligomers provide insights into the changes in the structure brought about in the protomers on oligomerization, including swapping of the N-terminal stretch in one instance. The regions which undergo changes also tend to exhibit dynamic flexibility during MD simulations. The internal symmetries of individual oligomers are substantially retained during the calculations. Simulations were also carried out on models using all possible oligomers employing the four different protomers. The unique dimerization pattern observed in calsepa could be traced to unique substitutions in a peptide stretch involved in dimerization. The impossibility of a specific mode of oligomerization involving a particular protomer is often expressed in terms of unacceptable steric contacts or dissociation of the oligomer during simulations. The calculations also lead to a rationale for the observation of a heltuba tetramer in solution although the lectin exists as an octamer in the crystal, in addition to providing insights into relations among evolution, oligomerization and ligand binding. The known crystal structures of banana lectin in its native and ligand bound forms revealed interesting features including the presence of two functional carbohydrate-binding sites per subunit. However, some confusion remained on the role of glycosidic linkage in carbohydrate-binding. The three crystal structures reported in this thesis provide information on details of the interactions of mannose and mannosylα-1,3-mannose with banana lectin and evidence for the binding of glucosyl-α-1,2glucose to the lectin. The known structures involving the lectin include a complex with glucosyl-β-1,3-glucose. Modelling studies on the three disaccharide complexes with the reducing end and the non-reducing end at the primary binding site are also presented here. The results of the X-ray and modelling studies show that the disaccharides with an α-1,3 linkage prefers to have the non-reducing end at the primary binding site while the reducing end is preferred at the site when the linkage is β-1,3 in mannose/glucose specific β-prism I fold lectins. In the corresponding galactose-specific lectins, however, α-1,3 linked disaccharides cannot bind the lectin with the non-reducing end at the primary binding site on account of steric clashes with an aromatic residue which occurs only when the lectin is galactose-specific. MD simulations based on the known structures involving banana lectin enrich the information on lectin-carbohydrate interactions obtained from crystal structures. They demonstrate that conformational selection as well as induced fit operate when carbohydrates bind to banana lectin. Snake gourd seed lectin (SGSL) isolated from Trichosanthes anguina is a glycosylated, galactose-specific, non-toxic lectin similar to type II ribosome inactivating proteins (RIPs) with a molecular weight of ~53kDa. It was established through preliminary X-ray studies that chain A with molecular weight of ~23kDa adopts the same fold as that of type I RIPs and the toxic chain of type II RIPs. Chain B with molecular weight ~32kDa has two β-trefoil fold domains and is responsible for the lectin activity of the protein. The two chains are connected with a disulphide bond. The sequence of the protein could not be determined using conventional methods despite extensive effort. It was derived from X-ray data at 2.4 Å resolution, which was used for structure analysis. The non-toxicity of SGSL appears to result from a combination of changes in the catalytic site in chain A and sugar-binding site in chain B. Detailed analysis of the sequences of type II RIPs of known structure and their homologues with unknown structure, provide valuable insights into the evolution of this class of proteins. It also indicates some variability in carbohydrate-binding sites, which appears to contribute to different levels of toxicity exhibited by lectins from various sources. In addition to the work on plant lectins, the author was also involved in studies on the crystal structures of the adipic acid complexes of L- and DL-Lysine. This investigation is presented in an appendix. A part of the work presented in the thesis has been reported in the following publications. Sharma, A., Thamotharan, S., Roy, S., & Vijayan, M. (2006). X-ray studies of crystalline complexes involving amino acids and peptides. XLIII. Adipic acid complexes of L- and DL-lysine. Acta Cryst, C62, o148-o152. Sharma, A., Chandran, D., Singh, D.D., & Vijayan, M. (2007). Multiplicity of carbohydrate-binding sites in beta-prism fold lectins: occurrence and possible evolutionary implications. J Biosci, 32, 1089-1110. Sharma, A., Sekar, K., & Vijayan, M. (2009). Structure, dynamics, and interactions of jacalin. Insights from molecular dynamics simulations examined in conjunction with results of X-ray studies. Proteins, 77, 760-777.

Orientadores: Lea Rodrigues Simioni,Yoko Oshima Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T13:41:21Z (GMT). No. of bitstreams: 1 Bueno_LilianGobbodeFreitas_M.pdf: 986839 bytes, checksum: 048ec75c1469d547de311f325aab2cc3 (MD5) Previous issue date: 2006 / Resumo: Manganês, um bloqueador neuromuscular com atividades pré e pós-sinápticas, foi utilizado neste trabalho com o objetivo de estudar a neurotoxicidade e miotoxicidade induzida pelos venenos das serpentes Crotalus durissus terrificus (Cdt) e Bothrops jararacussu (Bjssu) em preparações biventer cervicis de pintainho (BCp). O pré-tratamento das preparações com Mn2+ (0,66 e 1,6 mM) não impediu o bloqueio neuromuscular induzido pelo veneno de Cdt, reduziu parcialmente a resposta contraturante à adição exógena de ACh, mas não à de KCl. Embora o bloqueio neuromuscular induzido pelo veneno de Bjssu seja irreversível, o pré-tratamento com ambas as concentrações de Mn2+ (0,66 e 1,6 mM) tornou o bloqueio parcialmente reversível após a lavagem das preparações com solução de Krebs, e somente nas preparações pré-tratadas com 1,6 mM Mn2+ houve redução do bloqueio da resposta contraturante à adição exógena de ACh (p<0,05). Os tipos de miotoxicidade induzidos pelos venenos de Cdt e Bjssu interferiram diferentemente na resposta contrátil. Em preparações pré-tratadas com Mn2+ (1,6 mM) houve redução parcial da porcentagem de dano muscular e da atividade miotóxica (CC, U/L) induzida por ambos os venenos. Os resultados desta pesquisa indicam que: Mn2+ interfere na contratura induzida pela ACh nos receptores nicotínicos; Mn2+ não evitou a neurotoxicidade do veneno de Cdt, mas reduziu parcialmente sua miotoxicidade in vitro, por sua ação estabilizante da membrana muscular; Mn2+ reduziu parcialmente a miotoxicidade e o bloqueio neuromuscular induzido pelo veneno de Bjssu. A dupla ação do Mn2+, pré- e pós-sináptica, é considerada útil para o estudo dos venenos de serpentes, uma vez que a maioria desses venenos apresenta também estas ações. Além disso, com a utilização deste cátion foi possível resgatar a coerência entre a interpretação dos resultados experimentais e clínicos / Abstract: In this study, we examined the effects of Mn2+, a neuromuscular blocker with pre and postsynaptic actions, on the neurotoxicity and myotoxicity induced by Crotalus durissus terrificus and Bothrops jararacussu venoms in chick biventer cervicis preparations. Pretreating the preparations with Mn2+ (0.66 or 1.6 mM) did not affect the blockade induced by C. d. terrificus venom or KCl-induced contractures, but partially reduced ACh-induced contractures. On the other hand, both concentrations of Mn2+ partially prevented the blockade induced by B. jararacussu venom (seen after washing the preparations with Krebs solution), whereas only 1.6 mM Mn2+ significantly restored ACh-induced contractures. Pretreatment with Mn2+ (1.6 mM) partially prevented the muscle damage and the release of creatine kinase induced by both venoms. These results show that Mn2+ did not prevent the neurotoxicity of Cdt venom, but partially reduced its myotoxicity, whereas this metal partially attenuated the myotoxicity and neuromuscular blockade caused by B. jararacussu venom. The pre- and postsynaptic actions of Mn2+ may be useful for studying snake venoms that show one or both of these activities / Mestrado / Mestre em Farmacologia

Se estudia la presencia de actividad fibrinolítica en los venenos de cinco serpientes peruanas empleando coágulos de fibrina formados a partir de fibrinógeno bovino y una enzima semejante a trombina de L. muta muta parcialmente purificada Los cinco venenos estudiados presentaron actividad fibrinolítica en ausencia de plasmina (rango: 16,3 a 57,5 U/h mg protefua), lo que demuestra la existencia de enzimas proteolíticas en los venenos de vipéridos peruanos, que actúan directamente sobre la fibrina. El veneno de L. muta muta presentó la mayor actividad enzimática específica (57 ,5), seguido en orden decreciente por B. barnetti (45,2),B.pictus (29,4), C. durisus terrificus (22,5) y B. brazilü (16,3). / We studied the presence of fibrinolytic activity in five dried peruvian snake venoms using fibrin clots formed by one partially purified fraction of Thrombin-like enzyme from L. muta muta. In all venoms studied, fibrinolysis were detected in absence of plasmin in the range from 16,3 to 57,5 U/h mgprotein. This results demonstrate the existence of proteolytic enzymes in Peruvian Viperid snake venoms that act upon fibrin. The L. muta muta venom showed the higher activity (57 ,5), followed in down order by B. barnetti (45,2) B. pictus (29,4), C. durisus terrificus (22,5) and B. brazilü venom (16,3).

A Bradicinina, a mais importante das cininas plasmáticas, foi encontrada pela primeira vez em mamíferos. Pouco foi estudado sobre o sistema calicreína-cininas em serpentes, mas o que se sabe é que, provavelmente, são deficientes do Fator XII, um ativador da pré-calicreína em mamíferos. Com o objetivo de identificarmos novos peptídeos, submetemos plasmas das serpentes Bothrops jararaca (SBJ) e da Crotalus durissus terrificus (SCDT) tratados por duas metodologias simplificadas de determinação de bradicininogênio. O estudo com os plasmas das SBJ e da SCDT liberou uma substância com efeito semelhante à cinina de mamíferos que produziu hipotensão na serpente e no rato; esse efeito foi potencializado com o uso do captopril (0,05/0,010 mg/Kg), que inibe a cininase II, confirmando resultados diferentes das literaturas existentes sobre esse assunto. Com o resultado obtido, fracionamos este produto em aparelho de HPLC, e obtivemos seqüências peptídicas que não apresentaram homologia com a bradicinina. / The Bradykinin (BK), most important of the plasmatic kinins, was encountered for the first time in mammals. Little was studied in the kallikrein-kinin system in snakes, but it is known that, it is probably deficient of Factor XII, an activator of the daily prekallikrein in mammals. In order to identify new peptides, we submit plasma of the snakes Bothrops jararaca (SBJ) and e Crotalus durissus terrificus (SCDT) in two simplified methodologies for bradykininnogen determination, to analyze in bioassays of average arterial pressure of mammals and isolated ileum of guinea pig. The study with plasma of the SBJ and the SCDT showed that the kinin of mammals released a substance with effect similar to that producing hypotension in snakes and rats; this effect was potentiated with the use of captopril (0,05/0,010 mg/Kg), wich inhibits kininase II, confirming results different from literature?s obtained on this subject. With the obtained result, we fractioned this product in HPLC equipment and obtained peptides sequences with no homologie with the bradykinin.

La première partie de cette thèse s’inscrit dans le domaine des cartes aléatoires, qui est un sujet à la frontière des probabilités, de la combinatoire et de la physique statistique. Nos travaux complètent une série de résultats de convergence de différents modèles de cartes aléatoires vers la carte brownienne, qui est un espace métrique compact aléatoire. Plus précisément, on montre que la limite d’échelle d’une carte de loi uniforme sur l’ensemble des cartes biparties enracinées à n arêtes, munie de la distance de graphe renormalisée par (2n)^(−1/4), est, au sens de Gromov–Hausdorff, la carte brownienne. Pour prouver ce résultat, les arguments importants sont d’une part l’utilisation d’une bijection combinatoire entre cartes biparties et arbres multitypes, et d’autre part des théorèmes de convergence pour les arbres de Galton–Watson multitypes étiquetés. Dans un deuxième temps, le but est de présenter une théorie des excursions pour le mouvement brownien indexé par l’arbre brownien. De manière analogue à la théorie d’Itô des excursions pour le mouvement brownien, chaque excursion correspond à une composante connexe du complémentaire des zéros du mouvement brownien indexé par l’arbre, et l’excursion est définie comme un processus indexé par un arbre continu. On explique comment mesurer la longueur de la frontière de ces excursions, de sorte que la famille de ces longueurs coïncide avec les sauts d’un processus de branchement à temps continu de mécanisme de branchement stable d’indice 3/2. De plus, conditionnellement aux longueurs des frontières, les excursions sont indépendantes et leur loi conditionnelle est déterminée à l’aide d’une mesure d’excursion explicite que l’on introduit et décrit. Dans ce travail, le serpent brownien apparaît comme un outil particulièrement important. / The first part of this thesis concerns the area of random maps, which is a topic in between probability theory, combinatorics and statistical physics. Our work complements several results of convergence of various classes of random maps to the Brownian map, which is a random compact metric space. More precisely, we prove that the scaling limit of a map which is uniformly distributed over the class of rooted planar maps with n edges, equipped with the graph distance rescaled by (2n)^(−1/4), is, in the Gromov-Hausdorff sense, the Brownian map. To establish this result, the main arguments are the use of a combinatorial bijection between bipartite maps and multitype trees, together with convergence theorems for Galton-Watson multitype trees. We then aim to develop an excursion theory for Brownian motion indexed by the Brownian tree. Analogous to the Itô excursion theory for Brownian motion, each excursion corresponds to a connected component of the complement of the zero set of the tree-indexed Brownian motion, and the excursion is defined as a process indexed by a continuous tree. We explain how to measure the length of the boundary of these excursions, in a way that the collection of these lengths coincides with the collection of jumps of a continuous-state branching process with a 3/2-stable branching mechanism. Moreover, conditionally on the boundary lengths, the excursions are independent and their conditional distribution is determined in terms of an excursion measure that we introduce and study. In this work, the Brownian snake appears as a particularly important tool.

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