Venoms of the Sicariidae spider family contain phospholipase D (PLD) enzyme toxins that can cause severe dermonecrosis and even death in humans. PLD toxins are known to cleave the substrates sphingomyelin (SM) and lysophosphatidylcholine (LPC) in mammalian tissues, releasing a choline headgroup and a reported monoester phospholipid formed via a hydrolytic reaction. However, some PLD toxins have demonstrated the ability to utilize substrates besides SM and LPC and other PLD toxins have demonstrated no activity against either SM or LPC. Given that the etiology of the disease state following envenomation is not well understood, we postulated that PLD toxins could be utilizing other phospholipid substrates in vivo. To determine the level of promiscuity among the PLD toxins, we developed a novel ³¹P-NMR assay to measure phospholipase activity against a panel of potential phospholipid substrates. While developing the assay, we made the surprising discovery that recombinant PLD toxins, as well as whole venoms from diverse Sicariidae species, exclusively generates cyclic phosphate rather than hydrolytic products. We also found that a distantly related PLD toxin from a pathogenic bacterium, with low sequence identity to the spider PLDs, exclusively generates cyclic phosphate products. We then established that St_βIB1i, a PLD with extremely diminished activity toward SM and LPC, actually demonstrates large preferential specificity towards ethanolamine phospholipid substrates. We solved the crystal structure of St_βIB1i to compare to PLD toxins of known structure, toward an understanding of the molecular basis of substrate specificity. The cyclic phosphate products generated by the PLD toxins have extremely different biochemical properties than their monoester counterparts and may be relevant to the pathology following envenomation or bacterial infection. In addition the specificity St_βIB1i has for ethanolamine substrates may have biological implications, as insects have high concentrations of ethanolamine-containing phospholipids.

Thesis (M.Tech.: Homoeopathy)--Durban University of Technology, 2008 / The proving substance Hemachatus haemachatus commonly known as the Rinkhals belongs to the family of Elapidae. This spitting-cobra is a local snake found only in Southern Africa. This proving tested the effects of the thirtieth centesimal (30CH) potency of venom from Hemachatus haemachatus on healthy provers. OBJECTIVES It was hypothesised that Hemachatus haemachatus 30CH would produce clearly observable signs and symptoms in healthy provers, and that the comparison of Hemachatus haemachatus to those yielding the highest numerical value and total number of rubrics on repertorisation of the proving symptoms would highlight differences and similarities between the remedy symptoms so that confusion as to the indication is eliminated. It was hypothesised that a fuller understanding of Hemachatus haemachatus and its relationship to other remedies would be gained following this comparison. METHODOLOGY A double blind, placebo controlled proving of Hemachatus haemachatus 30CH was conducted on thirty healthy volunteers who met the inclusion criteria. Six of these thirty provers randomly received placebo, with neither prover nor researcher knowing whom received placebo. Provers had a homoeopathic case history taken and a physical examination performed on them prior to commencement of the proving. The provers recorded their signs and symptoms 6 by means of a journal before, during and after administration of the remedy. On completion of the proving, the information obtained was correlated and assessed by the two researchers, De la Rouviere and Cahill. The symptoms elicited during the proving were translated into materia medica and repertory language, and a homoeopathic picture of the remedy was subsequently formulated. Data from the case histories, physical examinations and group discussions were also considered in the assessment. RESULTS During the period of investigation, provers experienced a variety of symptoms on the mental, emotional and physical spheres. On the mental emotional sphere there was a marked degree of irritability and changeability in moods as is commonly seen in many of the snake remedies. Along with this, it was noted that there were feelings of anxiety for reasons unknown, a sense of having lost something or someone close, and a desire to be left alone. There were also a great number of feelings regarding the home, where there were feelings of the home being a place of safety and wanting order in the home. On a physical level, many of the provers noted varying degrees of abdominal discomfort and headaches. Along with anxiety, provers experienced palpitations and sensations of chest restriction or constriction with shortness of breath. There were a variety of musculoskeletal symptoms ranging from painful joints in the fingers to stiffness and tightness in the neck and back. Provers noted flushes of heat and alterations of their internal thermostat. Provers experienced marked dryness of the mucus membranes and the skin, and there was also a general feeling of weakness and heaviness as well as a marked aggravation in the mornings on waking. 7 CONCLUSIONS Symptoms obtained from the proving of Hemachatus haemachatus 30CH were studied and evaluated. Those symptoms that appeared to represent the remedy picture of Hemachatus haemachatus most accurately in the researchers‟ opinion were used in the repertorisation of the remedy. The investigation supported the hypothesis that Hemachatus haemachatus 30CH would produce clearly observable signs and symptoms in healthy provers. The subsequent comparison of the proving symptoms of Hemachatus haemachatus to Lycopodium (Club moss), Sulphur, Alumina (Aluminium oxide), Sepia (Cuttle fish) and Calcarea carbonica (Carbonate of Lime) highlighted differences and similarities between these remedies and Hemachatus haemachatus. The further comparison of remedies that came up on repertorisation restricted to the plant, mineral and animal kingdoms respectively provided a further comparison of remedies, which aimed at enhancing the differentiation of Hemachatus haemachatus to other similar remedies.

ClC proteins encompass a large protein family consisting of both voltage-dependent chloride channels and chloride/proton exchangers that are found in both eukaryotes and prokaryotes. These proteins mediate Cl- flux across the plasma membrane or intracellular membranes of many cell types including neurons, epithelial cells, and skeletal muscle in mammals. Mutations in genes encoding these channels also contribute to several human diseases. The mechanism of ion conduction through ClC proteins is becoming better defined, largely due to the availability of a crystal structure of a bacterial ClC transporter. Because crystal structures only capture a snapshot a protein in a single conformation, however, the large conformational changes associated with channel opening and closing have remained largely undefined. In the cation channel field, ion conduction and conformational changes that occur during channel gating have been studied using peptide toxin inhibitors isolated from animal venoms. However, only one peptide toxin inhibitor of a chloride channel of known molecular identity has ever been identified. Georgia anion toxin 1 (GaTx1), inhibits the CFTR chloride channel, which is unrelated to ClC proteins on the levels of both three dimensional structure and primary sequence. Here, we describe the characterization of the inhibitory activity of Leiurus quinquestriatus hebraeus scorpion venom against the ClC-2 chloride channel. We found that the venom from this scorpion contains a peptide component that is capable of inhibiting the ClC-2 chloride channel. This component was isolated using standard chromatography techniques, and found that the active component is a 3.2 kDa peptide composed of 29 amino acids. We showed that the active toxin, Georgia anion toxin 2 (GaTx2), interacts with ClC-2 with an affinity in the picomolar range, and appears to slow channel opening. Finally, GaTx2 is not capable of inhibiting other members of the ClC protein family, other major chloride channels, or voltage-gated potassium channels. This toxin will provide a new tool for structure/function studies of ClC-2, and will hopefully serve as only the first toxin inhibitor available for this protein family.

A candidate protein from the venom gland of parasitoid wasp, Habrobracon hebetor, predicted to be responsible for the paralysis of lepidopteran caterpillars, was produced in baculovirus and bacterial expression systems. The function of both recombinant protein variants was confirmed by in vivo tests in Galleria mellonella larvae.

Submitted by FERNANDO KAMIMURA COCCHI null (fecocchi@gmail.com) on 2016-03-30T19:44:04Z No. of bitstreams: 1 Dissertação Fernando Kamimura Cocchi.pdf: 3236502 bytes, checksum: 436c4468ce72564ad0846615cbd9ce4a (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-04-01T20:12:00Z (GMT) No. of bitstreams: 1 cocchi_fk_me_rcla.pdf: 3236502 bytes, checksum: 436c4468ce72564ad0846615cbd9ce4a (MD5) / Made available in DSpace on 2016-04-01T20:12:00Z (GMT). No. of bitstreams: 1 cocchi_fk_me_rcla.pdf: 3236502 bytes, checksum: 436c4468ce72564ad0846615cbd9ce4a (MD5) Previous issue date: 2016-03-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os escorpiões são artrópodes da classe Arachnida, ordem Scorpiones. São animais peçonhentos, ou seja, possuem órgão secretório ou glândulas conectadas a um dispositivo de injeção, o qual pode inocular o veneno produzido pela glândula ou células especializadas. O veneno pode ser descrito como sendo uma mistura complexa, contendo muco, sais inorgânicos, moléculas orgânicas de baixa massa molecular, muitos peptídeos neurotóxicos e proteínas. O envenenamento humano por picada de escorpião é um importante problema de saúde pública em vários países tropicais e subtropicais. No Brasil, Tityus serrulatus é uma das espécies mais perigosas, que causam grave envenenamento, podendo levar ao óbito. Em geral, o envenenamento pode causar sintomas de dor, febre, agitação psicomotora, salivação, lacrimejamento, aumento da mobilidade do trato gastrointestinal, insuficiência respiratória e arritmia cardíaca, hipertensão arterial seguida de hipotensão, insuficiência cardíaca, edema pulmonar e choque anafilático. O veneno dos escorpiões tem sido estudado ao longo dos anos, a fim de elucidar seus compostos juntamente com suas sequências e suas funções biológicas. Algumas espécies de escorpiões possuem em seu veneno compostos antimicrobianos, mediadores que ativam os processos inflamatórios, causam a desgranulação de mastócitos, como também, podem ser capazes de induzir quimiotaxia de neutrófilos. Até o presente momento a toxinologia de escorpiões tem concentrado o foco nas neurotoxinas, que são peptídeos contendo entre 30 e 70 resíduos de aminoácidos em suas sequências, com quatro ou cinco pontes dissulfeto em suas estruturas. A despeito de existir muitos pequenos peptídeos (apresentando entre 5 e 20 resíduos de aminoácidos em suas sequências), que apresentam conformações lineares, por não apresentarem pontes dissulfeto em suas estruturas, este grupo de toxinas tem sido negligenciado pela literatura, devido à dificuldade em isolar e determinar suas estruturas moleculares. O presente trabalho teve como objetivo sintetizar alguns destes peptídeos (Typep-14, -15, -16, -17, and -18), previamente isolados e sequenciados a partir do veneno do escorpião T. serrulatus, por nosso grupo de pesquisas, e caracterizá-los de acordo com suas possíveis funções biológicas através de ensaios de desgranulação de mastócitos e liberação de lactato desidrogenase, hemólise, antibiose, efeitos hiperalgésicos e edematogênicos, e teste de campo aberto. Nenhum peptídeo apresentou atividade hemolítica, antibiótica; ou apresentou atividade desgranuladora de mastócito e liberação de LDH em ratos. Apenas o TyPep 18 apresentou alterações na locomoção de camundongos. Em geral, todos apresentaram ações de hiperalgesia ou formação de edemas localizados. Conclui-se portanto, que os peptídeos ensaiados no presente trabalho estão relacionados à produção de dor e inflamação. / Scorpions are arthropods of the class Arachnida, order Scorpiones. They are venonous animals, i. e, they have secretory organ or gland connected to an injection apparatus, which can inoculate the venom produced by specialized cells. The venom can be described as a complex mixture containing mucus, inorganic salts, organic molecules of low molecular mass, many neurotoxic peptides and proteins. The human enenvenoming by scorpion sting is an important problem of public health in many tropical and subtropical countries. In Brazil, Tityus serrulatus is one of the most dangerous species, which cause severe enenvenoming, that can lead to death. In general, the envenoming can cause symptoms of pain, fever, agitation, salivation, lacrimation, increasing motility of the gastrointestinal tract, respiratory failure and cardiac arrhythmia, hypertension followed by hypotension, heart failure, pulmonary edema and anaphylaxis. The venom of scorpions has been studied over the years in order to elucidate their biochemical composition, along with their sequences and biological functions. Some species of scorpions have antimicrobial compounds in their venom, mediators that trigger inflammatory processes, causing mast cell degranulation, and also may be capable of inducing neutrophil chemotaxis. To date, the toxinology of scorpions has concentrated the focus on neurotoxins, which are peptides containing between 30 and 70 amino acid residues in their sequences, with four or five disulfide bonds in their structures. Despite the existence of many small peptides (having between 5 and 20 amino acid residues in their sequences), that have linear conformations for not having disulfide bonds in their structures, this group of toxins has been neglected in the literature, due to the difficulty in isolating and determine molecular structures of these toxins. This study aimed to synthesize some of these peptides (Typep-14, -15, -16, -17, and -18) previously isolated and sequenced from T. serrulatus scorpion venom, by our research group, and characterize them according to their possible biological functions through mast cell degranulation and lactate dehydrogenase release test, hemolysis, antibiosis, hyperalgesic and edematogênics effects and open field test. None of the peptides presented hemolytic, antibiotic activity, mast cell degranulation or LDH release in rats. Only TyPep 18 influenced the pattern of locomotion in mice. In general, all peptides had presented hyperalgesic and edematogenic effects. It may be concluded that the peptides tested in this study are related to the production of pain and inflammation. / CAPES: 1208/2011

Resumo: O ensaio imunoenzimático de ELISA foi utilizado para avaliar, acompanhar e comparar a resposta imune humoral de camundongos SWISS durante o processo de hiperimunização com veneno nativo e irradiado com Cobalto-60 (60Co) obtido das serpentes Crotalus durissus terrificus, Bothrops jararaca, Bothrops jararacussu e Bothrops moojeni. A potência e a capacidade de neutralização foram avaliadas por meio de desafios "in vitro". A imunidade obtida ao final do processo de hiperimunização foi verificada mediante desafio "in vivo" e os efeitos colaterais foram avaliados. A imunização dos animais, com uma DL50 de cada veneno, ocorreu nos dias um, 15, 21, 30 e 45, quando foram colhidas amostras de sangue. No 60º dia ocorreram os desafios. Os resultados demonstraram que o teste de ELISA mostrou-se eficiente na avaliação, acompanhamento e comparação da resposta imune dos camundongos durante o processo de hiperimunização. Os títulos do soro produzido com veneno nativo foram semelhantes aos títulos do soro obtido com veneno irradiado. A capacidade imunogênica foi mantida após a irradiação com 60Co. O soro produzido a partir do veneno irradiado de Crotalus durissus terrificus apresentou potência e capacidade de neutralização maiores, quando comparado ao soro obtido a partir do veneno nativo.Os anticorpos antiveneno nativo e irradiado, mediante os desafios "in vitro", mostraram-se igualmente eficazes na neutralização dos venenos de Bothrops jararaca, Bothrops jararacussu e Bothrops. Todos anticorpos foram capazes de neutralizar cinco DL50 dos respectivos venenos. As alterações clínicas foram mínimas durante o processo de hiperimunização com veneno irradiado e evidentes com veneno bruto. / Abstract: ELISA was used to evaluate, follow, and compare the humoral immune response of Swiss mice during hyperimmunization with natural and Cobalt-60- irradiated (60Co) Crotalus durissus terrificus, Bothrops jararaca, Bothrops jararacussu, and Bothrops moojeni venoms. Potency and neutralization were evaluated by in vitro challenges. After hyperimmunization, immunity was observed by in vivo challenge and the side effects were assessed. The animals' immunization with one LD50 of each venom was on days one, 15, 21, 30, and 45, when blood samples were collected; the challenges occurred on the 60th day. Results showed that ELISA was efficient in evaluating, following, and comparing mouse immune response during hyperimmunization. Serum titers produced with natural venom were similar to those with irradiated venom. Immunogenic capacity was maintained after 60Co irradiation. Serum produced from Crotalus durissus terrificus irradiated venom showed higher potency and neutralization capacity than that from natural venom. Antibodies from natural and irradiated venom by in vitro challenges were also efficient in neutralizing Bothrops jararaca, Bothrops jararacussu, and Bothrops moojeni venoms. All antibodies were capable of neutralizing five LD50 from these venoms. Clinical alterations were minimum during hyperimmunization with irradiated venom and evident with natural venom. / Orientador: Domingos Alves Meira / Coorientador: Benedito Barraviera / Mestre

Orientador: Valmir Fadel / Banca: Claudia Munte / Banca: João Ruggiero Neto / Resumo: Os venenos de vespas socias são ricos em peptídeos biologicamente ativos que causam alguns males ao ser humano como: dores prolongadas, edema, eritema, reações alérgicas e sistêmicas. Possuem em sua composição vários tipos de aminas biogênicas, peptídeos e proteínas. Dentre eles, o que chama mais atenção na atividade farmacológica do veneno são os peptídeos policatiônicos. São diversas as atividades desses peptídeos como: neurotoxicidade, hemólise, liberação de histamina de mastócitos e antibatericida. Neste trabalho, foram estudados peptídeos catiônicos da família Polybine, sintetizados pelo Departamento de Biologia, Unesp, Rio Claro-SP. Os peptídeos Polybine I e II foram sintetizados na forma acetilada e não-acetilada para o estudo detalhado da sua estrutura. Com este objetivo, utilizamos as técnicas de Dicroísmo Circular (CD), para uma análise da estrutura secundária da amostra e espectroscopia de Ressonância Magnética Nuclear (RMN) para o estudo da estrutura tridimensional do peptídeo em solução. / Abstract: Social wasp venoms are rich of biologically active peptides that may cause some malady to human such as prolonged pains, edema, erythema, allergies and systemic reactions. They have, in its composition, many kinds of biogenic amines, series of polycationics peptides and proteins. Among them, the most interesting thing in pharmacological activity are the polycationics peptides. These peptides show several activities like neurotoxicity, hemolytic activity, histamine releasing activity and antimicrobial activity. In this project, cationic peptides of the Polybine family synthesized by the Department of Biology, CEIS/IBRC, UNESP, Rio Claro, SP were studied. The cationic peptides Polybine I and II were synthesized in acetylad and non-acetylad forms to the detailed study of the structure. This way, CD spectroscopy were performed to analyze secondary structure of the sample and, to analyze treedimension structure, NMR spectroscopy were used. / Mestre

Orientador: Sergio Marangoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T15:45:07Z (GMT). No. of bitstreams: 1 Calgarotto_AndranaKarla_M.pdf: 1035943 bytes, checksum: f942243a78f40f0df6fd6b1150c07714 (MD5) Previous issue date: 2008 / Resumo: Bothrops moojeni é uma espécie de serpente de grande importância devido a sua ampla distribuição na América do Sul e Central além dos quadros clínicos que o veneno causa. Dentre as espécies peçonhentas brasileiras o gênero Bothrops é o mais numeroso e é o que apresenta os maiores índices de notificações relacionados a acidentes ofídicos. Os venenos de serpentes possuem uma mistura de substâncias biologicamente ativas sendo a maior parte composta por proteínas. Fosfolipases A2 (PLA2), proteínas presentes no veneno de serpentes, agem hidrolisando fosfolipídios de membrana na posição sn2 liberando lisofosfolipídios e ácidos graxos, além de exibir uma ampla variedade de efeitos farmacológicos. A isoforma de fosfolipase A2 (PLA2), denominada BmTX-I foi isolada através de um sistema de cromatografia em HPLC utilizando uma coluna de fase reversa µ-Bondapak C18. O alto grau de pureza foi confirmado através de eletroforese em SDS-PAGE Tricina (16,5%) e também através da determinação da massa molecular (14,238.71 Da) por espectrometria de massas (MALDI Tof). A caracterização cinética da BmTX-I PLA2 (Asp49) mostrou que tal isoforma é altamente estável e apresenta um pH ótimo de 8,0 e temperatura de 37º C. Frente a diferentes concentrações do substrato ácido 4-nitro-3-(octanoyloxy) benzóico a BmTX-I mostrou um comportamento com tendência alostérica. Na ausência de Ca2+ e na presença de alguns íons divalentes tais como Mg2+, Mn2+ e Cd2+ (na concentração de 10mM) a atividade BmTX-I foi significativamente diminuída, já na presença de Ca2+ (1mM) e com os mesmos íons divalentes citados apresentou uma discreta atividade. Também foi demonstrado o efeito inibitório de crotapotinas crotálicas sobre a atividade PLA2 da BmTX-I. A análise de composição de aminoácidos mostra que se trata de uma proteína de caráter básico pela alta presença de Lys, His e Arg. A presença de 14 resíduos de cisteína sugere a formação de 7 pontes dissulfeto. O estudo de homologia seqüencial da região N-terminal entre BmTX-I com outras PLA2 (Asp49) revelou um alto grau de homologia. O efeito neurotóxico in vitro do veneno total e da BmTX-I foi analisado na preparação biventer cervicis de pintainho. Nossos resultados mostraram que a ação do veneno de Bothrops moojeni na junção neuromuscular é menos potente quando comparado com venenos crotálicos, já que estes últimos levam a um bloqueio muito mais rápido e usando-se baixas concentrações. No entanto, não se pode negar que houve uma ação neurotóxica in vitro. O completo bloqueio tanto do veneno total quanto da BmTX-I não foi acompanhado pela inibição das respostas ao potássio (KCl) e a acetilcolina (ACh), exceto em altas concentrações de veneno (50 e 100 µg/ml) o que demonstra uma ação pré-sináptica primordial. Os testes in vivo do veneno total e da fração BmTX-I demonstraram o efeito miotóxico através da liberação de creatina quinase (CK) e o efeito inflamatório através de edema de pata e liberação de interleucina-6 (IL-6). O comportamento de liberação de CK foi semelhante tanto para o veneno total como para a PLA2 BmTX-I, os quais tiveram o maior liberação de CK uma hora após a injeção i.m. Oito horas após a injeção os níveis de CK estavam similares aos do controle. Ocorreu liberação de IL-6 bem como ação edematizante tanto do veneno total como da BmTX-I. A reprodutibilidade dos efeitos farmacológicos, só é possível com a utilização de frações quimicamente homogêneas que mantenham a integridade da função biológica. Essas frações são obtidas com metodologias de alta eficiência como HPLC, através do qual podemos isolar a BmTX-I em um único passo cromatográfico e o grau de pureza confirmado por espectrometria de massa. Estes resultados podem ser associados com a sua atividade biológica, eliminando a subjetividade causada por veneno total ou frações impuras. Essa abordagem pode ser aplicada nos estudos bioquímicos, estrutura-função, fisiológicos e farmacológicos, podendo revelar mecanismos ainda desconhecidos na relação estrutura-função das PLA2 procedentes de veneno de serpentes / Abstract: Bothrops moojeni is a very important snake species due to its great distribution in the South and Central America besides the clinic alterations caused by the venom. Among the Brazilian species the Bothrops genus is the most numerous and shows the highest registrations related to ophidian accidents. The snake venoms are source of biologically active substances which main components are proteins. Phospholipases A2 (PLA2), proteins present in the snake venom, hydrolyze the sn-2 acyl groups of phospholipids liberating fatty acids and lysophospholipds, besides exhibiting many pharmacological effects. The fosfolipase A2 (PLA2) isoform, named BmTX-I was isolated through RP-HPLC performed on a C18 column. The high degree of purity was confirmed by SDS-PAGE and also by determining the molecular mass (14238.71 Da) in a MALDI TOF mass spectrometry. The kinetic characterization of BmTX-I PLA2 (Asp49) showed that the isoform was highly stable and presented an optimum pH of 8.0 and temperature of 37° C. At different substrate acid 4-nitro-3-(octanoyloxy) benzoic concentrations the BmTX-I showed allosteric behavior. In the absence of Ca2+ and in the presence of some divalent ions such as Mg+2, Mn+2, Cd2+ (at the concentration of 10 mM) the BmTX-I activity significantly decreased. But in the presence of 1mM Ca+2, under the same divalent ions conditions, the isoforms showed a discreet activity. An inhibitory effect of crotalic crotapotins on the activity PLA2 was also demonstrated. The analysis of amino acids composition showed a high content of basic amino acids such as Lys, His and Arg indicating a basic character for the BmTX-I. The presence of 14 cysteine residues suggests the formation of 7 disulfide bridges. N-terminal amino acid sequence revealed a high level of homology between BmTX-I and other Asp49 PLA2s. The neurotoxic effect of the whole venom and of BmTX-I was analyzed at chick biventer cervicis muscle preparation. Our results showed that the blockage of the muscle contraction was lesser when compared with crotalic venoms, which blockage at lower concentrations. Nevertheless it is sure that there was an in vitro neurotoxic action. The complete blockage, as much the whole venom as the BmTX-I, was not accompanied by any significant inhibition of the responses to KCl and to Ach, excepting at higher concentrations of the venom (50 e 100 µg/ml) suggesting the primordial presynaptic action. The tests in vivo with the whole venom and BmTX-I fraction showed the miotoxic effect through the creatine kinase (CK) releases and the inflammatory effect through edema-forming activity and interleukin-6 release. The CK release was similar for both whole venom and BmTX-I, which showed higher liberation one hour after the i.m injection. After eight hours of injection the CK levels were similar to the controls. There was the IL-6 release as well as edema-forming activity both in to the whole venom and BmTX-I. The reprodubility of pharmacological effects, is just possible with the utilization of chemically homogenous fractions that maintain the integrity of biological function. These fractions were obtained with high efficient methodologies as HPLC, through which this we the BmTX-I could be isolated in only one chromatography step, with purity direct by mass spectrometry. These results may be associated with the biological activities, eliminating the subjectivity caused by total venom or impure fractions. This approximation may be applied to the biochemical, structure-function, physiological and pharmacological studies, and it may reveal still unknown mechanisms in the structure-function relationship of PLA2 from the serpents venom / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Albetiza Lobo de Araujo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T13:35:19Z (GMT). No. of bitstreams: 1 Martins_LucimaraJulio_M.pdf: 11295607 bytes, checksum: 8492693ea03dda6f72a75c0c23ee0d9c (MD5) Previous issue date: 2006 / Resumo: O veneno de Bothrops lanceolalus exerce várias atividades biológicas, dentre elas a atividade hemolítica indireta. Neste trabalho verificamos algumas das características desta atividade in vitro. O veneno induziu hemólise indireta em eritrócitos de cavalo, boi, rato e carneiro, sendo que, dentre essas espécies, a primeira foi mais sensível; não houve atividade hemolítica direta. A hemólise foi concentração-dependente e atenuada em temperaturas >40°C. O tratamento do veneno com brometo de p-bromofenacil aboliu a atividade da fosfolipase A2 (PLA2) do veneno e impediu a hemólise. Corroborando estes achados, uma PLA2 ácida purificada deste veneno também induziu hemólise. Esta PLA2 reagiu contra o anti-soro do veneno de B. lanceolatus. Estes resultados indicam que atividade hemolítica do veneno de B. lanceolatus é mediada pela PLA2. A reação cruzada da PLA2 com o anti-soro sugere que a atividade desta enzima pode eficazmente ser neutralizada durante a terapia sorológica / Abstract: Bothrops lanceolatus venom exerts a variety of biological activities, including indirect hemolysis. In this work, we examined some of the characteristics of this activity in vitro. The venom caused indirect hemolysis in horse, ox, rat and sheep erythrocytes, with the first of these species being the most sensitive; there was no direct hemolysis. The hemolysis was concentration-dependent and was markedly attenuated at >40°C. Treatment of the venom with />-bromophenacyl bromide abolished the phospholipase A2 (PLA2) activity of the venom and prevented the hemolysis. In agreement with this finding, an acidic PLA2 purified from this venom also caused hemolysis. This PLA2 reacted with antivenom against B. lanceolatus venom. These results indicate that the hemolytic activity of B. lanceolatus venom is mediated by PLA2. The cross-reactivity of the PLA2 with antivenom suggests that the activity of this enzyme may be effectively neutralized during antivenom therapy / Mestrado / Mestre em Farmacologia

Orientador: Ricardo de Lima Zollner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T13:48:19Z (GMT). No. of bitstreams: 1 Trevizan_Giovanna_M.pdf: 992041 bytes, checksum: 788bad7f2b885488c459d8cc22f66468 (MD5) Previous issue date: 2010 / Resumo: As reações alérgicas à ferroada de inseto resultam de resposta exacerbada do sistema imune, com produção de elevados níveis de anticorpos IgE alérgeno-específicos e padrão de citocinas Th2. A diferenciação de linfócitos Th2 e a secreção de citocinas por estas células são reguladas pelo fator de transcrição GATA-3. A ligação da IgE ao seu receptor de alta afinidade (Fc?RI) em mastócitos e basófilos, promove a liberação de mediadores inflamatórios. Muitos estudos demonstram a eficácia da imunoterapia específica na dessensibilização e no desenvolvimento de tolerância em indivíduos com quadros graves de hipersensibilidade à ferroada de insetos, sobretudo da classe Hymenoptera. A histamina é o principal mediador liberado durante a resposta alérgica; através da ativação de diferentes receptores (HR1, HR2, HR3 ou HR4) as células do sistema imune podem ser tanto inibidas como estimuladas. Via HR1, a histamina estimula principalmente linfócitos Th1, enquanto inibe linfócitos Th2 via HR2. O receptor 4 desempenha papel central na diferenciação de linfócitos Th2 e também é capaz de modular a produção de citocinas. A liberação de histamina é regulada por fatores de liberação de histamina (HRF), sendo que os HRF dependentes de IgE induzem a liberação de histamina na fase tardia das reações alérgicas. Considerando-se todas essas informações, o objetivo do presente trabalho foi avaliar os efeitos modulatórios da imunoterapia na expressão gênica dos receptores de histamina (HR1, HR2 e HR4), fatores de liberação de histamina (HRF) e cadeia ? do receptor de alta afinidade de IgE, além da expressão protéica do fator de transcrição GATA-3, em células linfomononucleares de pacientes alérgicos ao veneno de abelha. As células foram isoladas de pacientes submetidos à imunoterapia, em diferentes períodos do tratamento (Pré, 1, 3, 6, 12, 18 e 24 meses), após injeção subcutânea, e submetidas à cultura por 72 horas, com estimulo de veneno de abelha a 1 ng/ml. Indivíduos não alérgicos foram estudados como grupo controle. Com objetivo de avaliar a expressão gênica foram realizadas extração de RNA, seguida de síntese de cDNA e PCR, para avaliação da expressão protéica, utilizou-se a técnica de imunofluorescência. A expressão gênica de HR1 e HR4, assim como de HRF e da cadeia gama do Fc?RI foi significativamente reduzida ao final do período analisado. O receptor 2 de histamina não apresentou alterações bem definidas após 24 meses de imunoterapia. O fator de transcrição GATA-3 apresentou diminuição significativa na expressão a nível protéico. Nossos resultados demonstram que a imunoterapia específica ao veneno de abelha foi capaz de modular elementos envolvidos na resposta imune / Abstract: Allergic disease is an abnormal response from the immune system, with high levels of allergen specific IgE antibodies and Th2 pattern of cytokines. Development of Th2 cells and the production of cytokines are regulated by transcription factor GATA-3. Binding of IgE to its high affinity receptor (Fc?RI) in mast cells and basophils induces inflammatory mediators' release. Many studies have shown the efficacy of specific immunotherapy. Histamine is an important mediator in allergy, through activation of distinct histamine receptors (HR1, HR2, HR3 or HR4) in the immune system; cells can be either stimulated or inhibited. Histamine can stimulate, by HR1 receptor, especially Th1 response, and inhibit particularly Th2 cells through HR2 activation. HR4 plays a central role in Th2 polarization and also modulates cytokine profile production. IgE-dependent histamine releasing factors (IgE-HRF) induce histamine release in late phase reaction. In regard of this information, the aim of this study was to evaluate the modulating effects of specific immunotherapy in gene expression of histamine receptors (HR1, HR2 and HR4), histamine releasing factor (HRF), in patients with allergy and the gama chain of Fc?RI, and also GATA-3 protein levels. Bee venom allergic subjects underwent specific bee venom immunotherapy (VIT) in different stages of treatment (Pre, 3, 6, 12, 18 and 24 months) were studied. Peripheral blood mononuclear cells were isolated after subcutaneous venom injection and submitted to culture for 24, 48 and 72 hours stimulated with 1ng/ml of bee venom. In parallel healthy subjects were studied as well. Total RNA extraction, followed by cDNA synthesis and PCR were used to evaluate gene expression; GATA-3 protein expression was analyzed by immunofluorescence assay. Data from all time of cell culture - 24, 48 and 72 hours - were grouped and analyzed. Gene expression from HR1 and HR4 and also HRF and ? chain of Fc?RI were significantly reduced at the end of 24 months of immunotherapy. Histamine receptor 2 didn't show well established alterations. For transcription factor GATA-3 significant decrease at protein level was observed. Together, our results indicate that bee venom specific immunotherapy was able to modulate some of the elements involved in the immune response / Mestrado / Ciencias Basicas / Mestre em Clinica Medica