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Investigação micológica do fluido vaginal de mulheres adultas da região de São José do Rio PretoCorrêa, Paula dos Reis [UNESP] 10 February 2009 (has links) (PDF)
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correa_pr_me_sjrp.pdf: 763319 bytes, checksum: 1d1304fe60a8bc1f5f7c29d67c166679 (MD5) / A candidíase vulvovaginal (CVV) é uma inflamação da mucosa genital decorrente de infecção por leveduras. Os principais sintomas são disúria, hiperemia, ardência, corrimento, prurido, dispareunia, fissuras. Aproximadamente 75% das mulheres sofrem ao menos um episódio de candidíase vulvovaginal, 40% apresentam mais de um episódio e menos de 5% tornam-se recorrentes (CVVR). A CVV tem como principal etiologia, Candida albicans, no entanto, episódios devido às espécies “não albicans” estão aumentando. O manejo das vaginites vem sendo realizado de modo empírico, levando a problemas quanto à terapêutica. Assim, este projeto teve como objetivos caracterizar fenotipicamente leveduras isoladas do conteúdo vaginal de 223 mulheres adultas, sintomáticas (S) e assintomáticas (A), atendidas pelo Serviço de Ginecologia do Hospital de Base de São José do Rio Preto e determinar os indicadores clínicos. Para tal, foi realizada análise micológica dos 223 isolados clínicos, sendo que, 87 amostras apresentaram cultura positiva. C. albicans foi a espécie mais prevalente nos dois grupos (A/S), seguida de Candida glabrata. A maior parte das mulheres era casada com média de idade de 30 a 40 anos, referiu uso de anticoncepcionais e ciclo menstrual regular. Em relação a práticas sexuais houve, para parte das pacientes, concomitância entre os hábitos, anal, oral e vaginal. Quando avaliado o tratamento prévio, não houve evidência de associação com sintomas particulares. A suscetibilidade a antifúngicos, realizada pelos dois métodos de referência disco-difusão e microdiluição, mostrou concordância, estatisticamente comprovada, apenas para o itraconazol. Em relação à produção de fator de virulência, apenas C. albicans produziu fosfolipase. Diferentemente, proteinase foi detectada em C. albicans, C. glabrata e Candida parapsilosis. Esse último fator de virulência... / Vulvovaginal candidiasis (CVV) is an inflammation of genital mucosa caused by infection by yeasts. The main symptoms are dysuria, hyperemia, burning, corrimento, pruritus, dyspareunia, cracks. Approximately 75% of women suffer at least one episode of vulvovaginal candidiasis, 40% have more than one episode and less than 5% become recurrent (CVVR). The main cause is to candidiasis is Candida albicans, however, episodes due to the species non albicans are increasing. The management of vaginitis has been done so empirically, leading to problems with therapy. Thus, this project aimed to phenotypically characterize yeasts isolated from vaginal content of 223 adult women, symptomatic (S) and asymptomatic (A), attended by the Service of Gynecology, Hospital de Base de São José do Rio Preto and determine the clinical indicators. This has mycological analysis of 223 clinical isolates, of which 87 samples showed positive culture. C. albicans was the species most prevalent in both groups (A / S), followed by Candida glabrata. Most women were married with an average age of 30 to 40 years, said use of contraceptives and menstrual cycle regularly. In relation to sexual practices was for the part of patients, concomitance between habits, anal, oral and vaginal. When assessing the previous treatment, there was no evidence of association with particular symptoms. The susceptibility to antifungal held by the two methods of reference disc-diffusion and microdilution, showed agreement, statistically proven, only to itraconazole. For the production of virulence factor, only C. albicans produced phospholipase. Conversely, proteinase was detected in C. albicans, C. glabrata and Candida parapsilosis. The latter factor was associated with virulence, mainly in isolates from symptomatic patients. Data from this study helped reveal association between virulence factors with clinical signs... (Complete abstract click electronic access below)
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Estudos funcionais e moleculares relativos às membranas apicais intestinais de Dysdercus peruvianus / Functional and molecular studies related to apical midgut membranes of Dysdercus peruvianusAndré Coppe Pimentel 12 August 2016 (has links)
Os insetos da ordem Hemiptera apresentam membranas lipoproteicas que revestem as microvilosidades das células intestinais, como se fossem dedos de luva, e formam expansões para o lúmen do intestino que parecem terminar em fundo cego. A presença das duas membranas no ápice dos enterócitos gera questões intrigantes de como se dá a formação da membrana perimicrovilar, como ocorre a absorção de nutrientes e como se dá o encaminhamento de enzimas digestivas para o lúmen intestinal. A digestão de proteínas baseada em enzimas originalmente lisossômicas é uma característica marcante nos Hemiptera, em especial os Heteroptera que evolutivamente voltaram a uma alimentação de polímeros. O presente estudo indica que os genes das proteinases tipicamente lisossômicas sofrem uma série de duplicações, havendo a manutenção de um gene para função puramente lisossômica e a divergência funcional dos demais genes para a função de digestão extracelular. O gene que mantém a função lisossômica não é modulado pela alimentação, além de ser expresso nos mais diversos tecidos. Já os genes que se especializaram na digestão extracelular têm a expressão aumentada com a ingestão de alimento, indicando sua função. Parece não haver diferença nas características relacionadas ao encaminhamento celular das proteínas produzidas por esses genes, indicando que o direcionamento para a rota secretória é devido à superexpressão dos genes relacionados à digestão. Enzimas como alfa-glicosidases, alfa-manosidases e aminopeptidases que participam da digestão extracelular seguem a rota secretória que envolve a formação de vesículas de dupla membrana. Foi possível ampliar o modelo de digestão incluindo a participação das catepsinas D, de uma alfa-glicosidase solúvel e a possível participação de uma tiolredutase além do definir o local de atuação das lipases. Temos agora uma visão global da participação das enzimas digestivas que atuam na digestão em D. peruvianus. / The insects of the order Hemiptera have lipoprotein membranes lining the microvilli of midgut cells, like glove fingers, and form expansions into the lumen of the intestine. The presence of two membranes on the apex of enterocytes thus generates intriguing questions about the formation of perimicrovillar membrane, the absorption of nutrients, and the targeting of digestive enzymes into the intestinal lumen. The digestion of proteins based on originally lysosomal enzymes is an important feature in Hemiptera, especially in Heteroptera that evolutionary returned to feed on polymers. The genes of typical lysosomal proteinases undergo a series of duplications followed by the maintenance of a gene for purely lysosomal function and functional divergence of other genes for extracellular digestion function. The gene that maintains the lysosomal function is not modulated by feeding, in addition to being expressed in diverse tissues. By the other hand, genes specialized in extracellular digestion are up regulated by food intake, indicating its function. No difference was found in the targeting in the proteins produced by these genes, which indicates that targeting to the secretory route is due to overexpression of digestion-related genes. Enzymes involved in extracellular digestion as alpha-glucosidase, alpha-mannosidase and aminopeptidase follow the secretory route that includes the formation of double membrane vesicles. In this study we increased the digestion model adding the participation of cathepsin D, a soluble alpha-glucosidase, and the possible participation of a tiolredutase, and also to defining the place of lipases operation. We now have a global view of the participation of digestive enzymes involved in digestion D. peruvianus.
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ProteÃnas inibidoras de fitopatÃgenos em fluidos laticÃferos: atividade e mecanismo de aÃÃo / Inhibitory proteins of plant pathogens in fluids latex: activity and mechanism of actionDiego Pereira de Souza 26 February 2010 (has links)
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Um relevante nÃmero de espÃcies vegetais à descrito como plantas produtoras de um fluido leitoso comumente denominado de lÃtex. Nestas espÃcies, o lÃtex à sintetizado e armazenado sob pressÃo em um sistema de canais formados por cÃlulas altamente especializadas denominadas de laticÃferas, em cujos citoplasmas estÃo presentes todas as estruturas eucariontes em meio à Ãgua, borracha e inÃmeras molÃculas, muitas das quais especÃficas deste conteÃdo. Muitos estudos tÃm sugerido que molÃculas produzidas nestes fluidos participam da defesa vegetal. Neste trabalho, o lÃtex de 5 espÃcies foi coletado e processado em laboratÃrio para obtenÃÃo de suas fraÃÃes protÃicas e estas foram avaliadas quanto a atividade sobre fungos fitopatogÃnicos atravÃs de ensaios de inibiÃÃo da germinaÃÃo de esporos e crescimento de hifas. ProteÃnas do lÃtex de C. procera (PLCp), Cryptostegia grandiflora (PLCg) e Carica candamarcensis (P1 G10) apresentaram atividade antifÃngica enquanto que Plumeria rubra (PLPr) e Euphorbia tirucalli (PLEt) nÃo apresentaram atividade sobre qualquer dos fungos avaliados (Colletotrichum gloeosporioides, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Neurospora sp. e Aspergillus niger). A atividade inibitÃria das fraÃÃes protÃicas se correlacionou diretamente com a presenÃa de atividade proteolÃtica do tipo cisteÃnica presente nas amostras de PLCp, PLCg e P1G10. A atividade antifÃngica foi aumentada na presenÃa de DTT, um ativador destas proteases e foi diminuÃda ou eliminada quanto Ãs amostras foram prÃ-tratadas com iodoacetamida (IAA), um inibidor especÃfico de proteases cisteÃnicas. AlÃm disso, a atividade antifÃngica foi observada quando papaÃna, uma protease cisteÃnica purificada do lÃtex de Carica papaya foi avaliada, mas tripsina e quimotripsina, duas proteases serÃnicas nÃo apresentaram atividade. AtravÃs de cromatografia em coluna de Mono-S Sepharose acoplada ao sistema de FPLC, uma protease cisteÃnica foi isolada de PLCg. A proteÃna purificada (Cg24-I) apresentou massa molecular de 24,118 KDa. A Cg24-I apresentou atividade proteolÃtica mÃxima em pH 8,0 e foi inibida por IAA e E-64, utilizando azocaseÃna e BANA como substratos, respectivamente. Cg24-I inibiu a germinaÃÃo de F. solani e foi capaz de alterar a permeabilidade das membranas dos esporos na concentraÃÃo de 90 ng/ml. Esse conjunto de resultados sugere que proteinases cisteÃnicas de fluidos laticÃferos participam da defesa das plantas contra fungos fitopatogÃnicos e que o provÃvel mecanismo de aÃÃo destas proteÃnas seja a alteraÃÃo da permeabilidade da membrana plasmÃtica destes microrganismos. A descriÃÃo de atividade antifÃngica de proteases cisteÃnicas oriundas de fluidos laticÃferos nÃo à ainda descrita em detalhes na literatura, sendo este um trabalho com carÃter original. / Canal systems containing secretions, such as latex, are widely disseminated in the plant kingdom. These fluids are chemically complex and exhibit intense metabolism. Despite their origin, latex is the cytoplasm of specialized cells growing intrusively into organized tissues and organs, forming an interconnected network allowing latex exudation immediately after tissue damage. Insecticidal effects of latex proteins have been described, however minor studies were devoted to investigate antifungal activities in latex. In this study proteins extracted from latex of Calotropis procera (Ait.) R.Br (PLCp), Plumeria rubra L.(PLPr), Carica candamarcensis Hook F.(P1 G10), Cryptostegia grandiflora (PLCg), and Euphorbia tirucalli L. (PLEt) were tested for antifungal activity against six phytopathogens (Fusarium solani, F. oxysporium, Aspergilus niger, Rhizoctonia solani, Neurospora sp. and Colletrotricum gloerosporioides). PLCp, PLCg and P1G10 exhibited antifungal activity and PLPr and PLEt were not efetive. Inhibitory activity of the protein fractions correlated with the cysteine-type proteolytic activity found in these fractions. The endogenous proteolytic activity and inhibitory activity on fungal growth were both increased when samples were first activated with DTT, a cysteine proteinase activator. Conversely, pre-treatment of samples with iodoacetamide, an inhibitor of these proteases rendered all samples deficient of both, proteolytic and antifungal activities. Antifungal of activity of cysteine proteinases of latex origin was also confirmed when papain, obtained from latex of Caryca papaya was tested while purified trypsin and chemotrysin, two serine-type proteases were not antifungal. A cysteine proteinase was thus, purified form PLCg by ion exchange chromatography on a Mono-S Sepharose matrix monitored by a FPLC system. The protein, named Cg24-I exhibited molecular mass of 26.118 KDa determined by MALDI spectrometry; maximum of proteolysis at pH 8.0 and inhibited by iodoacetamide and E-64 when assayed with azocazein or BANA as substrates. Cg24-I inhibited germination of F. solani and altered membrane permeability of spores at a minimum concentration of 90 ng/ml. Results present here suggest that cysteine proteinases of laticifer fluids are proteins with antifungal activity capable of damaging spore structure and inhibiting hyphae growth. Reports of antifungal activity of latex proteases are still scarce in literature and this work appears as an important contribution to this field. Furthermore, this work gives important evidence for the multiple defensive role of latex in plants.
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Estudo retrospectivo do tratamento ambulatorial da úlcera indolente em cães da raça Boxer / Retrospective study of clinical management of indolent ulcers in Boxer dogsAna Paula Franco do Amaral Hvenegaard 24 November 2010 (has links)
Úlceras indolentes são úlceras corneais superficiais, espontâneas, que apresentam curso prolongado e que tendem a recidivar. Comumente observadas em cães de meia idade, da raça Boxer, provoca dor de início agudo e necessita de tratamento específico, já que este, quando não realizado de forma correta, pode prolongar o curso da lesão por semanas a meses. A doença é explicada por diversas alterações da superfície ocular. Com o objetivo de avaliar a eficácia dos tratamentos ambulatoriais preconizados no Serviço de Oftalmologia do Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo (HOVET-FMVZ-USP), e as principais considerações observadas no levantamento dos prontuários, realizou-se estudo retrospectivo dos casos atendidos entre os anos de 1997 e 2008. Segundo os resultados, observou-se que a maioria dos cães da raça Boxer apresentaram úlcera indolente, distrofia corneal e catarata; que as úlceras indolentes foram mais frequentemente observadas em fêmeas de meia idade e que a maioria dos proprietários demoraram mais de 15 dias para levar seus animais ao HOVET-FMVZ-USP; que as alterações oculares mais frequentemente referidas pelos proprietários foram o blefarospasmo, olho vermelho e a secreção; que as principais características das lesões observadas após o exame oftalmológico foram que a maioria das úlceras eram transparentes, apresentando epitélio não aderido ou com algum grau de vascularização; unilaterais, mais frequentemente observadas no olho direito; de aparecimento espontâneo e localizadas no centro da córnea. Quanto ao tratamento, observou-se que os inibidores das proteinases foram as medicações mais frequentemente prescritas e que sua administração não interferiu no tempo de cicatrização corneal ou na formação de granuloma. Vitamina C, apesar de ter prolongado de maneira significante o tempo de cicatrização corneal, reduziu a inflamação, consideração observada pela diminuição da presença de granuloma. Debridamento/cauterização corneal, além de não interferir na formação de granuloma, acelerou, significativamente, o processo de cicatrização. A antibioticoterapia e a administração de Atropina 1 % não interferiu no tempo de cicatrização, mas se relacionaram diretamente, de forma estatisticamente significante, à presença de granuloma. O uso de anti-inflamatórios tópicos e sistêmicos também não interferiu no tempo de cicatrização, mas diminuíram, de maneira significante, a presença de granuloma nos cães em que foram administrados. Observou-se também que a não administração de atropina 1 %, antibióticos e anti-inflamatórios não interferiu no tempo de cicatrização, nem na formação de granuloma; que o tempo de alteração ocular, antes da primeira consulta e as características das lesões não interferiram, de maneira relevante, no tempo de cicatrização corneal. Portanto, conhecer os diversos tipos de tratamento se mostra fundamental para o sucesso da resolução da doença, já que este deve ser específico, realizado de forma cautelosa e por tempo indeterminado, cuidando para que a lesão não progrida e promovendo o retorno da transparência corneal. / Indolent ulcers are superficial corneal ulcers that occurs spontaneously, presents prolonged course and tend to relapse. Commonly observed in middle-aged Boxer dogs, causes pain of acute onset and requires appropriate treatment. The disease is explained by several changes on the corneal surface. Aiming to assess the effectiveness of clinical treatments, recommended by the Ophthalmology Service of the Veterinary Hospital, of the Veterinary College, of the University of São Paulo (HOVET-FMVZ-USP) and to evaluate major considerations registered on its medical records, a retrospective study was conducted (1997 2008). Results demonstrated that, during studied period: most Boxer dogs presented indolent ulcers, corneal dystrophy and cataracts; indolent ulcers were frequently observed in middle-aged female Boxers and most owners took more than 15 days to bring their animals to the hospital; blepharospasm, red eye and ocular discharge were the most owner´s referred ocular alterations at the primary consultation; main features of examined lesions were transparent ulcers presenting non adherent epithelium and/or some degree of vascularization; unilateral, often observed at the right eye, of spontaneous onset and located at the center of the cornea. Regarding treatment, proteinase inhibitors were the most often prescribed medications; its administration did not affect corneal healing or granuloma formation. Vitamin C prolonged, significantly, the corneal healing time, although, its administration reduced its inflammation, observed by the decrement on the granuloma frequency. Corneal debridement / cauterization, did not interfere on granuloma formation and was capable to accelerate, significantly, the healing process. Antibiotics and 1 % atropine did not affect the healing time, but were statistically related to the presence of granuloma. Topical and systemic antiinflammatories did not interfere at the healing time, but decreased, significantly, the presence of granuloma. Not to administer atropine 1%, antibiotics and antiinflammatories, did not interfere at the corneal healing time nor the formation of granuloma. Duration period of ocular alterations before the first consultation and characteristics of the lesions did not interfere at the corneal healing time. Therefore, to know the various types of treatments seems to be fundamental to the resolution of the indolent ulcer, as treatment must be specific, performed cautiously and for indefinitely period, preventing the progression of the lesion, and promoting the return of corneal transparency.
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Detection of a papaya cysteine proteinase inhibitor under different environmental conditionsBester, Christell 17 August 2012 (has links)
M.Sc. / Proteinases are involved in many cellular reactions involving protein degradation, such as degradation of storage proteins and protein degradation during senescence processes. Their action can be inhibited by proteinase inhibitors. Information is still limited about the regulation of these inhibitors in plants and their possible interaction with proteinases under stress conditions. To obtain a better understanding of the physiological role of a proteinase inhibitor in plants under stress, the expression of a papaya cysteine proteinase inhibitor (cystatin) and its relation to proteinase expression was investigated in more detail. For this purpose, expression of the inhibitor was studied in papaya plants exposed to different physiological stress conditions, such as high/low temperature, and treatment with selected chemicals, such as glutathione, OTC (L-2- Oxothiazolidine-4-carboxylate), bestatin ([(2S, 3R)-3-amino-2-hydroxy-4-phenyl butanoylj-L-leu) and 2.4-D (2,4-dichiorophenoxyacetic acid). Using detection tools like activity gel electrophoresis, immunoblotting and enzymatic assays, the production of the cystatin under stress was monitored in different papaya explants, such as roots, leaves and embryos. Inhibitor production increased under different stress conditions when compared to untreated controls. However, this increase was not dramatic in any of the stresses applied. Exact quantification of the increase by using immunoblotting as the only specific tool to determine cystatin expression, was difficult. Neither activity gel electrophoresis nor enzymatic assays were successful to further quantify the exact cystatin levels. Higher cystatin expression was accompanied with a decrease in proteinase activity. Transgenic tobacco plants carrying the gene for a rice cystatin had a significantly lower cysteine proteinase activity when compared to non-transgenic tobacco plants after prolonged cold stress. Furthermore, protein degradation and leaf yellowing as a consequence of cold treatment were prevented in transgenic plants. An attempt to obtain a transformed papaya plant to study silencing of cystatin expression under stress was unsuccessful. In this study, the protective role of a cystatin in cold stress was described for the first time.
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Rôle pro-inflammatoire et immunomodulateur de la proteinase 3 membranaire exprimée au cours de l'apoptose : implications dans la granulomatose avec polyangéite / Inflammatory and immunomodulatory role of proteinase 3 expressed at the membrane of apoptotic cells : application to granulomatosis with polyangiitisMillet, Arnaud 15 January 2014 (has links)
La granulomatose avec polyangéite est une vascularite systémique associée à une réaction auto-immune dirigée contre la protéinase 3, une serine protéase du neutrophile. Cette protéine présente un profil particulier d’expression dans le neutrophile, caractérisé notamment par une expression membranaire au cours de l’apoptose. Cette capacité de la protéinase 3 à se lier aux membranes repose sur l’existence de quatre acides amines formant un patch hydrophobe. Cette expression membranaire au cours de l’apoptose, qui dépend de l’existence de ce patch hydrophobe, conduit la protéinase 3 à interagir avec la calréticuline qui est une molécule impliquée dans la reconnaissance des cellules apopotiques par les macrophages. Nous avons démontré que cette interaction conduit la protéinase 3 à modifier le phénotype pro-résolutif des macrophages consécutif à la phagocytose de cellules apoptotiques. Le phénotype pro-inflammatoire résultant de cette expression de la protéinase 3 dépend de la voie de signalisation MyD88 mimant un signal danger. Cette activation des macrophages conduit à la sécrétion de chimiokines (MCP-1, KC, MIP-1α et MIP-1β) impliquées dans le recrutement de cellules exprimant la protéinase 3 participant ainsi au maintient de l’inflammation. Ce microenvironnement induit par les macrophages modifie le rôle immuno-modulateur de la clairance des cellules apoptotiques, influençant notamment l’interaction des cellules T naïves avec les cellules dendritiques plasmacytoïdes. La polarisation T résultante présente une distribution déséquilibrée vers les lymphocytes Th2/Th9 et une diminution de la génération de lymphocytes T régulateurs. La protéinase 3, qui est encodée par un gène de réponse au G-CSF, est de plus capable d’induire le recrutement de cellules sur-exprimant cette protéase capable à son tour de stimuler la sécrétion de G-CSF. La protéinase 3 apparait donc, par sa capacité à corrompre les mécanismes de résolution de l’inflammation et d’amplifier sa propre expression dans les cellules recrutées sur le site inflammatoire, comme un élément clé de la physiopathologie de la granulomatose avec polyangéite. / Granulomatosis with polyangiitis is a systemic vasculitides associated with an autoimmune response directed against proteinase 3 a neutrophil-derived serine protease. This protein presents a very specific pattern of expression in neutrophils, illustrated by its membrane expression during apoptosis. The ability of PR3 to bind membrane is based on the existence of four amino-acids involved in the formation of a hydrophobic patch. The membrane expression during apoptosis, which is dependent of the existence of the hydrophobic patch, leads to an interaction between proteinase 3 and calreticulin, a “eat-me” signal involved in the recognition of apopotic cells by macrophages. As a consequence, we have found that proteinase 3 expressed at the surface of apoptotic cells hampers the normal anti-inflammatory phenotype of macrophages following phagocytosis of apoptotic cells. The pro-inflammatory phenotype resulting from proteinase 3 expressing cells phagocytosis by macrophages is a MyD88 signaling pathway dependant mechanism mimicking an alarmin stimulation. This activation of macrophages through the secretion of chemokines (MCP-1, KC, MIP-1α and MIP-1β) implicated in the recruitment of cells expressing proteinase 3 participate in the maintenance of inflammation. This macrophage-induced microenvironment was also found to impact the immune silencing of apoptotic cells clearance by plasmacytoid dendritic cell-dependant T cell interaction leading to a skewed Th2/Th9 T cell distribution instead of the generation of regulatory T cells. Proteinase 3, which is encoded by a G-CSF responsive gene, was also able to recruit more over expressing proteinase 3 cells by its ability to stimulate G-CSF secretion. Then proteinase 3 through its corruption of inflammation resolving mechanisms and its self-induced over expression appears to be a central key in the physiopathology of granulomatosis with polyangiitis.
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Molecular Characterization Of Capsid Protein And Nuclear Inclusion Protein Of Pepper Vein Banding VirusRoy, Anindya 12 1900 (has links) (PDF)
No description available.
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New activity-based probes to detect matrix metalloproteases / Nouvelles sondes d'affinité pour la détection de metallo proteases de la matriceKaminska, Monika 14 December 2018 (has links)
Les Métallo Protéases Matricielles (MMP) en tant qu'endopeptidases à zinc ont une large gamme de fonctions biologiques allant du remodelage tissulaire à la modulation de la réponse cellulaire. Une modification de leur activité protéolytique est souvent associée à de nombreux désordres biologiques. In vivo, ces protéases sont soumises à de nombreuses modifications post-traductionnelles. Elles sont sécrétées sous formes latentes à l'extérieur des cellules pour être ensuite transformées en forme fonctionnelles. Ces dernières sont ensuite inhibées par des inhibiteurs endogènes. En raison de leur sécrétion dans l’espace extra cellulaire, les MMP sous formes actives ont longtemps été considérées comme de simples ciseaux moléculaires capable de dégrader uniquement la matrice extracellulaire. Cependant, le remodelage tissulaire ne constitue pas la fonction unique et encore moins la fonction principale de ces enzymes. Elles peuvent en effet cliver une grande variété de substrats non matriciels et à ce titre sont impliquées dans la progression tumorale, l'immunité et l'inflammation. Pour ajouter une complexité supplémentaire à la biologie des MMP, il a été récemment montré que certaines MMP ont une localisation intracellulaire associée à des fonctions non protéolytiques. Ces observations, mais aussi celles montrant que ces protease participent à la progression de la maladie alors que d'autres ont une fonction protectrice, soulignent la nécessité de mieux documenter leur activation spatiale et temporelle dans divers contextes biologiques.Le profilage protéique basé sur l'activité vise à analyser l'état fonctionnel des protéines dans des échantillons biologiques complexes. À cette fin, des sondes basées sur l'activité (ABP), qui réagissent avec les enzymes en s’appuyant sur leur mécanisme catalytique, ont été développées pour la détection d’enzymes sous formes actives, notamment dans le cas des protéases à sérine et à cystéine. Une sonde basée sur l’activité (ABP) est classiquement composée : i) d’un groupement réactif conduisant à la modification covalente de résidus au sein du site actif de l’enzyme, ii) d’un motif de liaison imposant la sélectivité au groupement réactif et iii) d’un groupement rapporteur permettant la détection des enzymes ciblées. Cette approche ne s’applique toutefois pas aux MMP, pour lesquelles il n’existe pas de résidus nucléophiles conservés au sein du site actif. À cet égard, tous les ABP ciblant les MMP comportent un groupement photo activable qui, sous irradiation UV, favorise la formation du complexe covalent. De telles sondes photo sensibles ont permis de détecter les MMP sous leurs formes actives dans des tissus et des fluides, mais pas chez les animaux vivants au sein desquels l’étape de photo-activation ne peut être réalisé.Dans ce contexte, en nous appuyant sur un contexte structural favorable et en exploitant la chimie de l'acyl imidazole (LDAI) dirigée par un ligand, nous avons identifié une nouvelle série de sondes capables de modifier de manière covalente les MMP sans recourir à la photo-activation. Nous avons ainsi validé la capacité de ces sondes à marquer de manière sélective et efficace la MMP12 humaine in vitro et dans des protéomes complexes. Dans ce dernier cas, jusqu’à 50ng de hMMP12 correspondant à 0,05% du protéome total peuvent être détectés. Nous avons également déterminé l'identité de l’unique résidu modifié de façon covalente au sein du site actif de la hMMP-12 et vérifié que cette modification avait peu d'impact sur l’activité protéolytique de cette dernière. Nous avons démontré que cette approche permettait de détecter des MMP endogènes. Enfin, nous avons étendu cette stratégie de marquage à un panel plus large de MMP.En développant la première stratégie de marquage des formes actives de MMP «sans photo-activation», il semble maintenant possible d’envisager la détection de ces enzymes à la fois dans les protéomes complexes et in vivo. / Matrix MetalloProteases (MMPs) as zinc endopeptidases have a wide range of biological functions, and changes in their proteolytic activity underlie many biological disorders. Since their proteolytic activity has to be tightly controlled to prevent tissue destruction, theses proteases are subjected to numerous posttranslational modifications in vivo. They are secreted under latent forms outside of the cells, and are subsequently processed into their functional form that can be further inhibited by endogenous inhibitors. Due to their delineated area of activation, MMP active forms have long been considered for their unique ability to degrade extracellular substrates. However, turnover and breakdown of the extracellular matrix are neither the sole nor the main function of MMPs. These enzymes can indeed process a wide variety of non-matrix substrates and are involved in the regulation of multiple aspects of tumor progression, immunity and inflammation. To add further complexity to MMPs biology, some members within the family were recently reported to have intracellular localization associated to non-proteolytic functions. These observations but also those evidencing that some MMPs participate in disease progression while others have a protective function, stress the need to better document their spatial and temporal activation in various biological contexts.Activity-based protein profiling (ABPP) aims to analyze the functional state of proteins within complex biological samples. To this purpose, activity-based probes (ABPs) that react with enzymes in a mechanism-based manner have been successfully developed for the profiling of several enzymes including serine and cysteine proteases. A typical Activity-Based probe (ABP) is composed of i) a reactive warhead, which reacts in a covalent manner with enzyme active site residues, ii) a targeting moiety that imposes selectivity upon the reactive group and iii) a detectable group for subsequent analyses. This approach is not applicable to MMPs, which lack a targetable nucleophile involved in the catalysis. In this respect, all ABPs directed to MMPs are affinity-based probes (AfBPs) containing within their structure a photo cross-linking group that promotes the formation of a covalent complex upon UV-irradiation. Such photoactivatable probes have been successfully developed for the detection of MMPs under their active forms in fluids and tissue extracts, but not in living animals where the photo-activation step is not feasible.By relying on a favorable structural context and by exploiting the ligand-directed acyl imidazole (LDAI) chemistry, we have identified a novel series of AfBPs capable of covalently modifying matrix metalloproteases without making use of photo-activation. These active-site-directed probes whose structure was derived from that of a MMP12 selective inhibitor harbored a reactive acyl imidazole in their P3' position. They demonstrated their labelling specificity in vitro by covalently modifying a single Lysine residue within the MMP-12 S3' region. We also showed that these probes only targeted functional states of hMMP-12 and spared forms whose active site was occluded either by a synthetic or a natural inhibitor. We have validated the ability of these chemical probes to efficiently label human MMP12 in complex proteomes. In this case, down to 50 ng of hMMP12 corresponding to 0.05% of the whole proteome can be labelled and detected by in-gel fluorescence analysis. We demonstrated that this approach also allowed detecting endogenous MMPs secreted by stimulated-macrophages. In addition, by modifying the nature of the targeting moiety, we have extended this affinity-labeling approach to six other MMPs.By developing the first “photo activation-free” strategy to covalently modify active forms of MMPs, the unresolved proteomic profiling of native MMPs should be now accessible both in complex proteomes and in preclinical model in which MMPs are potential relevant targets.
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Engineering α-1 Proteinase Inhibitor to Target Neutrophil Serine Proteinase PR3Al-Arnawoot, Ahmed January 2020 (has links)
Activated neutrophils release a neutrophil serine proteinase (NSP) called Proteinase 3 (PR3). In granulomatosis with polyangiitis (GPA), an autoimmune vasculitis, enhanced PR3 release results in endothelial damage. Serine proteinase inhibitors (serpins) such as α-1 proteinase inhibitor (API) inhibit NSPs through the serpin’s reactive center loop (RCL). However, API is known to bind PR3 with a low specificity, compared to its main inhibitory target Human Neutrophil Elastase (HNE). The current treatment for GPA is immunosuppression, which leaves patients immunocompromised. Thus, the overall aim of this study was to engineer an API variant with a higher specificity to PR3 than HNE, which could serve as a possible novel therapeutic strategy for GPA.
We created an API expression library, hypervariable at RCL residues A355-I356-P357-M358-S359, and expressed it in a T7 bacteriophage display system. This phage library was then biopanned for PR3 binding. Two conditions were used for each round of biopanning: experimental, with PR3, and the negative control, without PR3. The library was biopanned for a total of five consecutive rounds, with the product of one screen serving as the starting material for the next. A bacterial mass lysate screen was also employed to further probe the library with PR3.
The phage-display and bacterial lysate screens resulted in the selection of two novel variants API-DA (D357/A358) and API-N (N359). Serpin-proteinase gel complexing assays indicated that API-N formed complex with PR3 similar to API-WT (wild-type), while API-DA was mainly cleaved as a substrate. There was no significant difference between the second order rate constants of API-N and API-WT reactions with PR3. Rate constants for API-DA binding to PR3 or for API-HNE reactions were not completed due to novel coronavirus (COVID-19) restrictions. However, this project successfully demonstrated the ability to screen a hypervariable API phage library with PR3, yielding two new novel API variants. / Thesis / Master of Science in Medical Sciences (MSMS) / When harmful substances enter our body such as bacteria or viruses, we have ways of protecting ourselves from them. One of those ways is through a cell called the neutrophil. This is an immune cell that can release “fighting tools” into our blood to combat the harm. Some of these tools are called proteins. One of those proteins is Proteinase 3. However, sometimes our neutrophils can be activated without the presence of viruses or bacteria by products made in our bodies called autoantibodies. When this happens, too many of the “fighting tool” Proteinase 3 is released leading to damage to the tubes or vessels that our blood flows through. This project aimed to find a new possible way to stop these extra fighting tools from doing harm to our body. We did this by creating a library of different proteins that can stop Proteinase 3 once it is released by the neutrophil.
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The Utilization of the Hmg2 Inducible Promoter to Genetically Engineer Parasite Resistance in TobaccoWinston, Eugenia Michele 25 April 2003 (has links)
The cyst nematode, Globodera tabacum tabacum Behrens, and the parasitic angiosperm, Egyptian broomrape, Orobanche aegyptiaca Pers., are obligate root parasites that cause severe yield and quality loss of many important crop hosts. Although these represent two diverse classes of parasites, they have significant similarities in the modes of parasitism and complex interactions with their hosts. Conventional control methods have had limited success in controlling these parasites. The overall objective of this research was to engineer resistance to the cyst nematode and Egyptian broomrape by expressing genes encoding parasite specific toxins under the control of parasite-responsive promoters using tobacco (Nicotiana tabacum L. cv. Xanthi). For nematode resistance, an anti-feeding strategy was employed utilizing the tomato proteinase inhibitor I (PI-I) gene as a nematode specific toxin. Transgenic tobacco plants were generated that expressed genes encoding an intracellarly retained or secreted form of tomato PI-I under the control of the nematode-inducible promoter, derived from tomato (Lycopersicon esculentum L.) Hmg2 gene. Our goals were to determine the effectiveness of local PI-I expression on nematode resistance and to determine if intracellular or extracellular PI-I deposition enhances resistance. Two constructs were generated that contained either the coding region of the tomato PI-I gene, lacking the signal sequence (EM1), or the coding region of PI-I including the signal sequence (EM2), fused to the nematode-responsive Hmg2 promoter. Transgenic PI-I plants were inoculated with G. t. tabacum cysts and evaluated for nematode interactions. Our results suggest that local expression of intercellular of PI-I significantly reduced cyst production when compared to the nontransformed controls. For broomrape resistance, a well characterized R/avr gene pair, the tobacco N resistance gene and the tobacco mosaic virus replicase (TMV) gene, was utilized to create novel gene-for-gene resistance via a N gene-mediated hypersensitive response (HR) to limit broomrape parasitism. The bean (Phaselous vulgaris L.) chalcone synthase 8 (CHS8) promoter has been characterized as a broomrape–responsive promoter. We introduced the CHS8:TMV replicase gene construct into tobacco plants that contains an endogenous N gene. Transgenic tobacco plants were inoculated with O. aegyptiaca seeds and monitored for parasite attachment and development. The expression of the TMV replicase leads to a significant reduction in broomrape parasitism. These genetic engineering strategies show promise in enhancing resistance to these destructive parasites. / Ph. D.
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