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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

The poetry of Stanley Kunitz

Davis, Cynthia Ann. January 1900 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1972. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
2

Purificação e caracterização de inibidores de proteases de veneno de Bitis gabonica rhinoceros com potencial farmacológico / Purification and characterization of proteases inhibitors of Bitis gabonica rhinoceros venom with pharmacological potencial

Fucase, Tamara Mieco 17 May 2016 (has links)
Os venenos de serpentes são complexas misturas de proteínas e peptídeos que apresentam uma variedade de atividades biológicas. Estudos apontam para uma rica diversidade de moléculas bioativas de baixa massa molecular nos venenos, como a crotamina, miotoxina A, peptídeos potenciadores de bradicinina (BPPs) inibidores do tipo Kunitz de serinopeptidases e tripeptídeos inibidores de metalopeptidases. O interesse nestas moléculas está relacionado ao potencial uso como agentes terapêuticos contra diversas patologias, como distúrbios da coagulação e modulação da atividade de metalopeptidases, moléculas estas envolvidas com tumorigenêse e outros processos patológicos como inflamação crônica e distúrbios neurológicos. O veneno da serpente Bitis gabonica rhinoceros provoca alterações fisiopatológicas como severa desordem na coagulação sanguínea e danos teciduais seguidos de necrose. No presente estudo foram isoladas e caracterizadas metalopeptidases e serinopeptidases, além de componentes de baixa massa molecular como inibidor do tipo Kunitz e BPPs. Estes peptídeos foram testados quanto a sua capacidade inibitória frente as peptidases endógenas e sequenciados por espectrometria de massa. Os nossos dados mostram que as peptidases isoladas degradam caseína e não tem atividade sobre colágeno. A serinopeptidase tem atividade β-fibrinogenolítica e o inibidor tipo Kunitz isolado apresenta maior capacidade de inibir a quimotripsina, com valor de Ki= 0,07 μM, mostrando-se um promissor substituto ao fármaco aprotinina. Este peptídeo apresentou também atividade citotóxica em células B16F10 e tênue atividade antimicrobiana. Dentre os BPPs identificados, o peptídeo que possui sequência não canônica apresentou a capacidade de potencializar a ação da bradicinina tanto em ensaio edematogênico quanto de inibição da atividade enzimática da enzima conversora de angiotensina. Esses resultados indicam o potencial de peptídeos de venenos animais para o desenvolvimento de novos agentes terapêuticos para o tratamento de enfermidades como hipertensão e distúrbios de coagulação. / Snake venoms are complex mixtures of proteins and peptides with a wide array of activities. Some studies point towards a vast diversity of low molecular mass bioactive molecules in venoms such as crotamine, myotoxin A, bradykinin potentiating peptides (BPPs), Kunitz type serine peptidase inhibitors and tripeptides inhibiting metallopeptidases. The interest on these molecules is related to their potential use as therapeutic drugs against several pathologies such as coagulation disturbs and modulation of the activity of metallopeptidases, involved in tumorigenesis and other disease like chronical inflammation and neurological disorders. The venom of Bitis gabonica rhinoceros promotes severe blood clotting disorders and tissular damages followed by necrosis. In the present study we isolated and characterized metallo and serine-peptidases, as well as as low molecular mass components such as Kunitz inhibitors and BPPs. Those peptides were assayed for their ability to inhibit the venom ednogenous peptidases and were sequenced by mass spectrometry. Our data indicate that the isolated peptidases hydrolyze casein, but not gelatin, indicating that they have no activity on collagen. The isolated serine protase has β-fibrinogenolytic activity and is not inhibited by the endogenous Kunitz peptide isolated from the venom. The Kunitzlike peptide inhibits preferentially chymotrypsin with a Ki of 0.07 μM and appears as a promising substitute for the commercial drug aprotinin. Among the three bradykinin potentiating peptides, two displayed non-canonical sequences, a fact that might represent an interesting field for new studies for the development of new anti-hypertensives. Although displaying mutations in highly conserved regions, the non-canonical BPP potentialized bradykinin in both edematogenic and angiotensin converting enzyme inhibition assays. These results indicate the potential of animal venom peptides for the development of new drugs against Diseases such as hypertension and coagulopathies.
3

Purificação e caracterização de inibidores de proteases de veneno de Bitis gabonica rhinoceros com potencial farmacológico / Purification and characterization of proteases inhibitors of Bitis gabonica rhinoceros venom with pharmacological potencial

Tamara Mieco Fucase 17 May 2016 (has links)
Os venenos de serpentes são complexas misturas de proteínas e peptídeos que apresentam uma variedade de atividades biológicas. Estudos apontam para uma rica diversidade de moléculas bioativas de baixa massa molecular nos venenos, como a crotamina, miotoxina A, peptídeos potenciadores de bradicinina (BPPs) inibidores do tipo Kunitz de serinopeptidases e tripeptídeos inibidores de metalopeptidases. O interesse nestas moléculas está relacionado ao potencial uso como agentes terapêuticos contra diversas patologias, como distúrbios da coagulação e modulação da atividade de metalopeptidases, moléculas estas envolvidas com tumorigenêse e outros processos patológicos como inflamação crônica e distúrbios neurológicos. O veneno da serpente Bitis gabonica rhinoceros provoca alterações fisiopatológicas como severa desordem na coagulação sanguínea e danos teciduais seguidos de necrose. No presente estudo foram isoladas e caracterizadas metalopeptidases e serinopeptidases, além de componentes de baixa massa molecular como inibidor do tipo Kunitz e BPPs. Estes peptídeos foram testados quanto a sua capacidade inibitória frente as peptidases endógenas e sequenciados por espectrometria de massa. Os nossos dados mostram que as peptidases isoladas degradam caseína e não tem atividade sobre colágeno. A serinopeptidase tem atividade β-fibrinogenolítica e o inibidor tipo Kunitz isolado apresenta maior capacidade de inibir a quimotripsina, com valor de Ki= 0,07 μM, mostrando-se um promissor substituto ao fármaco aprotinina. Este peptídeo apresentou também atividade citotóxica em células B16F10 e tênue atividade antimicrobiana. Dentre os BPPs identificados, o peptídeo que possui sequência não canônica apresentou a capacidade de potencializar a ação da bradicinina tanto em ensaio edematogênico quanto de inibição da atividade enzimática da enzima conversora de angiotensina. Esses resultados indicam o potencial de peptídeos de venenos animais para o desenvolvimento de novos agentes terapêuticos para o tratamento de enfermidades como hipertensão e distúrbios de coagulação. / Snake venoms are complex mixtures of proteins and peptides with a wide array of activities. Some studies point towards a vast diversity of low molecular mass bioactive molecules in venoms such as crotamine, myotoxin A, bradykinin potentiating peptides (BPPs), Kunitz type serine peptidase inhibitors and tripeptides inhibiting metallopeptidases. The interest on these molecules is related to their potential use as therapeutic drugs against several pathologies such as coagulation disturbs and modulation of the activity of metallopeptidases, involved in tumorigenesis and other disease like chronical inflammation and neurological disorders. The venom of Bitis gabonica rhinoceros promotes severe blood clotting disorders and tissular damages followed by necrosis. In the present study we isolated and characterized metallo and serine-peptidases, as well as as low molecular mass components such as Kunitz inhibitors and BPPs. Those peptides were assayed for their ability to inhibit the venom ednogenous peptidases and were sequenced by mass spectrometry. Our data indicate that the isolated peptidases hydrolyze casein, but not gelatin, indicating that they have no activity on collagen. The isolated serine protase has β-fibrinogenolytic activity and is not inhibited by the endogenous Kunitz peptide isolated from the venom. The Kunitzlike peptide inhibits preferentially chymotrypsin with a Ki of 0.07 μM and appears as a promising substitute for the commercial drug aprotinin. Among the three bradykinin potentiating peptides, two displayed non-canonical sequences, a fact that might represent an interesting field for new studies for the development of new anti-hypertensives. Although displaying mutations in highly conserved regions, the non-canonical BPP potentialized bradykinin in both edematogenic and angiotensin converting enzyme inhibition assays. These results indicate the potential of animal venom peptides for the development of new drugs against Diseases such as hypertension and coagulopathies.
4

An immunohistochemical study of the cortex in Alzheimers's disease

Bielby-Clarke, Keren Elizabeth January 2000 (has links)
No description available.
5

Hranljiva vrednost sirovog i termički obrađenog zrna soje u ishrani tovnih pilića u zavisnosti od nivoa tripsin inhibitora / Nutritive value of raw and heat treatedsoybean in broilers nutrition dependingof the level of tripsin inhibitor

Beuković Dejan 19 September 2014 (has links)
<p>Soja predstavlja jedan od osnovnih hraniva koje se koristi u ishrani pilića u tovu, za<br />zadovoljenje potreba u proteinima i esencijalnim aminokiselinama. Po ukupnom<br />sadržaju proteina i njihovoj biolo&scaron;koj vrednosti soja se svrstava u jedno od<br />najkvalitetnijih proteinskih hraniva, čija aminokiselinska struktura može potpuno da<br />zadovolji potrebe pilića u skoro svim esencijalnim aminokiselinama. Prisustvo<br />Kunitz tripsin inhibitora (KTI), i drugih termolabilnih antinutritivnih faktora<br />ograničava upotrebu soje uz obaveznu termičku obradu. Selekcijski napredak<br />omogućio je stvaranje novih sorti koje imaju niži nivo antinutritivnih faktora, među<br />kojima je i sorta &bdquo;Lana&ldquo;. Prema ostvarenim rezultatima upotreba soje sorte &bdquo;Lana&ldquo; u<br />sme&scaron;ama (grover i fini&scaron;er) za ishranu brojlera u koncentraciji od 30% dala je<br />značajno lo&scaron;ije proizvodne parametre u odnosu na termički tretiranu, ali i značajno<br />bolje u odnosu na termički neobrađenu sortu sa standardnim nivoom KTI.<br />Zabeležena ekonomska efikasnost i indeks cena u SL grupi je skoro isti kao i kod<br />grupa sa termičkim tretmanom. Najveća povr&scaron;ina i visina crevnih resica, kao i<br />dubina kripte je zabeležena u SL grupi. Konstatovana je statistički vrlo značajna<br />hipertrofija pankreasa i uvećanje jetre kod SS grupe ali i značajno uvećanje<br />pankreasa u SL grupi u odnosu na grupe sa termičkim tretmanom. Evidentna je i<br />značajno lo&scaron;ija svarljivost hranljivih materija (marker metoda, i metoda totalne<br />kolekcije) u SL i SS grupama nasuprot onih grupa koje su bile na termičkom<br />tretmanu. Kod upotrebe različitih nivoa sorte &bdquo;Lana&ldquo; u fini&scaron;er (peletiranim)<br />sme&scaron;ama za ishranu brojlera, kada su u pitanju proizvodni parametri, dobijene<br />vrednosti su zadovoljavajuće uz sporadična odstupanja u grupama SL14% i SL21%.<br />Takođe Indeks cena i ekonomske efikasnosti je vrlo približan kontrolnoj grupi.<br />Svarljivost u distalnom ileumu je bila značajno bolja u kontrolnoj i 7%SL grupi, za<br />razliku od proksimalnog ileuma gde ne postoji statistička značajnost. Svarljivost<br />azota u distalnom ileumu je bila značajno lo&scaron;ija u 21% SL grupi, dok u<br />proksimalnom ileumu nije bilo statistički značajnih razlika. Retencija azota je bila<br />značajno bolja u kontrolnoj i 7%SL grupi. Razmatrajući sve rezultate, upotreba soje<br />&bdquo;Lana&ldquo; u ishrani brojlera bez prethodne termičke obrade nije preporučljiva u ishrani<br />od 11 dana u koncentraciji od 30% , ali je moguća u peletiranim fini&scaron;er sme&scaron;ama u<br />nivou do 14% udela u sme&scaron;i.</p> / <p>Soybean is one of the basic feed used in the chickens diet for , to satisfy the protein<br />and essential amino acids needs. By total protein content and their biological value,<br />the soybean is classified as one of the best protein sources, whose amino acid<br />structure can fully meet the needs of chickens in almost all essential amino acids.<br />The presence of Kunitz trypsin inhibitor (KTI), and other thermolabile<br />antinutritional factors limiting the use of soy with necessary thermal processing.<br />Selection progress has enabled the creation of new varieties that have lower levels of<br />anti-nutritional factors, including the variety &quot;Lana&quot;. Our results showed that the use<br />of soybean variety &quot;Lana&quot; in mixtures (grower and finisher) for broilers at the<br />concentration of 30% gave a significantly lower production parameters, as compared<br />to heat-treated, but also a significant better, compared to the thermally non-treated<br />soybean variety with standard level of KTI . Noted Economic efficiency and the<br />Price index in the SL group was almost the same as in the group with thermal<br />treatment. Largest surface and height of the villi and crypt depth was observed in the<br />SL group. We found a statistically significant hypertrophy of the pancreas and<br />enlargement of the liver in the SS group and a significant increase in pancreatic at<br />SL group compared to the groups with thermal treatment. Evident is significantly<br />worse digestibility of nutrients (marker method , and the method of total collection )<br />in the SL and SS groups opposed to those groups that were on the thermal treatment.<br />When using a different levels of variety &quot; Lana&quot; in the finisher ( pelleted ) diets for<br />broilers , in terms of production parameters, values obtained were satisfactory with<br />sporadic deviations in groups and SL14% SL21% . Also index price and economic<br />efficiency is highly approximate to the control group. The digestibility at the distal<br />ileum was significantly better in the control and the SL 7%, group in contrast to the<br />proximal ileum, where is no statistical significance. The digestibility of nitrogen in<br />the distal ileum was significantly inferior to 21% SL group, and in the proximal<br />ileum, there is no statistically significant differences. Nitrogen retention was<br />significantly better control, and in 7%SL group. Considering all the results, the use<br />of variety &quot;Lana&quot; in diet for broilers without heat treatment is not recommended in<br />the grower mixture (from 11 day) at the concentration of 30%, but it is freely<br />available for use in pellet for of finisher mixture, at levels up to 14% of mixture.</p>
6

Purificação e caracterização bioquímica de um inibidor de tripsina de sementes de Cassia leiandra Benth. e avaliação de sua atividade inseticida contra Aedes aegypti (Diptera: Culicidae) / Purification and Biochemical Characterization of a Trypsin Inhibitor of Seeds Cassia leiandra Benth. and Evaluation of its Insecticide Activity Against Aedes aegypti (Diptera: Culicidae)

Dias, Lucas Pinheiro January 2016 (has links)
DIAS, Lucas Pinheiro. Purificação e caracterização bioquímica de um inibidor de tripsina de sementes de Cassia leiandra Benth. e avaliação de sua atividade inseticida contra Aedes aegypti (Diptera: Culicidae). 2016. 81 f. Tese (Doutorado em Bioquímica)- Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Weslayne Nunes de Sales (weslaynesales@ufc.br) on 2017-03-29T16:22:03Z No. of bitstreams: 1 2016_tese_lpdias.pdf: 1964581 bytes, checksum: 311be97d5f40d2773e56367b158412b4 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-03-29T17:07:47Z (GMT) No. of bitstreams: 1 2016_tese_lpdias.pdf: 1964581 bytes, checksum: 311be97d5f40d2773e56367b158412b4 (MD5) / Made available in DSpace on 2017-03-29T17:07:47Z (GMT). No. of bitstreams: 1 2016_tese_lpdias.pdf: 1964581 bytes, checksum: 311be97d5f40d2773e56367b158412b4 (MD5) Previous issue date: 2016 / The World Health Organization (WHO) considers dengue virus infection one of the most important public health problems. Aedes aegypti (Diptera: Culicidae), a blood-sucking insect that lives and cohabits with the human population in tropical and subtropical areas, is the principal vector responsible for dengue virus transmission [3]. Ae. aegypti is also the main transmitter of Chikungunya and Zika viruses. To eliminate dengue mosquitoes, chemical control with insecticides is widely used. However, prolonged and indiscriminate application of these compounds has favored the appearance of resistant insects. This issue has driven the search for natural compounds with insecticidal activity, and plants are potential sources of such constituents. This present study reports the purification and biochemical characterization of a trypsin inhibitor (ClTI) from the seeds of Cassia leiandra Benth. (Fabaceae, Caesalpinioideae), a plant species native to the Amazon rainforest. In addition, the ClTI action on Ae. aegypti midgut proteases and its effect on the development and survival of this mosquito are assessed. ClTI was purified by DEAE-Cellulose and trypsin-Sepharose 4B chromatography, with a 15.5-fold purification and 2.4% yield. ClTI is composed of one polypeptide chain of 19,484 Da, as revealed by ESI mass spectrometry, is non-glycosylated, and comprises 35% β-sheets, 14% β-turns, and 50% disordered structures. ClTI is an uncompetitive inhibitor of bovine trypsin (IC50 of 33.81 x 10-8 M), with a Ki of 6.25 x 10-8 M, and has an amino acid sequence similar to other inhibitors of the Kunitz-type family. ClTI was stable over a broad range of pH (2.2–10.0) and temperature (30–70 C), but DTT incubation caused a partial loss of inhibitory activity. ClTI (4.65 x 10-6 M) promoted 50% activity reduction of Ae. aegypti midgut proteases, showed a dose-dependent acute toxicity on Ae. aegypti 3rd instar larvae, with an LC50 of 2.28 x 10-2 M, and after ten days of exposure, caused a 24-h delay of larval development and 44% mortality. Our results are an important contribution to a better understanding of the insecticidal activity of protease inhibitors, suggesting that ClTI has biotechnological potential as an alternative strategy to control this multiple disease vector, which may be used alone or in combination with other insecticidal compounds to produce enhanced toxicity. / A Organização Mundial da Saúde considera a dengue como um dos mais importantes problemas de saúde pública. Aedes aegypti (Diptera: Culicidae), um inseto sugador de sangue que se desenvolve em áreas tropicais e subtropicais, é o principal vetor do vírus da dengue, além de ser um importante transmissor das viroses Zica e Chikungunya. O controle químico através de inseticidas é bastante usado no combate do Ae. aegypti, mas sua aplicação indiscriminada e prolongada tem favorecido o surgimento de insetos resistentes. Isso tem motivado a busca por compostos naturais com atividade inseticida e as plantas têm se sobressaído como potenciais fontes de tais substâncias. Este estudo teve como objetivo a purificação e caracterização bioquímica de um inibidor de tripsina, denominado ClTI, de sementes de Cassia leiandra Benth. (Fabaceae, Caesalpinoideae), uma espécie vegetal nativa da Amazônia. A ação do ClTI sobre proteases intestinais do Ae. aegypti e seus efeitos no desenvolvimento e sobrevivência desse inseto foram também investigados. O ClTI foi purificado por cromatografias em DEAE-Celulose e tripsina-Sepharose 4B, com índice de purificação de 15,5 vezes e rendimento proteico de 2,4%. O inibidor puro é composto de uma cadeia polipeptídica de 19.484 Da, como mostrado por espectrometria de massas com ionização por “electrospray” (ESI), não glicosilado e apresenta estrutura secundária constituída por 35% de folhas-β, 14% de β-voltas e 50% de estruturas desordenadas. O ClTI é um inibidor incompetitivo de tripsina bovina (CI50 de 33,81 x 10-8 M), com Ki de 6,25 x 10-8 M e sequência de aminoácidos similar às de outros inibidores da família Kunitz. ClTI se mostrou estável em ampla faixa de pH (2,2-10,0) e temperatura (30-70 C), mas a incubação com ditiotreitol causou perda parcial de sua atividade inibitória. In vitro, ClTI (4,65 x 10-6 M) promoveu redução de 50% na atividade das proteases intestinais de Ae. aegypti. In vivo, esse inibidor apresentou toxicidade aguda dose-dependente para larvas de 3 estágio de Ae. aegypti, com uma CL50 de 2,28 x 10-2 M. O tratamento crônico de Ae. aegypti com ClTI (1,54 x 10-5 M concentração final) não interferiu na eclosão dos ovos, mas retardou o desenvolvimento larval do inseto em 24 h e causou mortalidade de 44%. Os resultados obtidos contribuem para um melhor entendimento da atividade inseticida dos inibidores de proteases e sugerem que ClTI tem potencial biotecnológico para ser usado como estratégia alternativa no controle desse vetor de várias doenças, o qual pode ser usado sozinho ou em combinação com outros compostos inseticidas na perspectiva de potencializar a toxicidade
7

Exprese rekombinantního proteinu Kunitzova typu, potenciálního toxinu ze slinných žláz klíštěte \kur{Ixodes ricinus} / Expression of Kunitz-type recombinant protein, potential toxin from the salivary glands of the tick \kur{Ixodes ricinus}

CERMANOVÁ, Tereza January 2009 (has links)
Ticks feed on their vertebrate hosts for number of days, providing enough time for development of an effective immune response. To overcome this, tick saliva contains a complex mixture of active substances which mediate host defense mechanisms. Extremely important role at the tick-host interface is played by the protease inhibitors. In this work, we have focused on the protein named Ixocludin 2, a member of the Kunitz type/Bovine trypsin inhibitors of serine proteases, related also to the potassium channel blockers toxins. Three different expression systems (Escherichia coli, Chinese hamster ovary and Pichia pastoris) were tested to prepare an active recombinant Ixocludin 2, out of which, only bacterial system was in part successful.
8

In vitro study of the structure / function relationship of the proteins GASP-1 and GASP-2 : Involvement of the second Kunitz domain in the functional duality of the GASP proteins / Etude in vitro de la relation structure/ fonction des protéines GASP-1 et GASP-2 : Implication du second domaine kunitz dans la dualité fonctionnelle des protéines GASP

Al Mansi, Montasir 14 December 2018 (has links)
Les muscles squelettiques, responsables des mouvements volontaires tels que la locomotion ou le maintien de la posture, représentent environ 40% de la masse corporelle.Cette masse musculaire est maintenue par plusieurs voies de signalisation qui régulent entre autres l’équilibre entre la synthèse et la dégradation des protéines myofibrillaires. En ciblant la voie de signalisation Akt/mTOR, la myostatine est un régulateur négatif de la myogenèse. Elle inhibe la différentiation myogénique et le renouvellement cellulaire. Parmi les différents facteurs moléculaires extracellulaires qui régulent la myostatine, les protéines GASP (Growth and differentiation factor Associated Serum Protein) ont été décrites comme des antagonistes de son activité. L’Unité de Génétique Animale a développé plusieurs stratégies qui permettent d’appréhender les mécanismes moléculaires qui régissent le(s) rôle(s) des protéines GASP au cours du développement musculaire. Ainsi, la création de la lignée murine appelée surGasp-1-20 a permis de montrer que la surexpression de Gasp-1 entraîne un phénotype hypermusclé associé à une hypertrophie des myofibrilles. Une analyse de l’expression génique dans des myoblastes dérivés des cellules satellites montre une surexpression de la myostatine corrélant avec une absence d’hyperplasie chez les souris surGasp-1-20. Des études similaires actuellement en cours pour la protéine GASP-2 devraient permettre de préciser son rôle dans le contexte musculaire. Les protéines GASP sont également définies comme des inhibiteurs composés hétérotypiques caractérisés par plusieurs domaines inhibiteurs pouvant moduler l’activité de différentes protéases. Parmi ces différents domaines,le second domaine Kunitz de GASP-2 a été précédemment décrit comme pouvant inhiber la trypsine. Dans ce travail, nous avons pu montrer que les deux protéines entières conservent cette capacité d’inhibition. Nos résultats indiquent cependant que GASP-1 et GASP-2 présentent une différence de spécificité due à la composition du second domaine Kunitz et non à l’environnement moléculaire présent dans chacune des protéines. Enfin, nous proposons un modèle structural du second domaine Kunitz impliqué dans la dualité fonctionnelle dans l'inhibition anti-trypsine de GASP-1 et GASP-2. / Skeletal muscles, responsible for voluntary movements such as locomotion or posture maintenance, represent about 40% of body mass. This muscle mass is maintened by several signaling pathways that regulate , among other things, the balance between synthesis and degradation of myofibrillar proteins. By targeting the Akt/mTOR pathway, myostatin is anegative regulator of myogenesis. It inhibits myogenic differentiation and cell turnover. Among the various endogenous molecular factors that regulate myostatin, proteins GASP (Growth and differentiation factor Associated Serum Protein) have been described as antagonists of its activity. The Animal Genetics Unit has developed several strategies to understand themolecular mechanisms that govern the role (s) of GASP proteins during muscle development.Thus, the creation of the transgenic mouse line named surGasp-1-20 has shown that overexpression of Gasp-1 results in a hypermuscular phenotype associated with myofibril hypertrophy. An analysis of gene expression in myoblasts derived from satellite cells showed overexpression of myostatin correlating with an absence of hyperplasia in Gasp-1-20 mice.Similar studies currently underway for the protein GASP-2 should clarify its role in the muscular context. Proteins GASP are also defined as compound heterotypic inhibitors characterized by several inhibitory domains that can modulate the activity of different proteases. Among these different modules, the second Kunitz domain of GASP-2 was previously been described asable to inhibit trypsin. In this work, we have shown that the two whole proteins conserve this capacity of inhibition. However, our results indicate that GASP-1 and GASP-2 exhibit a difference in specificity due to the composition of the second Kunitz domain and not to the molecular environment present in each of the proteins. Finally, by modeling, we propose a structural model of the second Kunitz domain of GASP-1 and GASP-2 implicated in the antitrypsin inhibition specificity
9

Miraculinas de citrus sinensis: modelagem molecular de estruturas e predição funcional / Miraculins of citrus sinensis: molecular modeling of structures and functional prediction

CAETANO, Érica Renata Nogueira Sá 12 July 2018 (has links)
Submitted by Rosana Amâncio (rosana.amancio@ufcg.edu.br) on 2018-07-12T22:11:47Z No. of bitstreams: 1 ERICA RENATA NOGUEIRA SÁ CAETANO - DISSERTAÇÃO PPGCNBio 2016..pdf: 2506925 bytes, checksum: 2aee1b855d59914fe68902bb6ec5b3fe (MD5) / Made available in DSpace on 2018-07-12T22:11:47Z (GMT). No. of bitstreams: 1 ERICA RENATA NOGUEIRA SÁ CAETANO - DISSERTAÇÃO PPGCNBio 2016..pdf: 2506925 bytes, checksum: 2aee1b855d59914fe68902bb6ec5b3fe (MD5) Previous issue date: 2016-07-14 / CNPq / Miraculina é uma glicoproteína que possui uma incrível propriedade de converter o sabor amargo em doce. Como a miraculina não apresenta sabor algum e tem um baixo teor calórico, esta proteína pode ser usada como adoçantes direcionados para pacientes com doenças relacionadas ao consumo excessivo de açúcar. Estudos comprovaram que membros da família de proteínas miraculinas também possuem atividade de inibidor de tripsina do tipo Kunitz, atuando como agentes naturais de defesa da planta contra pragas e predadores. Diante disso, proteínas do tipo miraculina são de grande relevância para aplicações biotecnológicas. Esse estudo teve como objetivo geral realizar a caracterização estrutural e funcional comparativa de duas miraculinas de Citrus sinensis, por meio de modelagem e docking molecular. Modelos 3D foram gerados e validados para as miraculinas CsMir1 e CsMir4, tripsina de Acryrthosiphon pisum e para os receptores de sabor doce mT1R2 e T1R3 de Mus musculus. Modelos homodiméricos foram gerados para CsMir1 e CsMir4 e modelo heterodimérico foi gerado para mT1R2-T1R3. Estudos da atividade de inibidor de tripsina foram feitos para CsMir1 e CsMir4 por interação com tripsina. Para analisar a atividade de modificação de sabor doce, foi realizada a interação das miraculinas com o receptor mT1R2-T1R3. Como resultados, os modelos dos monômeros e dímeros criados foram considerados bons modelos, válidos e confiáveis, com representações muito próximas das estruturas nativas dessas proteínas. A miraculina CsMir1, na forma monomérica ligou-se a tripsina de A. pisum e na sua forma dimérica ligou-se ao receptor heterodimérico mT1R2-T1R3 através do domínio ATD da subunidade T1R2, entretanto o potencial para as atividades de inibição de proteases e de indução ou inibição a modificação de sabor amargo/azedo em doce é menor do que para a CsMir4. A miraculina CsMir4, na sua forma monomérica ligou-se a tripsina de A. pisum, possivelmente apresentando atividade de inibição de proteases. CsMir4, na sua forma dimérica, ligou-se ao receptor heterodimérico mT1R2-T1R3, através do domínio ATD da subunidade T1R2, potencialmente apresentando atividade de indução ou inibição a modificação de sabor amargo/azedo em doce em M. musculus. / Miraculins are glycoproteins that displays a remarkable property in bitter to sweet taste conversion. As miraculin does not have any taste and has a low calorie, this protein can be used as sweeteners targeted to patients with diseases related to excessive sugar consumption. Studies have shown that members of miraculins protein family also display inhibitor activity against the Kunitz trypsin, acting as natural agents of plant defense against pests and predators. In this context, miraculin proteins are of great relevance for biotechnological applications. The aim of this research was to characterize structurally and functionally two miraculins of Citrus sinensis using in silico tools. Tridimensional models were built and validated for CsMir1 and CsMir4 miraculins, Acryrthosiphon pisum trypsin and for Mus musculus mT1R2-T1R3 receptor. Homodimeric and hetrodimeric models were generated for miraculins (CsMir1, CsMir4) and mT1R2-T1R3, respectively. Molecular docking simulations were performed to investigate the trypsin inhibitory activity and taste conversion activity of CsMir1 and CsMir4. The results showed that the predicted models were reliable and presented good quality parameters. The monomeric CsMir1 miraculin bound to A. pisum trypsin, while its dimeric form bound to ATD domain of the mT1R2-T1R3, although its potential as trypsin inhibitor and bitter/sweet taste modifier were minor than that presented by its homologous CsMir4. The dimeric form of CsMir4 bound to mT1R2-T1R3 receptor in the ATD domain, which strongly suggests bitter/sweet taste modifier activity in M. musculus.
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Purifica??o, caracteriza??o e avalia??o da atividade antiproliferativa de um inibidor de quimotripsina tipo kunitz de semente de Eryhrina velutina

Lucena, Sheila Varela 19 February 2010 (has links)
Made available in DSpace on 2014-12-17T14:03:33Z (GMT). No. of bitstreams: 1 SheylaVL_DISSERT.pdf: 1154692 bytes, checksum: c69fe71ef1d6a77b98930d0442bc2f7e (MD5) Previous issue date: 2010-02-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 &#956;g/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer / Um inibidor de quimotripsina do tipo Kunitz foi purificado de sementes de Erythrina velutina por fracionamento com sulfato de am?nio, cromatografias de afinidade Tripsina-Sepharose, Quimotripsina-Sepharose e cromatografia de Fase Reversa C- 18 no sistema FPLC/AKTA. O inibidor, denominado de EvCI, apresentou uma massa molecular de 17 kDa, determinada por SDS-PAGE. A an?lise por eletroforese bidimensional (2D) revelou quatro isoinibidores (valores de pI: 4,42, 4,63, 4,83 e 5,06). Todos os isoinibidores apresentaram massa molecular de 17 kDa. A sequ?ncia aminoac?dica dos pept?deos oriundos da digest?o enzim?tica de EvCI analisada por MALDI-TOF-MS apresentou 100% de identidade com o inibidor de quimotripsina ECl de Erythrina variegata. EvCI inibiu competitivamente a atividade de quimotripsina com Ki de 4 x10-8 M, mas n?o inibiu tripsina, elastase pancre?tica, bromela?na ou papa?na. Contudo, inibiu elastase de neutr?filos em 35,91 %. A atividade inibit?ria de EvCI sobre quimotripsina foi est?vel em uma ampla faixa de pH e temperatura. Na presen?a de 100 mM de DTT por 120 min, o inibidor perdeu 50% de sua atividade. A citotoxicidade do inibidor foi avaliada nas linhagens de c?lulas HeLa, MDA, HepG2, K562 e PC3 ap?s exposi??o a concentra??es variando de 0,0005 a 200 &#956;g/mL, por 72 horas. O EvCl inibiu o crescimento celular de c?lulas HeLa com um IC 50 de 50 &#956;g/mL. Os resultados obtidos na avalia??o de indu??o de morte celular e efeitos sobre o ciclo celular em c?lulas HeLa, indicam que o principal efeito do inibidor ? a indu??o de parada do ciclo celular, sendo, portanto, citost?tico. Os dados sugerem que o EvCl pode ser um composto promissor para ser estudado no futuro como agente citost?tico na terapia antitumoral combinada

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