Made available in DSpace on 2014-12-17T15:38:50Z (GMT). No. of bitstreams: 1 Silvia RSMP.pdf: 485662 bytes, checksum: c0e1bb8853ddea493b71396d1c9a5b2a (MD5) Previous issue date: 2004-04-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Cancer goes on to be a frightening disease by humanity, simetimes,it is considered as death, suffering and stigma synonym. Occurring at childhood, this meaning seems to acquire a more intense conotation, having in view of the perplexity and godliness feeling in the presence of the precocity of events, nearly always associated to the death. A psychologist co-existence with the cancer children is going acquiring, thus, a permeated sense by incognitas , fears and fantasy, which raised us the following question: how does the psychologist that answers children with cancer lives this experience? Therefore, the aim this research was to understand this co-existence experience. Our theoretical perspective comes from an existencial fenomenology and, more specifically, the Humanistic Approach and Martin Heidegger Existencial Ontology. The metodology is qualitative of phenomenological character. The access instrument to the experience was the narrative, such as purpose by Walter Benjamin. They were carried out nine semi-open interviews with psychologists who work on pediatric oncology services of Natal-RN city. Such interviews were recorded in cassette, transcripted and later, re-educated. These interviews were recorded, transcribed and later on edited with the help of the interviewee and turned into a text. The narrative comprehension was carried out on Heidegger Existencial Ontology, on dada exaustive reading and the clipping of indicative passages of experience sense of being psychologist on this area. The research suggests that the experience is oriented of clinic kowing-doing, being crossed by implications of key thematics which indicate the care as central ontologic element that orientates the way as these professionals come being in the world in association with the client?le. Besides, the caring experience of these children acquire the sense of true living experience, since the cancer undoes the immortality illusion, launching the psychologist to his/her condition of being to the death and with that, calling him/her the authenticity. Is is only not dealt with to experience the anguish and the death imminence, but above all, re-meaning them in favour of a continual learning, of quality answering , besides other possibilities. Working with child cancer brings news perspectives and world views, making the psychologist a more human people and sensitive to the distracted needs. And we believe that, regardless of area which actuates, being psychologist is a particular way which choose to be citizen. Is is a project that will be delimited by society, history and culture and after all, by us like human being. Therefore, we understand that the results this research suggest the discussed thematic deepening on this intervention field in order to new sense possibilities can arise giving origin to other reflections about the clinical practice, the professional formation in Psychology and other possible developments / O c?ncer continua a ser uma doen?a temida pela humanidade; n?o raro, ? considerado como sin?nimo de morte, sofrimento e estigma. Ocorrendo na inf?ncia, esse significado parece adquirir uma conota??o ainda mais intensa, tendo em vista o sentimento de piedade e perplexidade dos adultos diante da precocidade do evento quase sempre associado ? morte. A co-exist?ncia do psic?logo com a crian?a portadora de c?ncer,vai adquirindo, assim, um sentido permeado por inc?gnitas, medos e fantasias, que nos suscitou o seguinte questionamento: como o psic?logo que atende crian?as com c?ncer vivencia esta experi?ncia? O objetivo desta pesquisa foi, portanto, compreender esta experi?ncia de co-exist?ncia. Nossa perspectiva te?rica adv?m da fenomenologia existencial e, mais especificamente, da Abordagem Centrada na Pessoa e da Ontologia Existencial de Martin Heidegger. A metodologia ? qualitativa, de car?ter fenomenol?gico. O instrumento de acesso ? experi?ncia foi a narrativa, tal como proposta por Walter Benjamin. Foram realizadas nove entrevistas semi-abertas com psic?logas que trabalham nos servi?os de oncologia pedi?trica da cidade do Natal-RN. Tais entrevistas foram gravadas em fitas cassete, transcritas, e posteriormente, literalizadas. A compreens?o das narrativas foi realizada com base na ontologia heideggeriana, na leitura exaustiva dos dados e no recorte de trechos indicativos do sentido da experi?ncia de ser psic?logo nessa ?rea. A pesquisa sugere que a experi?ncia ? norteadora do saber-fazer cl?nico, sendo atravessada pelas implica??es de tem?ticas-chaves que indicam o cuidado como elemento ontol?gico central que orienta o modo como estes profissionais v?m-sendo-no-mundo, juntamente com sua clientela. Al?m disso, a experi?ncia de cuidar dessas crian?as adquire o sentido de verdadeira li??o de vida, uma vez que o c?ncer desfaz a ilus?o de imortalidade, lan?ando o psic?logo ? sua condi??o de ser-para-a-morte, e com isso, convocando-o a autenticidade. N?o se trata apenas de vivenciar a ang?stia e a imin?ncia de morte, mas, sobretudo, de ressignific?-las em prol de um aprendizado cont?nuo, de atendimentos de qualidade, dentre outras possibilidades. Trabalhar com c?ncer infantil traz novas perspectivas e vis?es de mundo, fazendo do psic?logo uma pessoa mais humana e sens?vel ?s necessidades alheias. E acreditamos que independente da ?rea de atua??o, ser psic?logo ? uma maneira particular que escolhemos para ser cidad?o. ? um projeto que ser? delimitado pela sociedade, pela hist?ria e pela cultura, e enfim, por n?s enquanto seres-no-mundo. Por isso, entendemos que os resultados desta pesquisa sugerem o aprofundamento das tem?ticas discutidas neste campo de interven??o, a fim de novas possibilidades de sentido possam emergir, dando margem a outras reflex?es sobre a pr?tica cl?nica, a forma??o profissional em Psicologia, dentre outros poss?veis desdobramentos

Central infusion of the hexapeptide angiotensin IV (Ang IV) and its analogs have been demonstrated to markedly enhance memory retention and retrieval in rats using a range of learning and memory paradigms. This effect is mediated by the binding of the peptide to the specific binding site previously described as the AT4 receptor. The AT4 receptor has been isolated and identified as insulin-regulated aminopeptidase (IRAP), a type II transmembrane protein belonging to the M1 family of zinc-dependent aminopeptidases. Subsequently, AT4 receptor ligands, including Ang IV and its analogues and the unrelated peptide LVV-hemorphin-7, were demonstrated to be peptide inhibitors of IRAP. These findings suggest that AT4 ligands may exert their cognitive effects by inhibiting the catalytic activity of IRAP in the brain. Therefore, IRAP is an important target for the development of a new class of therapeutic agents for the treatment of memory loss. / To characterize IRAP at the molecular level and identify non-peptide inhibitors of IRAP for drug development, the aims of this study were to: 1) determine whether IRAP exists as a homodimer; 2) identify cysteine residue(s) involved in IRAP dimerization; 3) investigate the roles of the conserved residues of the HEXXH(X)18E Zn2+-binding motif and the GAMEN motif in substrate/inhibitor binding using site-directed mutagenesis; 4) use a molecular model of the catalytic domain of IRAP based on the crystal structure of a related M1 family metallopeptidase to: (i) identify key residues required for substrate/inhibitor binding; (ii) identify and characterize non-peptide IRAP inhibitors from a compound database by in silico virtual screening based on the homology model of IRAP. / Co-immunoprecipitation followed by Western blotting of IRAP under reducing and non-reducing conditions showed IRAP exists both as covalently- and non-covalently-bound homodimers. Serine scanning of cysteine residues potentially involved in forming inter-molecule disulfide-bonds was performed. Mutational analyses indicated that covalent homodimerization of IRAP is due to more than one cysteine residue. Limited trypsin digestion followed by co-immunoprecipitation suggests that non-covalent homodimerization of IRAP involves residues/regions within the last 130 amino acids of the protein. / The catalytic site of IRAP contains two consensus motifs, the H464EXXH468(X)18E487 Zn2+-binding motif and the G428AMEN432 motif. The role of conserved residues with these motifs was investigated using site-directed mutagenesis and pharmacological analyses. The conserved His and Glu residues of the Zn2+-binding motif were shown to be essential for IRAP catalytic activity. This was also observed for the Met and Glu residues of the GAMEN motif, while Asn mutant retained some catalytic activity. Residues important for substrate or inhibitor binding were identified as Gly, Ala and Asn. / A molecular model of the catalytic domain of IRAP based on the crystal structure of a homologous M1 metallopeptidase, leukotriene A4 hydrolase (LTA4H) was used to compare the catalytic sites of IRAP and LTA4H, and identified two amino acids at the putative substrate-binding pocket: Ala427 and Leu483 in IRAP, and the corresponding residues Tyr267 and Phe314 in LTA4H. A mutational analysis involving substitution of Ala427 and Leu483 with the corresponding residues revealed Ala427 and Leu483 characterize the enzyme S1 subsite, influencing the affinity and placement of substrates and peptide inhibitors in the catalytic site. / The molecular model of IRAP was also used for virtual screening of compound databases to identify novel non-peptide inhibitors. After two rounds of in silico screening, a family of compounds was identified and shown to be specific and competitive inhibitors of IRAP. Preliminary results suggest that one of these inhibitors, referred to as HFI 142, may possess memory-enhancing properties. The identification of non-peptide IRAP inhibitors will assist in pharmacological studies aimed at understanding the molecular mechanisms of IRAP aminopeptidase activity and physiological role of IRAP. In addition, the new inhibitors have the potential to form the basis for the development of a novel class of drugs useful for treating memory disorders.

No description available.

Central infusion of the hexapeptide angiotensin IV (Ang IV) and its analogs have been demonstrated to markedly enhance memory retention and retrieval in rats using a range of learning and memory paradigms. This effect is mediated by the binding of the peptide to the specific binding site previously described as the AT4 receptor. The AT4 receptor has been isolated and identified as insulin-regulated aminopeptidase (IRAP), a type II transmembrane protein belonging to the M1 family of zinc-dependent aminopeptidases. Subsequently, AT4 receptor ligands, including Ang IV and its analogues and the unrelated peptide LVV-hemorphin-7, were demonstrated to be peptide inhibitors of IRAP. These findings suggest that AT4 ligands may exert their cognitive effects by inhibiting the catalytic activity of IRAP in the brain. Therefore, IRAP is an important target for the development of a new class of therapeutic agents for the treatment of memory loss. / To characterize IRAP at the molecular level and identify non-peptide inhibitors of IRAP for drug development, the aims of this study were to: 1) determine whether IRAP exists as a homodimer; 2) identify cysteine residue(s) involved in IRAP dimerization; 3) investigate the roles of the conserved residues of the HEXXH(X)18E Zn2+-binding motif and the GAMEN motif in substrate/inhibitor binding using site-directed mutagenesis; 4) use a molecular model of the catalytic domain of IRAP based on the crystal structure of a related M1 family metallopeptidase to: (i) identify key residues required for substrate/inhibitor binding; (ii) identify and characterize non-peptide IRAP inhibitors from a compound database by in silico virtual screening based on the homology model of IRAP. / Co-immunoprecipitation followed by Western blotting of IRAP under reducing and non-reducing conditions showed IRAP exists both as covalently- and non-covalently-bound homodimers. Serine scanning of cysteine residues potentially involved in forming inter-molecule disulfide-bonds was performed. Mutational analyses indicated that covalent homodimerization of IRAP is due to more than one cysteine residue. Limited trypsin digestion followed by co-immunoprecipitation suggests that non-covalent homodimerization of IRAP involves residues/regions within the last 130 amino acids of the protein. / The catalytic site of IRAP contains two consensus motifs, the H464EXXH468(X)18E487 Zn2+-binding motif and the G428AMEN432 motif. The role of conserved residues with these motifs was investigated using site-directed mutagenesis and pharmacological analyses. The conserved His and Glu residues of the Zn2+-binding motif were shown to be essential for IRAP catalytic activity. This was also observed for the Met and Glu residues of the GAMEN motif, while Asn mutant retained some catalytic activity. Residues important for substrate or inhibitor binding were identified as Gly, Ala and Asn. / A molecular model of the catalytic domain of IRAP based on the crystal structure of a homologous M1 metallopeptidase, leukotriene A4 hydrolase (LTA4H) was used to compare the catalytic sites of IRAP and LTA4H, and identified two amino acids at the putative substrate-binding pocket: Ala427 and Leu483 in IRAP, and the corresponding residues Tyr267 and Phe314 in LTA4H. A mutational analysis involving substitution of Ala427 and Leu483 with the corresponding residues revealed Ala427 and Leu483 characterize the enzyme S1 subsite, influencing the affinity and placement of substrates and peptide inhibitors in the catalytic site. / The molecular model of IRAP was also used for virtual screening of compound databases to identify novel non-peptide inhibitors. After two rounds of in silico screening, a family of compounds was identified and shown to be specific and competitive inhibitors of IRAP. Preliminary results suggest that one of these inhibitors, referred to as HFI 142, may possess memory-enhancing properties. The identification of non-peptide IRAP inhibitors will assist in pharmacological studies aimed at understanding the molecular mechanisms of IRAP aminopeptidase activity and physiological role of IRAP. In addition, the new inhibitors have the potential to form the basis for the development of a novel class of drugs useful for treating memory disorders.

Made available in DSpace on 2014-12-17T15:12:09Z (GMT). No. of bitstreams: 1 DaxFMPN_TESE.pdf: 2164638 bytes, checksum: ccd3b7aedb84ae6ada999a52ddec9d0b (MD5) Previous issue date: 2011-09-30 / Common understanding about what freedom means has always been more or less related to the power to realize something intended, desired, a capability. Therefore, being free is commonly interpreted under the concept of free-will and the category of possibility to act. Although there are predecessors in History of Philosophy, Schopenhauer refuses the thesis of free will proposing otherwise the denial of willing (to live) as the ultimate possibility for human freedom, if not the only one left. The thesis that would make him famous was deeply misunderstood and so miscarried somewhat due to the way it was many times presented by the means of exotic examples wrapped in a mystical mood besides exaltations to Eastern traditions, which may satisfy anthropological curiosity instead of being capable to satisfy the reader in a philosophical way. It seems to result from Schopenhauer s thought a kind of pessimism against life. Otherwise, typical readings on the Schopenhauerian thesis are found full of inconsistencies once closely regarded, which blame does not belong to the author but to his interpreters. A new reading about the denial of willing as the ultimate possibility for human freedom demands a criticism on the inconsistencies and prejudgments deep grounded. For this, we firstly clarify the ways of understanding the willing nothing , which cannot be reduced to the mere refusal or conformism, being instead positively understood as a special manner of willing: the admission of oneself for the sake of one is. A few more than a century later The world as will and representation came to light, Heidegger proposes in his fundamental ontology that the proper being-free concerns to originary decision by which, in anguish of being suspended in nothingness, Dasein renders itself singular as the being who is in-a-world and to-death, concluding that the ultimate possibility of freedom is being-free-to-death. Developing the hypothesis that freedom, properly understood, concerns to nothingness as to indeterminate possibilities, we seek for a dialogue between Schopenhauer s thought and existential philosophy aiming to reconstitute and overcome Metaphysics tradition turning the question about freedom into a matter of Ontology. From the factual existence perspective, as we must show, every human activity (or inactivity) is ordinarily mediated by representations, in which me and world appear as distinct entities. So, each one among determininate individuals finds itself connected to the things in the world by interest, which proper concept must be sufficiently explored. Starting from this point, we may proceed to detailed analysis of usual representations of freedom aiming their destruction by Ontology and then reaching existential thesis according to Kierkegaard and Heidegger. Turning back to the analysis of Schopenhauer s work, we conclude existential understanding of freedom as will-to-be can also be found in Schopenhauer. In this way, denial of willing means ultimate freedom once the Will turns back to its own essence by suppressing the world as representation, which means the originary absolute indetermination of the extreme possibility to-be / A compreens?o usual de liberdade sempre esteve mais ou menos vinculada ao poder de efetiva??o ou realiza??o, de uma inten??o, de um desejo, de uma capacidade. Assim, ser livre ? comumente interpretado ? luz do conceito de livre-arb?trio e sob a categoria da possibilidade de agir. Embora n?o sem precedentes na Hist?ria da Filosofia, Schopenhauer, refutando a tese do livre-arb?trio, prop?e a nega??o da vontade (de viver) como possibilidade m?xima, se n?o ?nica, da liberdade humana. A tese que o deixou famoso foi, contudo, profundamente mal compreendida e mesmo mal recebida um tanto gra?as ? pr?pria forma como ? apresentada, por meio de exemplos muitas vezes ex?ticos envoltos em ares de misticismo e exalta??es a tradi??es orientais que, incapazes de satisfazer filosoficamente o leitor, s?o antes curiosidades antropol?gicas. O saldo final do pensamento schopenhaueriano parece ser um pessimismo inimigo da vida. No entanto, examinada de perto, a leitura t?pica da tese schopenhaueriana se mostra repleta de inconsist?ncias que, deve-se mostrar, n?o pertencem ao autor, mas a seus int?rpretes. Uma nova leitura sobre a nega??o da vontade como possibilidade m?xima da liberdade humana exige uma cr?tica das inconsist?ncias e preconceitos j? enraizados. Para tanto, em primeiro lugar, elucida-se as maneiras de se compreender o nada querer , que n?o se reduz ? mera recusa ou ao conformismo, podendo ser positivamente interpretado como um modo especial de querer: a admiss?o de si mesmo pelo que se ?. Pouco mais de um s?culo ap?s vir ? luz O mundo como vontade e representa??o, Heidegger prop?e em sua ontologia fundamental que o serlivre pr?prio diz respeito ? decis?o origin?ria pela qual, na ang?stia da suspens?o no nada, o Dasein singulariza-se como o ente que ? em-um-mundo e para-a-morte, concluindo que a possibilidade extrema da liberdade ? ser-livre-para-a-morte. Desenvolvendo a hip?tese de que a liberdade, propriamente compreendida, ? pertinente ao nada e a possibilidades indeterminadas, busca-se um di?logo entre o pensamento de Schopenhauer e a filosofia existencial em um movimento de reconstitui??o e supera??o da tradi??o metaf?sica por meio de que o problema da liberdade converte-se em uma quest?o de Ontologia. Do ponto de vista da exist?ncia de fato , conforme se mostra em seguida, toda atividade (ou inatividade) humana ? ordinariamente mediada por representa??es, segundo as quais eu e mundo aparecem como entidades distintas, encontrando-se cada indiv?duo dado ligado ?s coisas do mundo pelo interesse, cujo conceito adequado deve ser suficientemente explorado. Partindo-se desta base, procede-se ao exame suficientemente pormenorizado das representa??es usuais da liberdade em vista de sua destrui??o pela Ontologia, atingindo-se, enfim, a proposta existencial conforme as formula??es de Kierkegaard e Heidegger. Retomando a an?lise da obra de Schopenhauer, chega-se ao resultado de que a compreens?o da liberdade como querer-ser, peculiar ?s filosofias da exist?ncia, tamb?m se aplica ? filosofia de Schopenhauer. Nesse sentido, a nega??o da vontade corresponde ao m?ximo de liberdade na medida em que a Vontade, pela ruptura como o mundo como representa??o, retorna a si mesma naquilo que tem de mais essencial: a absoluta indetermin?ncia origin?ria da possibilidade extrema para-ser

The purpose of the study is to examine how the image of the TV series Skam was constructed in the Swedish press during the winter of 2016-2017, with a focus on how Skam’s importance for individuals and society was expressed in the texts. To analyze how the discourse of Skam was constructed in the Swedish press, a discourse theoretical perspective based on Ernesto Laclau and Chantal Mouffe’s discourse theory was used, along with various theories of the media, the audience's production of meaning and pleasure, realism and psychology of religion. Different discursive tools from Laclau and Mouffe’s conceptual world are put into practice during the analysis part. Metaphors are also used as a method to understand and analyze the material.  In the analysis of the media discourse of Skam, four main themes emerged in the analyzed texts; Realism, identification, moralism and Norway and the Norwegian language. The discourse of realism raised the question whether Skam is realistic or not, and whether it is possible to understand teenagers by watching the series. The discourse of identification included questions like; whether participants could identify with the characters and situations in Skam or not, often from a nostalgic point of view. The discourse of moralism focused much on the different characters in the series and how they acted, or were portrayed. Topics such as feminism and homosexuality were discussed, along with other things. The discourse of Norway and the Norwegian language was a lot about the Swedish relations with Norway and the Norwegian language spoken in Skam. The discourse was very homogenous and notably it concerned the phenomena that Swedes have begun to speak Norwegian, and Swedish people creates a closeness and understanding of Norway and the Norwegians through the tv-series.  Skam was also constructed as something sacred that brings people together, in discussions as well as in cultural identity. A great majority of the texts discussed however adults should watch the tv-series or not.

第一章導論。說明本文寫作之動機、研究範圍、研究方法及其限制。以及對於「主義 」的體認。 第二章趙素昂先生與 孫中山牛生二人生平及其主要政治思想之比較研究。 第三章三均主義與三民主義的時代背景及其思想浦源之比較研究。 第四章三均主義與三民主義的哲學基礎及思想體系之比較研究。 第五章三均主義與三民主義的實錢方案之比較研究。 第六章結論。以三均主義與三民主義之精神義及比較研究之結果（包含兩主義之思想 形態及評價），做為本論文之總結。

Uma elastase-2 foi recentemente descrita como a principal enzima formadora de angiotensina (Ang) II no perfusato do leito arterial mesentérico (LAM) isolado de rato. Investigamos a interação dessa elastase-2 do perfusato do LAM isolado de rato (E-2LAMR) com alguns substratos e inibidores de elastases-2 e de quimases formadoras de Ang II. Os precursores de Ang II, [Pro11-D-Ala12]-Ang I e substrato tetradecapeptídeo de renina (TDP), foram convertidos em Ang II pela E-2LAMR com eficiências catalíticas de 8,6 min-1mM-1 e 5,1 min-1mM-1, respectivamente, enquanto os substratos cromogênicos N-succinil-Ala-Ala-Pro-Leu-p-nitroanilida e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida foram hidrolisados pela enzima com eficiências catalíticas de 10,6 min-1mM-1 e 7,6 min-1mM-1, respectivamente. O inibidor peptídico CH 5450 inibiu as atividades da E-2LAMR sobre os substratos Ang I (IC50=49 mM) e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida (IC50=4,8 mM), enquanto Acetil-Ala-Ala-Pro-Leu-clorometilcetona (Ac-AAPL-CK), um efetivo inibidor de elastases-2 pancreáticas, bloqueou eficientemente a atividade formadora de Ang II da E-2LAMR (IC50=4,5 mM). Em conjunto, esses dados confirmaram e estenderam as similaridades enzimológicas entre elastases-2 pancreáticas e a E-2LAMR. Além disso, a interação até então desconhecida da E-2LAMR com [Pro11-D-Ala12]-Ang I e CH 5450, ambos considerados como reagentes seletivos para quimases, sugere que as evidências para a formação de Ang II in vivo por quimases podem ter sido superestimadas em investigações prévias sobre vias geradoras de Ang II. Experimentos realizados com o LAM isolado de rato analisando o efeito vasoconstritor de Ang II, Ang I, TDP e [Pro11-D-Ala12]-Ang I mostraram a existência de uma via geradora de Ang II independente da ECA, a qual é sensível à quimostatina e Ac-AAPL-CK. Entre os possíveis candidatos para essa via alternativa à ECA aparece a E-2LAMR, uma enzima que não é inibida por captopril e que é sensível à quimostatina e Ac-AAPL-CK. Embora quimases, que também são sensíveis à quimostatina, também possam ser candidatos a essa via independente da ECA, com base nos fatos de que a quimase I de rato tem uma atividade predominante de degradação da Ang II e que não existem relatos na literatura de que quimases sejam sensíveis ao inibidor Ac-AAPL-CK, esses dados em conjunto sugerem um possível papel para a E-2LAMR, mas não quimases, como uma via alternativa à ECA para a geração de Ang II no LAM isolado de rato. A clonagem e o seqüenciamento do cDNA para a E-2LAMR foram alcançados pela combinação de transcrição reversa e reação da polimerase em cadeia. A seqüência do cDNA mostrou-se idêntica à do cDNA para a elastase-2 pancreática de rato; o cDNA tem 909 nucleotídeos mais uma cauda poli (A) e codifica uma preproenzima de 271 amino ácidos. A análise dos supostos amino ácidos no sítio de ligação da Ang I revelou características que poderiam explicar a atividade do tipo carboxidipeptidase necessária para a eficiente conversão de Ang I em Ang II. Adicionalmente, a seqüência revela características estruturais que poderiam contribuir para a ausência de atividade dessa enzima sobre a Ang II. O RNAm para a E-2LAMR foi expresso em LAM, pâncreas, pulmão, coração, rim, fígado e baço, mas não em aorta de rato. Células endoteliais do LAM em cultura expressaram o RNAm para a E-2LAMR e sintetizaram a enzima. A localização intravascular dessa enzima e sua habilidade em formar Ang II e não clivar esse peptídeo indicam que ela poderia ter uma participação significativa como um agente formador de Ang II no sistema cardiovascular. Esses resultados também podem indicar que a E-2LAMR é expressa em vasos de resistência, mas não em vasos de condutância. / An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursors [Pro11-D-Ala12]-Ang I and renin substrate tetradecaptide (TDP) were converted into Ang II by the rat MAB elastase-2 with catalytic efficiencies of 8.6 min-1mM-1 and 5.1 min-1mM-1, respectively, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min-1mM-1 and 7.6 min-1mM-1, respectively. The noncleavable peptide inhibitor CH 5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50=49 mM) and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (IC50=4.8 mM), whereas N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK), an effective active site-directed inhibitor of pancreatic elastases-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC50=4.5 mM). Altogether, these data confirm and extend the enzymological similarities between pancreatic elastases-2 and their rat MAB counterpart. Moreover, the thus far unrealized interaction of rat MAB elastase-2 with [Pro11-D-Ala12]-Ang I and CH 5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways. Experiments carried out in the isolated rat MAB analyzing the vasoconstrictor effect of Ang II, Ang I, TDP, and [Pro11-D-Ala12]-Ang I showed the existence of an ACE-independent pathway for Ang II generation, which is sensitive to chymostatin and Ac-AAPL-CK. Among the possible candidates for this ACE-independent pathway is rat MAB elastase-2, an enzyme that is not inhibited by captopril, and that is sensitive to chymostatin and Ac-AAPL-CK. Although chymases, which are also chymostatin-sensitive enzymes, might also be other possible candidates for this ACE-independent pathway, based on the fact that rat chymase I has a predominant Ang II-degrading activity, and because there are no reports in the literature that chymases are sensitive to Ac-AAPL-CK, altogether these data suggest a possible role for rat MAB elastase-2, but not chymases, as an alternative pathway to ACE for Ang II generation in the isolated rat MAB. The cloning and sequencing of the cDNA for the rat MAB elastase-2 was accomplished by reverse transcription-polymerase chain reaction. The sequence of this cDNA was found identical to the sequence of the rat pancreatic elastase-2; the cDNA is 909 nucleotides in length plus a poly (A) tail and encodes a preproenzyme of 271 amino acids. Analysis of the putative amino acids in the extended Ang I binding site of the rat MAB elastase-2 reveals features that could explain the dipeptidyl carboxypeptidase-like activity required for efficient Ang I to Ang II conversion. Additionally, the sequence reveals structural features that could contribute to the lack of activity of this enzyme toward Ang II. Rat MAB elastase-2 mRNA was expressed in rat mesenteric arteries, pancreas, lung, heart, kidney, liver, and spleen but not in aorta. Cultured mesenteric endothelial cells expressed the mRNA for rat MAB elastase-2 and synthesized the enzyme itself. The intravascular localization of this enzyme and its ability to generate Ang II and not destroy this peptide indicate that it might play a role in the rat cardiovascular system as an Ang II-forming agent. These results may also indicate that rat MAB elastase-2 is expressed in resistance vessels but not in conduit vessels.

This study is an investigation of the importance of institutional goals as perceived by the faculty and students in Chung-ang University in Seoul, Korea. The purposes of this study are (1) to determine if significant differences exist between the perceptions of faculty and students as to the present and ideal institutional goals of the university, (2) to determine if significant differences exist between the perceptions of faculty and students of different colleges as to the present and ideal institutional goals of the university, and (3) to determine if significant differences exist between the present and ideal institutional goals as perceived by the faculty and students within each of the colleges of the university.

Uma elastase-2 foi recentemente descrita como a principal enzima formadora de angiotensina (Ang) II no perfusato do leito arterial mesentérico (LAM) isolado de rato. Investigamos a interação dessa elastase-2 do perfusato do LAM isolado de rato (E-2LAMR) com alguns substratos e inibidores de elastases-2 e de quimases formadoras de Ang II. Os precursores de Ang II, [Pro11-D-Ala12]-Ang I e substrato tetradecapeptídeo de renina (TDP), foram convertidos em Ang II pela E-2LAMR com eficiências catalíticas de 8,6 min-1mM-1 e 5,1 min-1mM-1, respectivamente, enquanto os substratos cromogênicos N-succinil-Ala-Ala-Pro-Leu-p-nitroanilida e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida foram hidrolisados pela enzima com eficiências catalíticas de 10,6 min-1mM-1 e 7,6 min-1mM-1, respectivamente. O inibidor peptídico CH 5450 inibiu as atividades da E-2LAMR sobre os substratos Ang I (IC50=49 mM) e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida (IC50=4,8 mM), enquanto Acetil-Ala-Ala-Pro-Leu-clorometilcetona (Ac-AAPL-CK), um efetivo inibidor de elastases-2 pancreáticas, bloqueou eficientemente a atividade formadora de Ang II da E-2LAMR (IC50=4,5 mM). Em conjunto, esses dados confirmaram e estenderam as similaridades enzimológicas entre elastases-2 pancreáticas e a E-2LAMR. Além disso, a interação até então desconhecida da E-2LAMR com [Pro11-D-Ala12]-Ang I e CH 5450, ambos considerados como reagentes seletivos para quimases, sugere que as evidências para a formação de Ang II in vivo por quimases podem ter sido superestimadas em investigações prévias sobre vias geradoras de Ang II. Experimentos realizados com o LAM isolado de rato analisando o efeito vasoconstritor de Ang II, Ang I, TDP e [Pro11-D-Ala12]-Ang I mostraram a existência de uma via geradora de Ang II independente da ECA, a qual é sensível à quimostatina e Ac-AAPL-CK. Entre os possíveis candidatos para essa via alternativa à ECA aparece a E-2LAMR, uma enzima que não é inibida por captopril e que é sensível à quimostatina e Ac-AAPL-CK. Embora quimases, que também são sensíveis à quimostatina, também possam ser candidatos a essa via independente da ECA, com base nos fatos de que a quimase I de rato tem uma atividade predominante de degradação da Ang II e que não existem relatos na literatura de que quimases sejam sensíveis ao inibidor Ac-AAPL-CK, esses dados em conjunto sugerem um possível papel para a E-2LAMR, mas não quimases, como uma via alternativa à ECA para a geração de Ang II no LAM isolado de rato. A clonagem e o seqüenciamento do cDNA para a E-2LAMR foram alcançados pela combinação de transcrição reversa e reação da polimerase em cadeia. A seqüência do cDNA mostrou-se idêntica à do cDNA para a elastase-2 pancreática de rato; o cDNA tem 909 nucleotídeos mais uma cauda poli (A) e codifica uma preproenzima de 271 amino ácidos. A análise dos supostos amino ácidos no sítio de ligação da Ang I revelou características que poderiam explicar a atividade do tipo carboxidipeptidase necessária para a eficiente conversão de Ang I em Ang II. Adicionalmente, a seqüência revela características estruturais que poderiam contribuir para a ausência de atividade dessa enzima sobre a Ang II. O RNAm para a E-2LAMR foi expresso em LAM, pâncreas, pulmão, coração, rim, fígado e baço, mas não em aorta de rato. Células endoteliais do LAM em cultura expressaram o RNAm para a E-2LAMR e sintetizaram a enzima. A localização intravascular dessa enzima e sua habilidade em formar Ang II e não clivar esse peptídeo indicam que ela poderia ter uma participação significativa como um agente formador de Ang II no sistema cardiovascular. Esses resultados também podem indicar que a E-2LAMR é expressa em vasos de resistência, mas não em vasos de condutância. / An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursors [Pro11-D-Ala12]-Ang I and renin substrate tetradecaptide (TDP) were converted into Ang II by the rat MAB elastase-2 with catalytic efficiencies of 8.6 min-1mM-1 and 5.1 min-1mM-1, respectively, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min-1mM-1 and 7.6 min-1mM-1, respectively. The noncleavable peptide inhibitor CH 5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50=49 mM) and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (IC50=4.8 mM), whereas N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK), an effective active site-directed inhibitor of pancreatic elastases-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC50=4.5 mM). Altogether, these data confirm and extend the enzymological similarities between pancreatic elastases-2 and their rat MAB counterpart. Moreover, the thus far unrealized interaction of rat MAB elastase-2 with [Pro11-D-Ala12]-Ang I and CH 5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways. Experiments carried out in the isolated rat MAB analyzing the vasoconstrictor effect of Ang II, Ang I, TDP, and [Pro11-D-Ala12]-Ang I showed the existence of an ACE-independent pathway for Ang II generation, which is sensitive to chymostatin and Ac-AAPL-CK. Among the possible candidates for this ACE-independent pathway is rat MAB elastase-2, an enzyme that is not inhibited by captopril, and that is sensitive to chymostatin and Ac-AAPL-CK. Although chymases, which are also chymostatin-sensitive enzymes, might also be other possible candidates for this ACE-independent pathway, based on the fact that rat chymase I has a predominant Ang II-degrading activity, and because there are no reports in the literature that chymases are sensitive to Ac-AAPL-CK, altogether these data suggest a possible role for rat MAB elastase-2, but not chymases, as an alternative pathway to ACE for Ang II generation in the isolated rat MAB. The cloning and sequencing of the cDNA for the rat MAB elastase-2 was accomplished by reverse transcription-polymerase chain reaction. The sequence of this cDNA was found identical to the sequence of the rat pancreatic elastase-2; the cDNA is 909 nucleotides in length plus a poly (A) tail and encodes a preproenzyme of 271 amino acids. Analysis of the putative amino acids in the extended Ang I binding site of the rat MAB elastase-2 reveals features that could explain the dipeptidyl carboxypeptidase-like activity required for efficient Ang I to Ang II conversion. Additionally, the sequence reveals structural features that could contribute to the lack of activity of this enzyme toward Ang II. Rat MAB elastase-2 mRNA was expressed in rat mesenteric arteries, pancreas, lung, heart, kidney, liver, and spleen but not in aorta. Cultured mesenteric endothelial cells expressed the mRNA for rat MAB elastase-2 and synthesized the enzyme itself. The intravascular localization of this enzyme and its ability to generate Ang II and not destroy this peptide indicate that it might play a role in the rat cardiovascular system as an Ang II-forming agent. These results may also indicate that rat MAB elastase-2 is expressed in resistance vessels but not in conduit vessels.