Дисертації з теми "SERPINH1"

Made available in DSpace on 2016-08-29T15:34:42Z (GMT). No. of bitstreams: 1 tese_8279_Tese_Clara Fernanda Barbirato.pdf: 794712 bytes, checksum: 13290894bcf3053215336989ea116592 (MD5) Previous issue date: 2015-02-13<br>A Osteogênese Imperfeita (OI) é uma doença clínica e geneticamente heterogênea caracterizada, predominantemente, por fragilidade e deformidade ósseas e por fraturas recorrentes. A maioria dos casos de OI resulta de mutações autossômicas dominantes nos genes COL1A1 e COL1A2, que codificam as cadeias formadoras do colágeno tipo I, principal proteína dos ossos. Nos últimos anos, um número crescente de casos decorrentes de mutações recessivas vem sendo relatado em genes associados à biossíntese do colágeno tipo I ou à formação e a mineralização óssea, como os genes LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 e SERPINF1. Mutações nesses genes, em geral, levam ao desenvolvimento de fenótipos graves e letais de OI. Neste trabalho, foram analisados os genes LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 e SERPINF1 de 25 pacientes com OI utilizando-se as técnicas de SSCP e sequenciamento. Ao todo, 29 variações genéticas foram detectadas, entre mutações e polimorfismos. Das onze variações encontradas no gene LEPRE1, estão a já bem descrita c.1080+1G>T e as mutações potencialmente deletérias c.2024G>A / p.Lys363Glu e c.1501C>T / p.Arg501Trp. No gene FKBP10, foi encontrada a também descrita duplicação c.831dupC, além da c.1546G>A / p.Leu516Phe, predita como causadora da doença. Observou-se que os genes FKBP10 e LEPRE1 contêm as principais mutações encontradas neste trabalho e sugere-se que os mesmos sejam preferencialmente analisados em estudos de triagem e identificação de mutações em OI. Até o momento, não existem relatos de mutações nos genes LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 e SERPINF1 em pacientes brasileiros e este trabalho fornece novas informações sobre os aspectos genéticos da OI recessiva<br>Osteogenesis Imperfecta (OI) is a clinically and genetically heterogeneous disease predominantly characterized by bone fragility and deformity and recurrent fractures. Most cases of OI result of autosomal dominant mutations in COL1A1 and COL1A2 genes that encode the chains forming type I collagen, the main protein in bones. In the past few years, an increasing number of cases due to recessive mutations has been reported in genes associated with the biosynthesis of type I collagen or to the formation and bone mineralization, such as LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 and SERPINF1. Mutations in these genes, in general, lead to the development of severe and lethal OI phenotypes. In this work, LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 and SERPINF1 of 25 OI patients were analyzed using SSCP and automated sequencing. Altogether, 29 genetic variations were detected, mutations and polymorphisms. Among the eleven variants found in LEPRE1 gene, there are the already well described c.1080 + 1G> T and the potentially deleterious mutations c.2024G> A / p.Lys363Glu and c.1501C> T / p.Arg501Trp . In FKBP10 gene, the previously described duplication c.831dupC, and c.1546G>A / p.Leu516Phe, predicted to be disease causing, were detected. It was observed that FKBP10 and LEPRE1 contain the most important mutations found in the patients studied in this work and it is suggested that LEPRE1 and FKBP10 should be preferably analyzed in studies of screening and identification of mutations in patients with OI. To date, there are no reports of mutations in LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 and SERPINF1 genes in Brazilian patients and this study provides new information on the genetic aspects of recessive OI.

Age related macular degeneration is the commonest cause of blindness in the western world and current treatment regimens represent a significant output for national health services. The disease process is multifactorial in origin and has a variable progression and response to current methods of treatment. A targeted approach with individualized therapy based on recognized biomarkers to predict disease outcome would be the ideal treatment modality. We plan to investigate the role of genes known to influence the progression of early AMD to the advanced stage (wet AMD) with emphasis on genes involved in complement regulation (SERPING1, CFB, CFI, CFH, C2 and C3). The investigation will also encompass other genotypes involved in AMD pathogenesis. Polymorphic variations in the gene SERPING1, which codes for complement 1 inhibitor (C1Inh), have been previously implicated in AMD pathogenesis. Many clinical trials involving complement antagonists are currently proceeding at various stages of development. We plan to investigate the role of C1Inh in AMD development and explore its potential as a therapeutic agent, utilising Ccl2-/-/Cx3cr1-/- (in the presence of an rd8 mutation in the Crb1 gene) and wild type C57BL/6 mice. In the neurodegenerative condition Alzheimer’s disease, acute or chronic and chronic systemic inflammation have been associated with progression of symptoms. Both chronic and acute inflammation have been implicated in choroidal angiogenesis and subsequent AMD development. We hypothesised that systemic inflammation may alter the response of AMD to the anti-vascular endothelial growth factor (VEGF) agent ranibizumab. Inflammatory markers may therefore offer an objective indicator of future treatment response. These experiments will provide a comprehensive analysis of prognostic indicators for AMD and investigate a potentially new therapeutic agent.

Les tiques sont des arthropodes ectoparasites obligatoires qui se nourrissent sur une grande variété de vertébrés sur une large partie du globe. Au cours de leur repas, les tiques sécrètent dans leur salive de nombreux facteurs leur permettant de contourner bon nombre des défenses de l’hôte. Bien que la littérature rapporte beaucoup d’informations au sujet des effets du repas de la tique sur l’hôte, la nature des facteurs actifs exprimés par les glandes salivaires de la tique est peu connue. Au cours d’anciens travaux au sein du laboratoire, le crible de deux banques d’ADN complémentaires - issues de la rétro-transcription des ARN messagers synthétisés par les glandes salivaires de la tique Ixodes ricinus – a permis l’identification de 27 protéines dont l'expression est spécifiquement induite ou régulée positivement pendant le repas sanguin de la tique I. ricinus. Parmi ces protéines, la protéine Seq24, induite au cours du repas sanguin, présente la capacité de moduler les immunités innée et acquise de l’hôte. En conséquence, la protéine Seq24 a été nommée Iris pour « Ixodes ricinus Immunosuppressor ». Au cours de la présente étude, notre but fût de caractériser le rôle d’Iris et de déterminer son importance dans le repas sanguin de la tique I. ricinus.<p>La protéine Iris appartient à la famille des inhibiteurs de sérine protéases et présente une homologie significative avec l’inhibiteur d’élastase de leucocytes. Une analyse in silico a confirmé qu’Iris présentait la structure des serpines, et notamment le RCL (Reactive Center Loop), boucle responsable de l’activité anti-protéasique. Comme attendu (sur base de l’analyse in silico), Iris inhibe de manière spécifique l’activité de plusieurs sérine protéases, et en particulier l’élastase de leucocyte. Ces tests effectués, nous avons essayé de comprendre quel(s) pouvai(en)t être le(s) rôle(s) d’Iris dans l’accomplissement du repas sanguin de la tique, c’est à dire dans la lutte contre les différents systèmes de défenses de l’hôte.<p>Tout d’abord, des tests ont démontré la capacité d’Iris à inhiber les mécanismes de l’hémostase. Des tests sur du plasma et du sang complet ont montré qu’Iris allonge le temps de fibrinolyse, la voie intrinsèque de la coagulation et l’adhésion plaquettaire. L’utilisation de mutants a également démontré que si les deux premières activités sont dépendantes du RCL, et donc d’un mode de fonctionnement anti-protéolytique, l’adhésion plaquettaire est indépendante de ce système. Ce résultat met en évidence l’existence d’autres sites actifs, isolés par analyse in silico, nommés Receptor Binding Domain (RBD).<p>Un travail antérieur du laboratoire avait permis d’indiquer la capacité de la protéine recombinante Iris semi-purifiée à inhiber la production de TNF-a, d’IL-6, et d’IL-8 (cytokines pro-inflammatoires) ainsi que l’IFN-g par des PBMCs (Peripherical Blood Mononuclear Cells) humaines. Ces résultats ont été confirmés avec de la protéine purifiée. Des analyses complémentaires ont démontré qu’un mutant d’Iris - dépourvu d’activité anti-protéasique - conserve l’activité pro-inflammatoire. Là encore, ce mécanisme semble impliquer un ou plusieurs RBD. L’utilisation d’anticorps dirigés contre ces zones a permis de déterminer le domaine d’interaction (aa :105-120) impliqué dans cette fonction. D’autre part, une analyse par FACS a permis de démontrer qu’Iris interagit uniquement avec les cellules d’origine monocytaire.<p>Enfin, nous avons également analysé l’importance d’Iris au cours du repas sanguin de la tique par une approche vaccinale. Les résultats observés indiquent que 30 % des tiques nourries sur des lapins immunisés par la protéine rIris ne survivent pas au repas.<br>Doctorat en sciences, Spécialisation biologie moléculaire<br>info:eu-repo/semantics/nonPublished

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1′) of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1′ glutamate (Glu379) with an arginine residue (Arg302) of the β-sheet C. A P1′ glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1′ residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin–protease interaction. Arg302 is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1′ Glu379, which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu379 and Arg302 influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.

Les inhibiteurs des protéases à sérine (Serpins) constituent une classe d'enzymes très peu étudiée chez les bactéries. Dans ce travail de thèse nous nous sommes intéressés à l'étude des serpins provenant du microbiote intestinal et l'investigation de leur potentiel anti-inflammatoire pour le traitement des maladies inflammatoires chroniques de l'intestin (MICI) chez l'homme. Pour cela nous avons identifié les serpins provenant du microbiote intestinal humain et analysé leur diversité ainsi que leur distribution entre les individus malades et sains. Ces données nous ont permis d'isoler les serpins significativement associées aux MICI. La purification de quarte d'entre elles nous a amené à démontrer qu'elles inhibent les protéases humaines impliquées dans les MICI. L'analyse biochimique et cinétique approfondie de ces protéines a montré qu'elles possèdent des propriétés originales notamment leur efficacité d'inhibition élevée. L'étude de l'effet protecteur de trois serpins chez un modèle animal de colite a démontré pour la première fois l'efficacité des serpins in vivo démontrant ainsi leur potentiel thérapeutique<br>Serine protease inhibitors (Serpins) are a class of proteins that reamin poorly studied in bacteria. In this thesis we are interested in the study of serpins originating from the intestinal microbiota and the investigation of their anti-inflammatory potential for the treatment of inflammatory bowel diseases (IBD) in humans. For this we have identified serpins from the human gut microbiota and analyzed their diversity as well as their distribution between healthy and IBD patients. These data allowed isolating serpins significantly associated with IBD. The purification of four of them led us to demonstrate that they inhibit human proteases involved in IBD. Biochemical and kinetic analysis of these proteins showed that they exhibit original properties, in particular their high inhibition efficiency. The study of the protective effect of three serpins in an animal model of colitis demonstrated for the first time the efficacy of serpins in vivo demonstrating thus their therapeutic potential

Orientador: Prof. Dr. Luciano Puzer<br>Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017.<br>As calicreinas teciduais humanas (KLKs) compreendem uma familia de quinze serino proteases encontradas em uma diversidade de fluidos e tecidos biologicos. Estas enzimas sao identificadas como possuindo papel em diferentes doencas como Alzheimer, cancer, dermatite atopica, esclerose multipla, Parkinson, psoriase e outras. Existe, portanto, uma crescente demanda por inibidores especificos para cada uma das calicreinas e este e o objetivo do nosso grupo de pesquisa na UFABC. Neste trabalho pretendemos gerar inibidores para as calicreinas teciduais humanas 3, 5 e 7, utilizando bibliotecas baseadas em duas serpinas diferentes: uma expressando a forma Pittsburgh do inibidor de proteinase-¿¿1 (IP-¿¿1 M358R), randomizada nos residuos 352-356 (P7-P3); e outra expressando a serpina bacteriana vioserpina, randomizada nos residuos 343-347 (P3-P2f). A abordagem do phage display foi eficaz para gerar as bibliotecas e o protocolo de bioselecao utilizado adequado para enriquecer diversas variantes reativas. Na selecao da biblioteca do IP-¿¿1 M358R, consensos PSEAL e PSRIL foram observados, para KLK5 e KLK7, respectivamente, e varias das sequencias selecionadas exibiram maiores taxas de inibicao para ambas as calicreinas, quando comparadas a molecula molde (IP-¿¿1 M358R). A variante HDVIL e o consenso PSRIL foram identificados como sendo altamente seletivos para a KLK7, com constantes de segunda ordem 14 e 33 vezes maiores que as para KLK5. Pudemos realizar uma selecao efetiva da biblioteca de vioserpina contra a KLK7, cujas variantes enriquecidos demonstraram uma preferencia geral pelo aminoacido Serina ocupando as posicoes P3, P1f, P2f e P1, seguido por uma Tirosina, tambem preferida em P2. A tecnica de phage display foi, portanto, eficiente como base para um estudo de especificidade, e para o desenvolvimento de melhores e mais especificos inibidores para as Calicreinas Teciduais Humanas, e pode ser utilizada para o desenvolvimento de novas bibliotecas, com outras regioes da RCL randomizadas, ou mesmo baseadas em outras serpinas.<br>The human tissue kallikreins (KLKs) comprise a family of fifteen serine proteases found in a diversity of biological fluids and tissues. These enzymes are identified as having a role in different diseases such as Alzheimer's, cancer, atopic dermatitis, multiple sclerosis, Parkinson's, psoriasis, and others. Thus there is a growing demand for specific inhibitors for each of these kallikreins, and this is the aim of our group at UFABC. In this work we intended to generate inhibitors for the human tissue kallikreins 3, 5 and 7, using libraries based on two different serpins: one expressing the Pittsburgh form of the human serpin á1-proteinase inhibitor (á1-PI M358R), randomized at residues 352-356 (P7-P3); and another one expressing the bacterial vioserpin, randomized at residues 343-347 (P3-P2¿). The phage display approach was effective to generate the libraries and the biopanning protocol used suitable to enrich numerous reactive variants. On the á1-PI M358R selection, loose consensus of PSEAL and PSRIL were observed, for KLK5 and KLK7, respectively, and several of the selected sequences exhibited higher inhibition rates when compared to the template molecule for both kallikreins. The variant HDVIL and consensus PSRIL were found to be highly selective for the KLK7, with second order constants 14- and 33-fold higher than the ones for KLK5. We could only perform an effective selection with the vioserpin library for the KLK7, whose enriched variants demonstrated a general preference for the amino acid Serine occupying the positions P3, P1¿, P2¿ and P1, followed by a Tyrosine, also preferred on the P2. The phage display approach was therefore effective as basis for a specificity study, and for the development of improved, more specific inhibitors for the Human Tissue Kallikreins, and can be used to develop new libraries, with other randomized RCL regions, or even based on other serpins.

L’hémophilie A est une maladie hémorragique rare caractérisée par un déficit du facteur de la coagulation VIII (FVIII). De nouvelles stratégies thérapeutiques ne reposant pas sur l’injection de FVIII se développent. Nous proposons une approche innovante qui consiste à cibler un anticoagulant naturel présent dans les plaquettes, la Protéase Nexine-1 (PN-1). La PN-1 est un inhibiteur puissant de la thrombine, enzyme clé de la coagulation. Bloquer la PN-1 favoriserait ainsi la génération de thrombine et donc la coagulation chez les patients hémophiles. Nous avons montré que bloquer la PN-1 améliore effectivement la génération de thrombine dans les plasmas de patients hémophiles. Nos données obtenues in vivo ont également montré une restauration de la coagulation et des temps de saignement chez des souris hémophiles déficientes en PN-1. L’ensemble de nos résultats indique que la PN-1 constitue une nouvelle cible thérapeutique intéressante pour le traitement de l’hémophilie.Le rôle potentiel de la PN-1 dans la régulation des mécanismes inflammatoires n’est pas connu. Au niveau des cellules inflammatoires, sa présence n’a été démontrée que dans les monocytes-macrophages et son expression n’a encore jamais été étudiée dans les polynucléaires neutrophiles. Nous avons ainsi mis en évidence pour la première fois la présence de PN-1 dans les neutrophiles et avons montré que le déficit en PN-1 limite le recrutement des neutrophiles in vivo. Ces travaux ont permis d’identifier la PN-1 comme un nouveau régulateur des mécanismes de recrutement des neutrophiles<br>Hemophilia is a disease caused by the lack of Factor VIII (FVIII), leading to insufficient thrombin production, and therefore bleeding. Recently, new therapeutic strategies for hemophilia treatment, that do not rely on clotting factor replacement, have emerged. We propose an innovative approach consisting in targeting a natural and potent thrombin inhibitor, expressed by platelets and called protease nexin-1 (PN-1). Our results demonstrated that blocking PN-1 increased, in vitro, thrombin generation in plasma from patients with hemophilia, and reduced blood loss in hemophiliac mice. Our study provides proof-of-concept that PN-1 neutralizing can be a a novel approach for future clinical care in hemophilia. The potential role of PN-1 in regulating inflammatory processes is not known. Vascular cells, platelets and inflammatory cells express PN-1, but no data are available concerning its expression and potential function in neutrophils. For the first time, we demonstrated the presence of PN-1 in neutrophils. Our data have shown that neutrophil recruitment was much less important in peritoneal cavity of PN-1-/- mice than in those of WT mice. These novel findings suggest that PN-1 is a serpin regulating positively PMNs functions

The adipokine vaspin (serpinA12) is mainly expressed in white adipose tissue and exhibits various beneficial effects on obesity-related processes. Kallikrein 7 is the only known target protease of vaspin and is inhibited by the classical serpin inhibitory mechanism involving a cleavage of the reactive center loop between P1 (M378) and P1′ (E379). Here, we present the X-ray structure of vaspin, cleaved between M378 and E379. We provide a comprehensive analysis of differences between the uncleaved and cleaved forms in the shutter, breach, and hinge regions with relation to common molecular features underlying the serpin inhibitory mode. Furthermore, we point out differences towards other serpins and provide novel data underlining the remarkable stability of vaspin. We speculate that the previously reported FKGx1Wx2x3 motif in the breach region may play a decisive role in determining the reactive center loop configuration in the native vaspin state and might contribute to the high thermostability of vaspin. Thus, this structure may provide a basis for future mutational studies.

Serpins are a unique breed of proteins due to their enzymatic mechanism. Two systems were closely monitored during fluorescent binding studies, the ACT-CHY along with the AT:TRY interaction. Four different conformational variants of each system were studied including the native, cleaved, latent and complex forms. Three different fluorescent dyes were used to identify the conformations including ANS, TNS, and bis-ANS. SI studies and protease assays utilizing both Suc-AAPF-pNA and L-BAPNA were instrumental in determining conformations along with gel electrophoresis studies. The hydrophobic dyes bound to the different serpins with varying KD and ΔFmax due to structural variations among the conformers and the complex. Both TNS and bis-ANS gave higher ΔFmax values than ANS. Bis-ANS gave significantly higher ΔFmax values for the ACT:CHY than the other conformations, while also exhibiting relatively low KD value. KD values for the bis-ANS complexes are relatively low when compared to other fluorophores. Bis-ANS is more specific for the AT system than either TNS or ANS. Bis-ANS displays a ΔFmax of 36 fold for the ACT:CHY complex, while TNS displays a 27 fold increase for AT:TRY system. Modulation studies using bis-ANS to alter the kinetics of latent ACT formation proved unsuccessful, suggesting that fluorescent dyes have little, if any effect on serpin variant formation.

Orientador: Prof. Dr. Luciano Puzer<br>Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, São Bernardo do Campo, 2017.<br>Serpina é o nome dado à superfamília de proteínas com uma vasta diversidade de funções biológicas, que tem como principal característica a inibição irreversível de serino proteases. A característica estrutural mais marcante das serpinas é a presença de uma alça na sua porção C-terminal, composta por 20 aminoácidos denominados alça do centro reativo (RCL). A RCL é a região da serpina que se liga covalentemente ao sítio ativo da serino protease, promovendo a inibição irreversível da enzima pela desarticulação de estruturas funcionais essenciais para a catálise enzimática. As calicreínas teciduais humanas (KLKs) são um grupo de 15 serino proteases (KLK1-KLK15) expressas em uma gama de tecidos. A rKLK5, estudada neste trabalho, é expressa abundantemente na pele humana, e parece que exerce importante papel no processo de descamação epidermal, podendo estar relacionada à patologias, como a psoríase e dermatite atópica. Assim, nesse trabalho foi realizada subclonagem, expressão e caracterização bioquímica da atividade inibitória dos mutantes (VSR344G e VSA345G) da serpina oriunda da cianobactéria Gloeobacter violaceus, identificada pelo nosso grupo e nomeada vioserpina. Os genes codificadores da vioserpina foram previamente clonados no vetor de expressão pET-28a e por meio da técnica de mutação sítio-dirigida foram produzidos dois mutantes substituindo os resíduos de aminoácidos arginina e alanina nas posições P1 e P2 da alça do centro reativo (RCL) da vioserpina, respectivamente, por um resíduo de aminoácido glicina (VSR344G e VSA345G). O sucesso das mutações foi avaliado através de sequenciamento de DNA. As vioserpinas mutantes foram expressas na cepa bacteriana E. coli BL21(D3), purificadas pela técnica de cromatografia de afinidade em resina de níquel (Ni-NTA) e obtidas na forma solúvel. Foi possível visualizar a formação do complexo covalente entre a vioserpina e a KLK5 por análise em SDS-PAGE 10% confirmados pela espectrometria de massas. Também foi analisada a ação inibitória dos mutantes (VSR344G e VSA345G) frente a KLK5. Os valores de SI (estequiometria de inibição) apresentaram valores altos o que indica que é preciso uma concentração grande de inibidor, no caso os mutantes da vioserpina, para que a enzima tenha sua atividade inibida, apresentando-se desta forma uma inibição ineficiente frente a rKLK5. Nesse estudo foi possível obter um inibidor específico (vioserpina) para a rKLK5, podendo assim contribuir para o desenvolvimento de novos procedimentos terapêuticos para patologias envolvidas com o processo de descamação epidermal.<br>Serpin is the name given to the superfamily of proteins with wide range of biological functions, and that has as main feature the irreversible inhibition of serine proteases. The most striking structural feature of serpins is the presence of a loop in the C-terminal portion of the protein, composed by 20 aminoacids called Reative Center Loop (RCL). The RCL is the region of the serpin that binds covalently to the serine protease active site, which causes the irreversible inhibition of the enzyme by the disarticulation of functional structures essencial for the enzymatic catalysis. Human tissue kalikreins (KLKs) are a group of 15 serine proteases (KLK1-KLK15) expressed in a plethora of tissues. The rKLK5, studied in this work, is abundantly expressed in human skin, and seems to play an important role in the epidermal desquamation process, and may be related to pathologies such as psoriasis and atopic dermatitis. Therefore, in this work the subcloning, expression and biochemical characterization of the inhibitory activity of serpin mutants (VSR344G e VSA345G) of the cyanobacteria Gloeobacter violaceus, recently described by our group and named vioserpin, was proposed. The genes coding for vioserpin were previously cloned into the pET-28a expression vector and through the site-directed mutation technique two mutants were produced by substituting the arginine and alanine amino acid residues at the P1 and P2 positions of the serine reactive center loop (RCL), respectively, by a glycine amino acid residue (VSR344G e VSA345G). The success of the mutations was assessed by DNA sequencing. The mutant vioserpin was expressed in the bacterial strain E. coli BL21 (D3), purified by the nickel resin affinity chromatography technique (Ni-NTA) and obtained in the soluble form. Covalent complex formation between vioserpin and rKLK5 could be visualized by 10% SDS-PAGE analysis confirmed by mass spectrometry. We also analyzed the inhibitory action of mutants (VSR344G and VSA345G) against rKLK5. The SI values (inhibition stoichiometry) presented high values indicating that a large inhibitor concentration is required in the case of the vioserpin mutants, so that the enzyme has its inhibited activity, thus presenting an inefficient inhibition in front To rKLK5. In this study it was possible to obtain a specific inhibitor (vioserpin) for rKLK5, thus contributing to the development of new therapeutic procedures for pathologies involved with the epidermal desquamation process.

La recherche d’abords thérapeutiques innovants est particulièrement d’actualité dans l’adénocarcinome canalaire du pancréas (ACP) tant le pronostic de ce cancer, dont l’incidence augmente, reste redoutable. Une des voies d’exploration réside dans la compréhension des mécanismes entrainant l’importante réaction stromale habituellement constatée dans l’ACP et prédisant la résistance à la chimiothérapie. Cette réorganisation tissulaire ou desmoplasie, résulte de l’activation de cellules normales présentes dans le tissu conjonctif de l’organe touché par les cellules tumorales d’origine épithéliale. Se développe ainsi un terreau fertile pour l’expansion des cellules cancéreuses, dont les fibroblastes activés sont un des acteurs prépondérants. Afin de mieux appréhender les interactions entre ces fibroblastes associés au cancer (CAF), les cellules tumorales et le reste du microenvironnement tumoral, l’étude de leur(s) mode(s) d’activation apparaît comme un préalable et pourrait permettre une stratification des patients dans leur orientation thérapeutique. L’identification d’un panel d’anticorps pertinent utilisable in situ en immunohistochimie (IHC) est donc un des enjeux pour la prise en charge future des patients atteints d’ACP et constitue l’objectif de ce travail<br>The search for innovative therapeutic approaches is particularly topical in pancreatic ductal adenocarcinoma (PDAC), as its prognosis remains daunting. One of the avenues of exploration lies in the understanding of the mechanisms leading to the important stromal reaction usually observed in PDAC predicting resistance to chemotherapy. This tissue reorganization or desmoplasia results from the activation of normal cells present in the connective tissue of the organ affected by tumor cells of epithelial origin. A fertile breeding ground for the expansion of cancer cells develops, of which activated fibroblasts are one of the most important players. In order to better understand the interactions between these cancer-associated fibroblasts (CAF), tumor cells and the rest of the tumor microenvironment, their characterization appears as a prerequisite and could allow stratification of patients in their therapeutic orientation. The identification of a relevant antibody panel that can be used in situ in immunohistochemistry (IHC) is therefore an issue for the future management of patients with PDAC and is the objective of this work

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.<br>Title from electronic title page (viewed Sep. 16, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology and Laboratory Medicine." Discipline: Pathology and Laboratory Medicine; Department/School: Medicine.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>Recurrent airway obstruction (RAO) and inflammatory airway disease (IAD) show high prevalence and are economically important in equine athletes. RAO is considered a multifactorial disease due to genetic and environmental components in their patophysiology. The aim of this study was to determine the presence of polymorphism at exons 2, 3, 4 and 5 of the SPi2 gene and a possible association between them and ORA or IAD on 51 thoroughbred horses through single-strand conformational polymorphism (SSCP). Exons 2, 3 and 4 of the Spi2 gene showed no polymorphism. On exon 5, 3 alleles and 6 genotypes were identified. Frequency of allele A (0.6388) and genotype AA (0.3888) were higher in horses affected by RAO but no association was found between any polymorphism and horses with RAO or IAD.<br>A obstrução recorrente das vias aéreas (ORA) e a doença inflamatória das vias aéreas (DIVA) são doenças de alta prevalência e economicamente importantes em cavalos atletas. A ORA é considerada uma enfermidade multifatorial por apresentar componentes ambientais e genéticos em sua fiosiopatologia. O presente trabalho teve por objetivo determinar a presença de polimorfismos nos éxons 2, 3, 4 e 5 do gene SPi2 e verificar uma possível associação destes com a ORA e/ou DIVA em 51 cavalos Puro Sangue de Corrida através da técnica de polimorfismo conformacional de fita simples (SSCP). Os éxons 2, 3 e 4 não apresentaram polimorfismo. No éxon 5 do gene Spi2 foram identificados três alelos e seis genótipos. Apesar do alelo A e o genótipo AA apresentarem freqüência (0,6388 e 0,3888, respectivamente) mais elevada nos animais com ORA, não houve associação entre os polimorfismos observados e ORA ou DIVA.

Orientador: Prof. Dr. Luciano Puzer<br>Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Ciência & Tecnologia - Química, 2014.<br>Serpina é o nome dado a uma superfamília de proteínas com grande diversidade de funções biológicas, e que tem como principal característica a inibição de serino proteases. Até meados do ano de 2002, acreditava-se que as serpinas eram exclusivas de organismos eucarióticos. No entanto, com o desenvolvimento das técnicas de sequenciamento de genomas, essas proteínas passaram a ser identificadas também em organismos procariotos. Assim com o objetivo de avaliar a capacidade de duas serpinas bacterianas (G. violaceus e M. xanthus) em inibir serino proteases foi realizado nesse trabalho a clonagem, expressão e caracterização bioquímica da atividade inibitória de duas serpinas bacterianas descritas no banco de dados de genoma "genbank". As serpinas que foram alvo de nosso estudo possuem diferentes sequências de aminoácidos na sua RCL (alça do centro reativo), visando produzir serpinas com diferentes especificidades para serino proteases. Os genes codificadores das duas serpinas foram clonados em vetor de expressão pET-28a(+). As serpinas S1 (Gloeobacter violaceus) e S2 (Myxococcus xanthus) foram expressas na cepa bacteriana E. coli Bl21 (D3), purificadas pela técnica de cromatografia de afinidade em resina de níquel Ni-NTA e obtidas na forma solúvel. Foram realizados ensaios para determinar a atividade inibitória das serpinas S1 (G. violaceus) e S2 (M. xanthus) contra as enzimas tripsina, quimotripsina, elastase, subtilisina, NS3 (dengue) e ainda da serpina S1 frente as calicreínas teciduais humanas 1, 3 e 5. A eficiência da reação de inibição da serpina S1 contra essas enzimas também foi testada na presença de glicosaminoglicanos (GAG¿s): heparina, sulfato de dermatan e sulfato de condroitina. A serpina S1 (G. violaceus) apresentou atividade inibitória frente à tripsina na ausência e também na presença dos GAG¿s heparina, sulfato de dermatan e sulfato de condroitina, sendo que na presença de heparina foi possível observar o menor valor de constante de velocidade de segunda ordem (k¿) em relação aos demais GAG¿s testados, sugerindo que a heparina atua de alguma forma na diminuição da velocidade na reação de inibição da tripsina pela serpina S1. Foi possível visualizar a formação do complexo covalente entre a serpina S1 (G. violaceus) e a tripsina por análise em SDS-PAGE 10%. Também analisamos a ação inibitória de compostos sintéticos derivados do isomanídeo frente às calicreínas teciduais humanas 1, 3, 5, 6 e 7. Nesse estudo foi possível obter inibidores seletivos para as calicreínas humanas teciduais 5 e 7, a partir dos compostos sintéticos derivados do isomanídeo e através dos estudos de docking, juntamente com a análise das estruturas das KLK5 e KLK7, foram fornecidas informações sobre as diferenças observadas entre as afinidades de ligação dos diferentes compostos derivados do isomanídeo refletidos nos resultados dos testes de inibição.<br>Serpin is the name given to the superfamily of proteins with wide range of biological functions, and that has as main feature the inhibition of serine proteases. Until mid-year 2002 it was believed that the serpins were unique to eukaryotic organisms. However, with the development of genome sequencing techniques, these proteins are now also been identified in prokaryotic organisms. Thus, with the objective of evaluate the ability of two bacterial serpins (G. violaceus and M. xanthus) to inhibit serine proteases was performed in this work the cloning, expression and biochemical characterization of the inhibitory activity of two bacterial serpins described in the database of genome "genbank". Serpins that have been the target of our study have different sequences of amino acids in RCL (the reactive center loop), to produce serpins with different specificities for serine proteases. The genes encoding the two serpins were cloned into the expression vector pET-28a (+). S1 (Gloeobacter violaceus) and S2 (Myxococcus xanthus) serpins were expressed in the bacterial strain E. coli BL21 (D3), purified by the technique of affinity chromatography on nickel Ni-NTA resin and obtained in soluble form. Assays were performed to determine the inhibitory activity of serpins S1 (G. violaceus) and S2 (M. xanthus) against the enzymes trypsin, chymotrypsin, elastase, subtilisin, NS3 (dengue) and also the serpin S1 against human tissue kallikrein 1, 3 and 5. The efficiency of the inhibition reaction against these enzymes serpin S1 was also tested in the presence of glycosaminoglycans (GAG's) heparin, dermatan sulfate and chondroitin sulfate. S1 serpin (G. violaceus) had inhibitory activity against trypsin in the absence and presence of GAGs heparin, dermatan sulfate and chondroitin sulfate, and in the presence of heparin was observed the lowest value of the rate constant of the second order (k') compared to the other GAG's tested, suggesting that heparin acts in some way in reducing the speed in inhibiting reaction of trypsin by serpin S1. It was possible to visualize the formation of a covalent complex between S1 (G. violaceus) serpin and trypsin by SDS-PAGE 10% analysis. We also analyzed the inhibitory activity of synthetic compounds derived from isomannide against human tissue kallikrein 1, 3, 5, 6 and 7. In this study it was possible to obtain specific inhibitors for human tissue kallikrein 5 and 7 from synthetic compound derived from the isomannide and through docking studies, together with the analysis of the structures of KLK5 and KLK7, were provided information about the differences between the various binding affinities of compounds derived from isomannide and reflected in the results of the inhibition tests.

L'antithrombine (at) et l'alpha-1-antitrypsine (1 at) appartiennent a la super famille des serpines. Nous decrivons les bases moleculaires de plusieurs deficits en at de type i et ii. La structure primaire du gene expliquerait certaines mutations par derapage replicatif, rendant compte de certaines deletions et mutations recurrentes. Une mutation phe 402 leu de l'at a ete analysee ; le variant a une stabilite accrue au sodium dodecyl sulfate, suggerant que la boucle du site actif pourrait etre inseree dans le feuillet ba. Trois autres mutations sont responsables de la formation de complexes de haut poids moleculaire, par liaison disulfure avec d'autres proteines plasmatiques. Nous rapportons enfin, un second cas d'1 at-pittsburgh (met 358 arg) chez un jeune patient de 15 ans. L'expression clinique moderee de ce dernier par rapport au premier cas rapporte, s'expliquerait par le profond deficit en proteine c associe. Ce dernier est un deficit acquis par la formation de complexes proteine c activee/1 at. Nous avons pu demontrer que la thrombomoduline etait un inhibiteur non competitif de l'inhibition de la thrombine par le variant pittsburgh. Ceci explique que la proteine c puisse etre activee, en depit de l'importante activite circulante antithrombine

La mort cellulaire est un processus indispensable à l'homéostasie des organismes. Les premières protéases impliquées dans l'apoptose furent les caspases. Cependant, leur participation n'est plus considérée comme une étape obligatoire, plusieurs effecteurs indépendants de l'activation des caspases ayant été mis en évidence. Un de ces effecteurs, caractérisé dans notre laboratoire, est la LEI/L-DNase II. Cette protéine appartient à la famille des serpines. Nous avons montré que la LEI (antiprotéase) change de conformation en découvrant un site endonucléase, la transformant ainsi en L-DNase II (endonucléase). En même temps, le changement de conformation découvre un site de nucléarisation qui permet sa translocation dans le noyau. Lorsque la LEI n'est pas transformée en L-DNase II, celle-ci protège les cellules de l'apoptose. Lors de l'induction par l'étoposide, la LEI empêche l'activation de la caspase-8 à travers l'inhibition de la cathepsine D dans deux modèles de mort indépendants des caspases : la dégénérescence rétinienne induite par la lumière et le paraptose des cellules somato-lactropes<br>The first proteases implicated in apoptosis were the caspases. But their participation in this process is no longer considered as indispensable. Other non-caspases proteases have been implicated in apoptosis, such as LEI/L-DNase II. LEI/L-DNase II belongs to the serpin superfamily. We show that LEI (antiprotease) is transformed into L-DNase II by a conformational modification. This conformational change also uncovers a nuclear translocation site, allowing this L-DNase II (endonuclease) to go to the nucleus to degrade DNA. When cells are induced with etoposide, wich does not permit the conformational change of LEI, this serpin protect cells from caspase-8 activation by indirect inhibition of cathepsin D. We have also implicated L-DNase II into two models of caspase independent cellular death : light-induced retinel degeneration and paraptosis of somato-lactotropes cells

BACKGROUND SERPINB3 is a serine protease inhibitor belonging to the serpin family, characterized by pleyotropic functions. TGF-beta1 is the master cytokine in the pathogenesis of liver fibrosis. Inflammatory and mesenchymal cells, including hepatic stellate cells, have been identified as the principal source of this cytokine, however growing evidence also supports the ability of parenchymal hepatocytes to produce TGF-beta1. Recently, a direct correlation between TGF-beta1 and the ov-serpin SERPINB3 has been reported in idiopatic pulmonary fibrosis, whose pathological features recall cirrhosis pathogenesis. SERPINB3, a serine protease inhibitor up-regulated in cirrhosis, dysplastic nodules and hepatocellular carcinoma (HCC), can trigger epithelial to mesenchymal transition (EMT) in HepG2 cells. EMT is also triggered by hypoxia which is common in HCC and believed to favour selection of more invasive tumour cells and cancer progression. AIM Aim of the first study was to assess the potential correlation between SERPINB3 and TGF-beta1 in chronic liver diseases and to determine their relationship using in vitro models of cells transfected with SERPINB3. Aim of the second study was to investigated molecular mechanisms involved in SERPINB3-mediated EMT induction and the relationship between hypoxia and SERPINB3 expression in vitro, by employing normal and SERPINB3-transgenic HepG2 cells or in vivo by immunohistochemistry (IHC) on specimens from cirrhotic patients carrying HCC. METHODS In the first study SERPINB3 and TGF-beta1 expression in liver biopsies from 80 patients with chronic liver disease (59 chronic hepatitis and 21 liver cirrhosis) was evaluated by immunohistochemistry and by Real time PCR. To investigate whether SERPINB3 could affect TGF-beta1 expression, TGF-beta1 was determined in HepG2 cells transfected with SERPINB3 and in control HepG2 cells at transcription and protein level by Real time PCR and ELISA assay. In the second study SERPINB3 up-regulation, involvement of hypoxia inducible factors (HIFs) and signal transduction pathways, intracellular generation of reactive oxygen species (ROS), EMT and invasiveness were analysed by morphological, molecular and cell biology techniques in HepG2 cells, exposed to hypoxia and/or incubated with wild type and mutated SERPINB3 recombinant proteins. Moreover, liver specimens from either HCV cirrhotic patients carrying HCC or from transgenic mice over-expressing SerpinB3 were analyzed by immunohistochemistry. RESULTS Study I In chronic hepatitis and liver cirrhosis a strong positive correlation was observed between SERPINB3 and TGF-beta1 immunoreactivity. At transcription level, cDNA amplification profiles of SERPINB3 and TGFbeta1 were similar in the majority of the cases. A significant correlation was found between the degree of parenchymal TGF-beta1 1 and SERPINB3 expression and the severity of liver fibrosis. Neither of the two molecules were detectable in 20 normal livers. Time course analysis of TGF-beta1 1 over time in SERPINB3 transfected cells showed an increase of this cytokine, with a peak value at 48 hours for TGF-beta1 1 mRNA and at 72 h for protein production. Study II EMT and increased invasiveness triggered by wild-type and mutated recombinant SERPINB3 are independent of their anti-protease activity and involve intracellular generation of ROS and redox-dependent, phosphorylation of GSK3beta. SERPINB3 transcription, protein synthesis and release in the medium are up-regulated under hypoxia, almost abolished by specific silencing of HIF-2alpha (but not HIF-1alpha), significantly modulated by hypoxia-dependent intracellular generation of ROS and involve Ras/Erk and PI3K signalling pathways. In addition, liver specimens revealed co-localization of HIF-2alpha and SERPINB3 immunostaining in either HCC cells and in hepatocytes of surrounding cirrhotic liver. CONCLUSIONS Study I SERPINB3 is overexpressed in chronically damaged hepatocytes, where increased TGF-beta1 has also been documented. The positive correlation found between the expression of either TGF-beta1 and SERPINB3 and fibrosis grade supports the dynamic interplay of these molecules, suggesting that SERPINB3 could orchestrate the most peculiar aspects of chronic liver disease by inducing TGF-beta1 production, leading to enhanced collagen synthesis and by increasing the survival capacity of damaged hepatocytes. Study II Hypoxia, through a common redox sensitive signalling, can switch on both EMT program and SERPINB3 expression. Hypoxia may then sustain increased invasiveness in cancer cells by a release of HIF2-dependent SERPINB3 molecule. This serpin could elicit EMT and invasiveness even in normoxic surrounding cells, then enhancing its putative role in carcinogenesis.<br>INTRODUZIONE SERPINB3 appartiene alla famiglia delle serpine, inibitori delle proteasi seriniche implicati in molte funzioni biologiche e nei processi di controllo dell'omeostasi cellulare. E' espressa normalmente negli epiteli squamosi, ma si trova iper-espressa nelle cellule neoplastiche di origine epiteliale e nell'epatocarcinoma ma anche in noduli cirrotici ad alto grado di displasia. TGF-beta1 una citochina multifunzionale, coinvolta nella regolazione della proliferazione, differenziamento e migrazione di molti tipi cellulari ed il più importante fattore implicato nel processo di fibrogenesi epatica, dove regola la sintesi di collagene nelle cellule stellate epatiche. Recentemente in un'altra patologia fibrosante progressiva, quale la fibrosi polmonare idiopatica, è stata documentata una correlazione diretta tra entità di espressione di TGF-beta1 e della serpina SERPINB3, con co-localizzazione delle due molecole a livello cellulare. Recenti studi sperimentali condotti nel nostro laboratorio suggeriscono il coinvolgimento in vitro di SERPINB3 in caratteristiche tipiche della transizione epitelio mesenchimale, (EMT) un fenomeno chiave anche nell'epatocarcinoma. Inoltre, ulteriori risultati descritti in letteratura identificano l'ipossia, una delle condizioni più comuni che si instaurano nel microambiente tumorale, come condizione in grado di indurre in modo indipendente EMT e lo stesso fenomeno di transizione epitelio mesechimale è indotto anche dalla citochina TGF-beta1 . SCOPO Studio I Lo scopo della prima parte della tesi è valutare la potenziale correlazione tra SERPINB3 e TGF-beta1 nel fegato in pazienti con danno epatico cronico e valutare la relazione tra queste due molecole utilizzando modelli in vitro di colture cellulari trasfettate con SERPINB3. Studio II Lo scopo della seconda parte della tesi è quello di caratterizzare gli stimoli regolatori e i meccanismi dell'induzione di SERPINB3 come fattore solubile in grado di indurre l'invasività e la transizione epitelio mesenchimale. Inoltre, ulteriore scopo è valutare la correlazione tra ipossia e SERPINB3 ed i relativi meccanismi di regolazione sia in modelli in vitro che in vivo. MATERIALI E METODI Per quanto concerne il primo studio sono stati studiati 80 pazienti, di cui 59 con epatite cronica e 21 con cirrosi epatica. L'espressione di SERPINB3 e TGF-beta1 stata analizzata su biopsie epatiche di pazienti con epatite cronica a livello proteico mediante immunoistochimica e a livello trascrizionale mediante PCR quantitativa. Il modello in vitro prevedeva la trasfezione transiente e stabile di cellule HepG2 con il gene SERPINB3 wild type e con differenti vettori caratterizzati da estensioni diverse di delezione del sito attivo della serpina, responsabile della sua attività  antiproteasica, con successiva analisi dell'espressione genica e proteica. Per quanto riguarda il secondo studio sono stati effettuate analisi di immunofluorescenza e invasività  in cellule HepG2 trattate con diverse forme di proteina SERPINB3 ricombinante per valutarne l'effetto di trasformazione epitelio-mesenchimale e la generazione intracellulare di specie reattive all'ossigeno (ROS). Inoltre le cellule sono state mantenute in condizioni di ipossia (3%O2 ) per studiare il comportamento di SERPINB3 ed è stato effettuato il silenziamento genico per i fattori inducibili in ipossia (HIF) al fine di determinare il loro coinvolgimento. L'espressione di SERPINB3 e dei fattori relativi all'EMT e all'ipossia sono stati valutati anche su biopsie epatiche in pazienti con epatocarcinoma. RISULTATI Studio I L'analisi immunoistochimica nel fegato ha documentato una significativa correlazione positiva con co-localizzazione di SERPINB3 e TGF-beta1 nelle cellule parenchimali del fegato. Anche a livello trascrizionale l'espressione di mRNA ha confermato un simile andamento, con livelli di espressione genica sostanzialmente sovrapponibili (r=0,7483, p=0,0070). Inoltre, l'espressione di entrambe le molecole è risultata e positivamente correlata con l'entità  della fibrosi. In cellule HepG2 trasfettate con il gene SERPINB3 è stata documentata un'induzione di TGF-beta1 sia dal punto di vista trascrizionale che della produzione proteica. I risultati ottenuti dalla trasfezione hanno dimostrato che è cruciale l'integrità  del sito attivo della serpina per l'induzione di TGF-beta1 , in quanto questa citochina è totalmente assente nei vettori con la delezione del sito antiproteasico di SERPINB3, mentre dove la delezione è minore, si riscontra una ridotta induzione, rispetto al controllo. Studio II SERPINB3 induce EMT e aumenta l'invasività  in cellule HepG2 mediante il coinvolgimento di ROS e la fosforilazione di GSK3b, indipendentemente dall'attività antiproteasica della serpina. In condizioni di ipossia si osserva un aumento della trascrizione e sintesi di SERPINB3, modulata dalle vie di segnale Ras/ERK e PI3K e completamente dipendente dal gene HIF-2alpha. Questi dati sono stati confermati nel topo transgenico per SERPINB3, dove è stata dimostrata un'iperespressione della molecola nelle zone ipossiche circostanti le vene centro globulari. Inoltre, in biopsie di pazienti con epatocarcinoma è stata confermata una colocalizzazione di SERPINB3 e HIF-2alpha CONCLUSIONI Studio I Nella malattia epatica cronica evolutiva esiste una correlazione diretta tra espressione di SERPINB3 e TGF-beta1. La correlazione di entrambe queste molecole con il grado di fibrosi epatica rafforza l'ipotesi di una loro potenziale interazione nel processo fibrogenetico epatico. In modelli in vitro SERPINB3 è in grado di indurre l'espressione di TGF-β e questo effetto dipende dalla sua attività  antiproteasica. Alla luce di queste considerazioni SERPINB3 potrebbe essere considerato un bersaglio terapeutico per contrastare il processo di fibrogenesi. Studio II L'ipossia può indurre SERPINB3 e conseguente trasformazione epitelio-mesenchimale mediante la produzione di radicali liberi dell'ossigeno. L'ipossia inoltre è il primo stimolo di rilevanza fisiopatologica descritto in letteratura capace di modulare la trascrizione e l'espressione di SERPINB3 attraverso un meccanismo dipendente dal gene HIF-2alpha.

Made available in DSpace on 2015-12-06T23:46:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2013<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>Objetivo: Descrever clinica e radiologicamente os individuos afetados pela Osteogenese Imperfeita (OI) (OMIM 613982) em uma familia endocruzada procedente de Bueno Brandao, MG; definir a mutacao genetica responsavel pela doenca e identificar variabilidade fenotipica intrafamiliar. Metodologia: Levantamento genealogico com visitas domiciliares de familia demonstrando consanguinidade em 8 geracoes, com tres individuos apresentando baixa estatura deformante diagnosticados com OI autossomica recessiva. Outro individuo afetado foi avaliado na regiao. Apos a avaliacao clinica morfologica classica e estudo radiologico do esqueleto foram coletadas amostras de sangue dos afetados e individuos saos da familia e encaminhadas para sequenciamento. Resultados: Os achados clinicos e radiologicos foram diferentes entre os afetados, ja que somente um deles apresenta ossos wormianos, afilamento de costelas e calcificacoes oem pipocao. As informacoes da genealogia permitiram levantar a hipotese de modelo de heranca autossomica recessiva. Nao foi possivel atraves dos dados clinicos e radiologicos definir um dos diversos tipos de OI descritos na literatura. No entanto, o sequenciamento do exoma revelou que o probando tem uma delecao de 19 pb em homozigose no exon 8 do gene SERPINF1, atualmente associado a OI tipo VI. Os demais afetados tambem apresentam a mesma delecao em homozigose e seus genitores apresentam esta delecao em heterozigose, o que foi confirmado pelo sequenciamento de Sanger. Discussao: Embora mutacoes no gene SERPINF1 tenham sido relacionadas a OI, esta mutacao nao havia sido descrita e a variabilidade fenotipica intrafamiliar provavelmente esta associada a interacao com outros genes e fatores epigeneticos ate o momento nao esclarecidos, sugerindo efeito fundador da doenca na populacao estudada Conclusao: Este estudo e pioneiro em identificar uma delecao parcial patogenica no exon 8 do gene SERPINF1 em brasileiros consanguineos de ancestralidade Portuguesa/Holandesa/Indigena causando OI autossomica recessiva deformante grave com variabilidade fenotipica intrafamiliar clinica e radiologica<br>BV UNIFESP: Teses e dissertações

L'[alpha][indice inférieur 1]-antitrypsine ([alpha][indice inférieur 1]-AT), un inhibiteur naturel de l'élastase, et l'[alpha][indice inférieur 1]-antichymotrypsine ([alpha][indice inférieur 1]-ACT) appartiennent à la superfamille des serpines, qui sont des inhibiteurs irréversibles de sérines protéinases. Nous avons développé une approche qui consiste à modifier la boucle du site réactif de ces serpines afin de cibler diverses sérines protéinases impliquées dans le clivage de précurseurs protéiques (furine) ou de protéines de la matrice extracellulaires (élastase). La furine est une sérine protéinase Ca[indice supérieur 2+]-dépendante appartenant à la famille des convertases de mammifères de type subtilisine/kexine. Elle réside principalement dans le réseau du trans-Golgi (TGN) de la voie de sécrétion constitutive, mais elle peut aussi se retrouver à la surface cellulaire. À partir de la spécificité de la furine qui reconnaît principalement des paires ou plusieurs acides aminés basiques d'une séquence donnée, nous avons construit une série de variants d'[alpha][indice inférieur 1]-AT et d'[alpha][indice inférieur 1]-ACT contenant différents motifs de reconnaissance pour la furine afin d'augmenter l'affinité et leur pouvoir inhibiteur envers cette convertase. De plus, nous avons aussi développé une méthode qui consiste à conjuguer chimiquement sur une cystéine de l'[alpha][indice inférieur 1]-AT recombinante un polyéthylène glycol (PEG) afin d'augmenter sa stabilité lors des études in vivo. Les résultats montrent que la boucle du site réactif de l'[alpha][indice inférieur 1]-AT peut être aisément modifiée en un inhibiteur irréversible de la furine, comme l'[alpha][indice inférieur 1]-AT-PDX, sans compromettre les propriétés inhibitrices essentielles de la serpine originale. Les divers variants de la boucle du site réactif de l'[alpha][indice inférieur 1]-AT et l'[alpha][indice inférieur 1]-ACT ont montré tous une capacité de former des complexes SDS-stables avec des stoechiométries d'inhibitions (SI) variables in vitro et d'abolir la maturation protéolytique du précurseur du facteur von Willebrand par la furine in cellulo. Toutefois, l'insertion de résidus basiques dans la région réactive de l'[alpha][indice inférieur 1]-AT et l'[alpha][indice inférieur 1]-ACT à des positions stratégiques a affecté l'ensemble des propriétés inhibiteurs de type serpine de certains variants. La reconnaissance de motifs canoniques furine (e.g. RXXR) par la furine est pH indépendante, alors que l'étape de déacylation est dépendante du pH, comme montré par AT-EK2. Par contre, les variants [alpha][indice inférieur 1]-ACT sont plus efficaces à former un complexe à pH acide alors que les variants [alpha][indice inférieur 1]-AT le sont à pH basiques. De plus, la conjugaison au PEG de l'[alpha][indice inférieur 1]-AT recombinante a permis de prolonger sa demie-vie dans le poumon et le sang et de contrecarrer les effets hémorragiques causés par l'élastase leucocytaire dans le poumon des souris. Étant donné que la farine est responsable de l'activation de plusieurs précurseurs protéiques bactériens, tel que l'anthrax, et les glycoprotéines virales, tel que le virus d'Ebola, ces inhibiteurs anti-furine contenant des séquences de résidus basiques déterminées pourraient potentiellement être utilisés pour combattre ces infections pathogènes. De plus, ils permettraient de contrecarrer l'acuité des effets de morbidité causés par des pathogènes opportunistes dans certaines maladies du système respiratoire (emphysème et fibrose kystique).

the serpins are a family of serine protease inhibitors involved in several biological functions and in the maintenance of cell homeostasis. SERPINB3 (known as SCCA1), a member of the ovalbumin family, is normally expressed in squamous epithelium, and it is over-expressed in tumours of epithelial origin and in hepatocellular carcinoma. The SERPINB3 involvement in the control of proteolytic processes has important implications in neoplastic transformation, whereas the balance between proteases and their inhibitors can affect cell motility, invasiveness, proliferation and apoptosis. Considering the potential role of SERPINB3 and the lack of human biological tissues for clinical research, the possibility to use a transgenic animal model to explore in vivo the role of this serpin in several cell processes could be of great value. The aim of the present study was to assess the SERPINB3 involvement in several pathologies by a transgenic mouse model for human SERPINB3, expressed in different organs as brain, lung and liver.<br>Le serpine sono una famiglia di inibitori delle proteasi seriniche implicata in molte funzioni biologiche e nei processi di controllo dell’omeostasi cellulare. SERPINB3 (chiamata anche SCCA1), appartenente alle ov-serpine, è espressa normalmente negli epiteli squamosi, ma si trova iper-espressa nelle cellule neoplastiche di origine epiteliale e nell’epatocarcinoma. Il coinvolgimento di SERPINB3 nella regolazione dei processi proteolitici ha importanti implicazioni a livello dei processi neoplastici, dal momento che l’equilibrio tra le proteasi ed i loro inibitori, può influenzare la mobilità, l’invasività, la proliferazione e la morte cellulare stessa. Dato il potenziale ruolo di SERPINB3 ed il limite determinato dalla disponibilità di materiale biologico per la ricerca clinica sull'uomo, risulta importante poter utilizzare un modello animale che permetta di sperimentare in vivo il ruolo che la proteina può rivestire in molteplici processi cellulari. Lo scopo dello studio è quello di sperimentare il coinvolgimento della serpina in diverse patologie utilizzando un modello animale, costituito da un topo transgenico per SERPINB3 umana, caratterizzato dall’espressione della serpina in diversi organi quali il cervello, i polmoni ed il fegato.

HIV/AIDS is a global health problem of immense magnitude, with 33 million people living with HIV and 2 million AIDS-related deaths per year. As the development of drug resistance undermines treatment efficacy, the long-term success of anti-retroviral therapy depends upon the introduction of novel drugs aimed at additional targets essential for the viral life cycle. With a critical role in many viral diseases including the proteolytic maturation of the HIV-1 envelope glycoprotein gp160, the secretory pathway proprotein convertases (PCs) represent a potential anti-viral target. Our laboratory has reported the identification of Spn4A, a potent naturally occurring secretory pathway serine protease inhibitor directed at the prototype PC member, furin. Because of the requirement for the PCs in the production of infectious HIV-1, we hypothesized that strategic manipulation of PC activity by Spn4A and Spn4A-engineered variants would provide a means of effectively limiting HIV-1 infection. This thesis details the investigation of the anti-proteolytic activities and anti-HIV-1 properties of recombinant adenoviruses expressing Spn4A and Spn4A bio-engineered variants, including a secreted recombinant Spn4A (Spn4A S). Our data shows that the expression of Spn4A S in MAGI-CCR5 cells and furin-deficient LoVo cells inhibited the PC-dependent processing of the HIV-1 envelope precursor gp160. Furthermore, inhibition of processing resulted in a nearly complete reduction of productive HIV-1 infection as determined by HIV-1 Tat-driven β-galactosidase activity and multinuclear activation of a galactosidase indicator (MAGI) assays. Complementing the previously described anti-furin activity of Spn4A, our studies indicate that Spn4A S inhibits additional PCs involved in gp160 maturation, and that PC inhibition can serve as an effective means of limiting HIV-1 infection. With the central role of the PCs in the replication and pathogenesis of numerous infectious agents, the identification of Spn4A S as an efficacious HIV inhibitor establishes Spn4A as a prospective broad-based agent for the inhibition of PC-related diseases.

Objective: Serpins are serine protease inhibitors that are involved in a wide variety of biological functions in nature. They are known to regulate inflammation processes as well as provide host defense against microorganisms. Recent evidence has associated many types of mucosal serpins with a protective phenotype against HIV infection in women. Our hypothesis is that serpins with known antiviral activity against HIV-1 are correlated with protection in a group of HIV exposed seronegative individuals (HIV-resistant) from the Pumwani sex worker cohort. Study design: Cervico-vaginal lavage (CVL) fluid was collected from 66 HIV-positive, 82 HIV-negative and 84 HIV-resistant sex workers from the cohort. Clinical and epidemiological information was recorded at the time of sample collection. CVL protein levels were determined by BCA assay and serpin (A1 and A3) concentrations by a commercially available ELISA kit. Mucosal serpin concentrations were compared against clinical and epidemiological factors as well as sexual practices. Results: Serpin A1 was significantly higher in the HIV-resistant group compared to the HIV-negative controls (Anova: p=0.0470*). Total concentration of serpin A3 did not reach statistical significance between groups. Serpins did not correlate with age, sexual practices, contraceptive use or number of pregnancies. Serpins were differentially abundant during different stages of the menstrual cycle whereas serpin A1 was elevated during the proliferation phase but not in secretory phase (p=0.0275*). Conclusion: Serpin A1 was correlated with HIV-protection in this group of HESN women. This work will contribute to a more complete understanding of mechanisms of resistance and susceptibility to HIV infection.

Master of Science<br>Division of Biology<br>Kristin Michel<br>To date malaria is the most important tropical disease, which is caused by Plasmodium sp. and vectored by anopheline mosquitoes. The mosquito’s immune system is one of the limiting factors of malaria transmission. Immune reactions, such as the prophenoloxidase (PPO) pathway result in the melanization of pathogens, and are effective at limiting parasite numbers. Novel strategies for malaria control aim to exploit the immune system to interrupt parasite transmission by boosting the immune responses in the mosquito vector. Serpins play a crucial role in regulating protease cascades involved in immunity of arthropods. In Anopheles gambiae, the major malaria vector in Sub-Saharan Africa, 18 SRPN genes encoding 23 distinct proteins have been identified. So far, two are identified as active inhibitors, and both affect parasite survival. This research aims to identify additional inhibitory serpins in An. gambiae and elucidate their potential function. Identification of such serpins will enhance our understanding of the immune system of this important vector species and may identify immunoregulators to be used in malaria control. SRPN7, 9, and 18 were tested for their ability to inhibit commercial proteases in vitro. Recombinant SRPN18 had no inhibitory activity, while SRPN7 and 9 inhibited several serine proteases. SRPN7, 9 and 18 were tested against two recombinant An. gambiae clip serine proteases (CLIPBs) that are required for activation of phenoloxidase and thus regulate melanization. Only SRPN9 strongly inhibited CLIPB9 in vitro, suggesting that this serpin is a potential negative regulator of melanization. This hypothesis is further supported by the finding that SRPN9 can inhibit PO activity in insect hemolymph, ex vivo. Taken together, this research identifies SRPN18 as the first non-inhibitory serpin described in mosquitoes. Additionally, this study describes the larval-specific SRPN7 as a functional inhibitor. Future studies on these proteins will elucidate their precise physiological functions. Finally, this thesis provides strong evidence that SRPN9 is a negative regulator of melanization in An. gambiae and may therefore affect pathogen survival within this important vector species.

Carrapatos são animais hematófagos transmissores de diversas doenças para animais e seres humanos. O Rhipicephalus (Boophilus) microplus é um ectoparasito específico de bovinos. É responsável por importantes perdas econômicas na pecuária de países produtores de carne e leite. O aumento de populações de carrapatos resistentes aos principais acaricidas usados e a possível contaminação ambiental e dos produtos derivados como carne e leite impõem estudos para desenvolver novos métodos de controle. O controle imunológico é uma estratégia comprovadamente viável, porém ainda falta encontrar antígenos suficientemente eficientes. Uma das estratégias para atingir esse fim é o estudo de proteínas participantes de processos fisiológicos de grande importância para o carrapato, como é a aquisição e digestão de sangue. Proteínas pertencentes à superfamília das serpinas (serine protease inhibitors) participam do controle de diversos processos fisiológicos em mamíferos, inclusive coagulação sanguínea, fibrinólise e ativação do sistema complemento. Uma vez que carrapatos possuem informação para a síntese de serpinas, supõe-se que algumas têm como função perturbar a homeostase de seus hospedeiros. Analisando banco de sequências de cDNA identificamos 16 sequências que codificam serpinas no carrapato bovino, que foram nomeadas RmS (R. microplus serpin). Análises por RT-PCR mostraram que na maioria dos tecidos e estágios de desenvolvimento do parasito há expressão das RmS. Todas as sequências identificadas mostram conservação com serpinas de outras espécies de carrapatos. Aproximadamente 41% das RmS (7 de 16) provavelmente são secretadas, pois possuem predição para peptídeo-sinal. Assim como em outras serpinas, todas RmS possuem entre 1 e 4 sítios para N-glicosilação. Modelos tridimensionais revelam conservação de estrutura terciária das RmS analisadas com outras serpinas de mamíferos. Devido à presença de peptídeo-sinal, conservação com serpinas de outros carrapatos e presença em saliva, que foi revelada pelo estudo proteômico preliminar da saliva do parasito, RmS-3 foi escolhida para estudos mais pormenorizados. Para isso, a sequência codificadora foi clonada e ela foi obtida em forma recombinante. Anticorpos policlonais produzidos contra a proteína recombinante revelaram a presença da proteína em todos os tecidos analisados, e também na saliva, o que sugere participar na modulação das respostas do hospedeiro.<br>Ticks are hematophagous animals vectors of several human and animal diseases. Rhipicephalus (Boophilus) microplus is a bovine-specific ectoparasite. It is causes significant economic losses in livestock-producing countries. Increasing tick acaricide-resistant populations and, possible contamination on the environmental and on bovine-derived products contamination such as meat and milk impel the study of new control methods. Immunological control is a strategy proved to be feasible, but it requires really efficient antigens. A strategy to achieve this purpose is the study of proteins acting in physiological processes of major importance for the tick, as blood intake and digestion. Proteins belonging to the superfamily of serpins (serine protease inhibitors) have a role in several mammalian physiological processes, including blood coagulation, fibrinolysis and complement activation. As ticks encode information for serpins synthesis it is assumed they disrupt homeostasis their hosts. Through analysis of the cDNA sequences database, we identified 16 sequences coding for serpins in cattle tick, which were named RmS (R. microplus serpin). RT-PCR analyses showed RmSs are expressed in most tick tissues and developmental stages. All identified sequences are conserved concerning other tick species serpins. Around 41% of RmS (7 out 16) are probably secreted, since they have sequences predicting for signal peptide. Similarly as other serpin, all RmSs have 1 to 4 N-glycosylation sites. Protein modeling showed all RmSs have a conserved tertiary structure with other mammal serpins. RmS-3 was chosen for further studies due to presence of signal peptide, conservation with serpin inform other ticks and the presence in saliva revealed by an initial a proteomic study of tick saliva. In these studies, its gene was cloned RmS-3 was obtained as a recombinant protein. Polyclonal antibodies were against the recombinant protein showed RmS-3 presence in all analyzed tissues and also in saliva, suggesting it have a role disturbing host responses.

Made available in DSpace on 2018-08-01T20:28:08Z (GMT). No. of bitstreams: 1 tese_9932_Dissertação_Maira Trancozo.pdf: 750603 bytes, checksum: b325e8b4e134bd6ec4eb5bc70315d8c5 (MD5) Previous issue date: 2016-03-02<br>A Osteogênese Imperfeita (OI) ou doença dos ossos frágeis é uma desordem hereditária dos tecidos conjuntivos que contém colágeno em sua formação. Mais de 15 genes relacionados com herança recessiva têm sido relatados dos últimos anos. A maioria destes genes codificam proteínas responsáveis por modificações pós traducionais do colágeno I. Com o objetivo de caracterizar o padrão de mutações em genes relacionados com a OI de padrão de herança autossômica recessiva no Espírito Santo foram estudados por meio das técnicas de Next Generation Sequencing (NGS) e Sequenciamento de Sanger os genes LEPRE1, RTAP, PPIB, SP7, SERPINF1, FKBP10 e WNT1 os quais apresentam maior frequência de mutações descritas atualmente de 22 pacientes não consanguíneos que apresentavam diagnóstico clínico compatível com a doença atendidos no Hospital Infantil Nossa Senhora da Glória de Vitória/ES. Foram encontradas alterações potencialmente patogênicas nos genes LEPRE1 e FKBP10 em cinco pacientes. Dois pacientes são heterozigotos para mutações missense no gene LEPRE1 (c.1087 A>G e c.2024 G>T). Os outros três pacientes são portadores de mutações no gene FKBP10, dois pacientes apresentam mutações frameshift em homozigose (c.825dupC e c.15dupC) e um pacientes é portador de duas mutações em heterozigose composta (c.A179C e c.1063+2T>C). A gravidade da doença nestes pacientes varia de moderado a grave. Os resultados deste trabalho sugerem que a maioria das mutações que causam OI de herança recessiva em pacientes do ES estão localizadas nos genes LEPRE1 e FKBP10. Além disto, de acordo com os resultados, mutações nos genes CRTAP, PPIB, SP7, SERPINF1 e WNT1 causando OI são raras na população estudada. A caracterização de mutações em genes relacionados com OI em diferentes populações ajuda a melhorar nosso conhecimento sobre o padrão de variações genéticas em OI e auxiliam no planejamento de estratégias mais eficientes que viabilizem o diagnóstico molecular da doença e o aconselhamento genético junto às famílias.

Made available in DSpace on 2018-08-01T21:35:02Z (GMT). No. of bitstreams: 1 tese_10966_CelenyFiguera_Dissertação_PPGBiotec (Final).pdf: 1556599 bytes, checksum: 51c85b164333a4f542d5ac9fbca3c088 (MD5) Previous issue date: 2017-02-22<br>O carcinoma epidermóide de cabeça e pescoço (CECP) representa o tipo histológico mais frequente nos cânceres de cabeça e pescoço. Apesar dos numerosos estudos realizados ainda não foi estabelecido um conjunto comum de biomarcadores para o CECP que facilitem o prognóstico e o diagnóstico precoce. O objetivo deste estudo foi avaliar no CECP o possível potencial de biomarcador da Serpina1 e Osteopontina (OPN) por serem moléculas pouco avaliadas neste câncer. Os dados clínicos- patológicos e amostras biológicas foram obtidos de 40 pacientes com CECP e 11 indivíduos saudáveis. Foi realizada a detecção da expressão relativa do mRNA de Serpina1 e OPN por ensaio de qPCR em amostras de tecido tumoral e tecido normal. A avaliação da expressão proteica de OPN e Serpina1 foi realizada pela técnica de Western Blot em amostras soro de pacientes com CECP e indivíduos saudáveis. Foi observado aumento significativo no mRNA da Serpina1 em tecido tumoral em relação ao tecido normal (p=0.0014) e em relação às diferentes características clinico-patológicas avaliadas (sexo, hábito de beber e fumar, estadiamento e desenvolvimento linfonodal). A expressão proteica da Serpina1 no soro foi significativamente maior (p=0.0007) em pacientes com CECP quando comparados aos controles, assim como as características clinico-patológicas avaliadas. Os níveis de OPN no mRNA não foram diferentes entre os tecidos. A expressão proteica da OPN foi maior nos pacientes com estadiamento avançado da doença (III/IV) (p=0.0181). A Serpina1 mostrou-se como um potencial biomarcador do CECP, seus níveis aumentados nos pacientes com CECP podem ser detectados tanto no tecido tumoral como no sangue o que pode facilitar sua aplicação no desenvolvimento da doença. A OPN deve ser melhor avaliada para estabelecer as possíveis diferenças significativas. Palavras-chave: Carcinoma epidermóide de cabeça e pescoço. Biomarcadores. Osteopontina. Serpina1.

Les Arthropodes se défendent contre les agressions microbiennes par un ensemble de réactions très efficaces, comprenant l'activation de cascades protéolytiques aboutissant à la coagulation, à la mélanisation et à la synthèse de peptides antimicrobiens. Ces cascades protéolytiques font intervenir des protéases à sérine et sont régulées par des serpines, qui sont des inhibiteurs de protéases à sérine. L'équipe au sein de laquelle j'ai effectué ma thèse étudie le rôle de serpines et de protéases à sérine dans la réponse immunitaire de la drosophile. La première cascade protéolytique mise en évidence dans l'immunité de la drosophile mène à l'activation de la voie Toll, qui contrôle l'expression de nombreux gènes dont les peptides antimicrobiens. Spaetzle, le ligand du récepteur Toll, est présent sous la forme d'un précurseur dans l'hémolymphe des drosophiles. Son activation lors des infections par les bactéries à Gram positif et par les champignons requiert une maturation protéolytique. Cette coupure est contrôlée par la serpine Necrotic, dont l'absence se traduit par l'activation de Toll en absence d'infection et le développement d'un phénotype de nécrose et de létalité des adultes quelques jours après leur émergence. Nous avons réalisé un crible génétique suppresseur de ce phénotype et identifié la première protéase à sérine (Perséphone) impliquée dans l'activation immunitaire de Spaetzle. L'analyse des mutants perséphone a révélé que, de façon tout à fait surprenante, les bactéries à Gram positif et les champignons activent deux cascades protéolytiques distinctes aboutissant à la coupure de Spaetzle. La serpine Necrotic possède dans sa région N-terminale une extension atypique riche en glutamine. Nous avons pu montrer que cette extension est coupée au cours de l'activation de la voie Toll. L'analyse de cette coupure a révélé une complexité et une relation inattendues entre les cascades protéolytiques activées respectivement par les bactéries à Gram positif et par les champignons. Une autre cascade protéolytique activée au cours de la réponse immunitaire des insectes mène à la coupure et à l'activation de l'enzyme de la mélanisation : la Phénoloxydase (PO). Nous avons isolé les premiers mutants de la cascade protéolytique aboutissant à l'activation de cette enzyme chez la drosophile. La première mutation affecte une serpine (la Serpine27A). Les mutants pour cette serpine présentent une activation constitutive de la PO en absence de stimulus immun. L'étude de la Serpine27A a révélé que l'activation de la PO nécessite une synthèse protéique préalable et qu'elle est sous le contrôle de la voie Toll. La deuxième mutation affecte une protéase à sérine, PAE1 (Prophenoloxidase Activating Enzyme 1) Les mutants PAE1 ne présentent aucune activation de la PO lors d'une infection. Leur étude nous a permis de montrer que l'activation de l'enzyme dépend d'une cascade protéolytique, ce qui n'était que supposé auparavant<br>Arthropods defend themselves against microbial aggression with an efficient battery of reactions, including activation of proteolytic cascades leading to coagulation, melanization and the production of antimicrobial peptides. These proteolytic cascades use serine proteases and they are regulated by serine protease inhibitors, such as the serpins. During my thesis, I studied the role of serpins and proteases in the Drosophila immune response. First proteolytic cascade found to be involved in the Drosophila immune response leads to activation of the Toll pathway, which controls the expression of numerous genes including those for the antimicrobial peptides. Spaetzle, the Toll ligand, is present as a precursor in Drosophila haemolymph. This precursor is proteolytically cleaved during Gram-positive bacterial or fungal infections, under the control of the serpin, Necrotic. Absence of Necrotic leads to constitutive activation of the Toll pathway and the development of a phenotype consisting of necrosis and adult mortality a few days after emergence. During a genetic suppressor screen using the Necrotic phenotype, Persephone, the first serine protease to be directly implicated in the immune activation of Spaetzle, was identified. Unexpectedly, the analysis of psh mutants revealed that two distinct proteolytic cascades can trigger immune activation of Spaetzle, dependant on either Gram-positive bacterial or fungal infection. In its N-terminal region, the serpin Necrotic has an atypically glutamine-rich extension. I was able to show that this section is removed during activation of the Toll pathway. A further analysis of this event revealed an unexpected level of complexity in relation with the proteolytic cascades activated by infections of Gram-positive bacteria or fungi. Another proteolytic cascade stimulated during the insect immune response leads to cleavage and activation of phenoloxidase (PO), the central enzyme of melanization. I isolated the first mutants involved in the proteolytic cascade controlling activation of PO in Drosophila. The first of these mutants, which displays a constitutive activation of PO, affects the Serpin27A gene. The study of Serpin27A revealed that protein production under the control of the Toll pathway must be underway before PO can be activated. The second mutant identified was in the gene encoding the serine protease Prophenoloxidase Activating Enzyme 1 (PAE1). This mutant has lost the capacity to activate PO during infections. The work carried out on PAE1 mutants allowed me to demonstrate that activation of PO in Drosophila depends on a proteolytic cascade

Orientador: Prof. Dr. Luciano Puzer<br>Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2018.<br>Serpinas sao proteinas inibidoras de serino-proteases, responsaveis pelo controle dos mais diversos processos fisiologicos e que cujo mecanismo de inibicao consite em formar um complexo covalente com a enzima alvo, em um processo de inibicao irreversivel. A alca de centro reativo (RCL) e responsavel por interagir com a protease, mimetizando um substrato, e quando clivada se insere como uma ¿À-fita no interior da serpina, trazendo a protease e causando uma distorcao no seu sitio ativo. Como as serpinas forma ligacoes covalentes com seus alvos durante a inibicao, essas proteinas tem sido utilizadas para o estudo do mecanismo de hidrolise das serino-proteases. No entanto, elas tambem oferecem a possibilidade do desenvolvimento de novas drogas contra enzimas relacionadas com patologias, como pode ser o caso da Vioserpina, uma serpina bacteriana capaz de inibir serino-proteases do tipo tripsina-like como a calicreina tecidual humana 5. Encontrar uma maneira de produzir uma Vioserpina fluorescente pode resultar no desenvolvimento de uma poderosa ferramenta para moniotramento e controle dessa e outras enzimas envolvidas com processos patologicos, como o antigeno prostatico especifico (PSA). Dessa maneira, realizamos mutacoes pontuais no gene da Vioserpina para a incorporacao do aminoacido cumarinico via par ortogonal tRNA/aaRS por supressao do codon ambar de parada. O aminoacido cumarinico e um aminoacido nao-canonico capaz de emitir fluorescencia na comprimento de onda de 450 nm quando excitado a 363 nm, permitindo o facil monitoramento e deteccao de uma proteina. Tres residuo distintos da Vioserpina foram escolhidos para a incorporacao do aminoacido cumarinico: Trp 208, Ile 342 (P4 da RCL) e Val 343 (P3 da RCL). A incorporacao nas posicoes Trp 208 e Ile 342 resultou em proteina fluorescentes, mas truncadas apos a insercao do aminoacido. Foi possivel obter uma Vioserpina completa com o aminoacido cumarinico na posicao Ile 342, embora com rendimento muito baixo. Tal mutante apresentou capacidade de inibicao e especificidade diferentes da Vioserpina wild type, em ensaios de inibicao enzimatica contra Tripsina e Quimotripsina, indicando que o aminoacido cumarinico em P4 parece causar diferentes interacoes no momento de inibicao da serpina. Para averiguar as melhores sequencias resultantes da incorporacao do aminoacido cumarinico, foi gerada uma biblioteca de genes da Vioserpina variando aleatoriamente as posicoes P4, P2, P1 e P1f da RCL, com incosporacao do aminoacido cumarinico na posicao P3 (Val 343). A biblioteca de Vioserpina foi apresentada por Phage Display no capsideo do fago M13 fusionada a proteina PIII, utilizando Biopanning para selecao das melhores sequencias com incorporacao do aminoacido nao-canonico, contra a serino-protease quimotripsina. A biblioteca de fagos gerou cinco sequencias com melhor inibicao que da Vioserpina, e mostrou que a insercao do aminoacido cumarinico parece acontecer com maior frequencia quando flanqueado por aminoacidos hidrofobicos ou sem cargas. A incorporacao do aminoacido cumarinico em sequencias da Vioserpina foi bem sucedida, sendo possivel avaliar a insercao de tal aminoacido na capacidade inibitoria dessa serpina. A investigacao da insercao de aminoacido nao-canonicos por Phage Display e Biopanning representa um grande passo para entender melhor como ocorre a incorporacao de tais aminoacidos, e quais residuos podem ocupar com maior naturalidade suas posicoes laterais.<br>Serpins are serine protease inhibitor proteins, responsible for the control of the most diverse physiological processes and whose mechanism of inhibition consists in forming a covalent complex with the target enzyme, in an irreversible inhibition process. The reactive center loop (RCL) acts as a bait for the protease, and when cleaved it inserts as a â-strand inside the molecule, distorting the protease active site. As serpins form covalent bonds with their targets, these proteins have been used to study the hydrolysis mechanism of serine proteases. However, they also offer the possibility of developing new drugs against disease-related enzymes, as is the case of Vioserpin, a bacterial serpin capable of inhibiting trypsin-like serine proteases such as human tissue kallikrein. Finding a way of producing fluorescent Vioserpin may result in the development of a powerful tool for monitoring and controlling this and other enzymes involved in pathological processes, such as prostate specific antigen (PSA). Thus, we performed point mutations in the Vioserpin gene for incorporation of the coumarin amino acid through tRNA/aaRS orthogonal pair by suppression of the amber stop codon. The coumarin amino acid is a non-canonical amino acid capable of emitting fluorescence at 450 nm when excited at 363 nm, allowing for easy detection of a protein. Three different residues of Vioserpin were chosen for incorporation of the coumarin amino acid: Trp 208, Ile 342 (P4 in RCL) and Val 343 (P3 in RCL). Incorporation at positions Trp 208 and Ile 342 resulted in fluorescent but truncated proteins following amino acid insertion. A complete Vioserpin bearing the coumarin amino acid at the Ile 342 position was possible, although at very low yield. Such a mutant exhibited different inhibition and specificity compared to Vioserpin wild type in enzymatic inhibition assays against trypsin and chymotrypsin, indicating that the coumarin amino acid at P4 appears to cause different interactions during serpin inhibition. To ascertain the best sequences resulting from coumarin amino acid incorporation, a Vioserpin gene library was generated by randomly varying the P4, P2, P1 and P1' positions in RCL, with coumarin amino acid incorporated at the P3 (Val 343) position. The Vioserpin library was exhibited by Phage Display in the M13 phage capsid fused to the PIII protein, using Biopanning against chymotrypsin to select the best sequences with incorporation of the non-canonical amino acid. The phage library generated five sequences with better inhibition than Vioserpin, and pointed to coumarin amino acid insertion appearing to occur more frequently when flanked by hydrophobic or uncharged amino acids. The incorporation of the coumarin amino acid into Vioserpin sequences was successful, and it is possible to evaluate the insertion of such amino acid in the inhibitory capacity of this serpin. The investigation of non-canonical amino acid insertion by Phage Display and Biopanning represents a great step to better understand how the incorporation of such amino acids occurs, and which residues may more naturally occupy their lateral positions