Thesis (doctoral)--Universität Hamburg, 1980.

Les kallicréines tissulaires humaines (KLK) ont récemment émergé comme une famille de protéases à serine pouvant jouer un rôle important dans la tumorigenèse. Le but de cette étude a été de mieux comprendre comment KLK6 et KLK12 pourraient intervenir dans la physiopathologie pulmonaire. Nous avons démontré qu’une expression ectopique de KLK6 induisait la prolifération des cellules A549 d’une manière dépendante de son activité enzymatique, une résistance à l’apoptose, ainsi qu’une translocation nucléaire de la β-caténine. Nous avons également montré une voie de signalisation impliquée dans la prolifération induite par KLK6 impliquant PAR2, EGFR et ERK. Nous avons identifié de nouveaux subtrats pour KLK12 : les CCN. Nous avons démontré que le clivage de CCN1 et CCN5 par KLK12 limitait leurs fixations avec le VEGF, BMP2 et le TGF-β1. / Recently, human tissue kallikreins (KLK) emerged as a new family of serine proteases which might play a major role in the tumorigenesis. The project aims at determining the contribution of KLK6 and 12 in lung pathophysiology. We showed that ectopic KLK6 promoted A549 cell proliferation in a protease activity-dependant manner, inhibited cell apoptosis and induced β-catenin nuclear translocation. Furthermore, this study uncovered a signaling pathway mediated by KLK6 in promoting A549 cell proliferation trough activation of the PAR2-EGFR-ERK pathway.We have also identified novel substrates of KLK12, the CCN family. We reported that KLK12-mediated proteolysis of CCN1 and CCN5 can reduce or abolish the binding of VEGF, BMP2, and TGF-β1.

O melanoma malígno está entre os cânceres que mais têm aumentado nas últimas décadas representando um grande desafio terapêutico. Quando diagnosticado precocemente, as chances de cura por excisão cirúrgica com margens de segurança adequadas são altas. Entretanto, casos avançados de melanoma são resistentes às formas atuais de terapia; assim, um dos maiores desafios para a pesquisa em melanoma é a identificação de alvos moleculares para o desenvolvimento de novas estratégias de tratamento. A capacidade de impedir o desenvolvimento de um tumor depende do melhor entendimento das vias celulares e moleculares que operam no microambiente tumoral. Uma inflamação crônica e persistente contribui para o desenvolvimento do câncer, e mesmo tumores que não são epidemiologicamente ligados a patógenos, são caracterizados pela presença de componentes inflamatórios em seu microambiente. O Sistema Calicreínas-Cininas (SCC) é responsável por uma série de efeitos biológicos, como vasodilatação, modulação da dor e inflamação, contração/relaxamento da musculatura lisa e efeitos sobre a proliferação celular. A participação do receptor B1 de cininas é bem relacionada a processos inflamatórios; contudo, a relação entre o SCC e câncer ainda é pouco descrita na literatura. Com relação ao melanoma, não existem na literatura, estudos que relacionam a participação do SCC e essa patologia. Portanto, a identificação de mecanismos genéticos e de vias de sinalização que levam à formação e progressão tumoral é de extrema importância para um desenho racional de terapias. Assim, o objetivo desse trabalho foi estudar a participação do receptor B1 de cininas no desenvolvimento de melanoma. Primeiramente realizou-se ensaios in vitro com células de melanoma murino, B16F10, verificando-se a presença dos componentes do SCC nesta linhagem celular, bem como a capacidade de migração das células quando estimuladas com o agonista e antagonista do receptor B1. Posteriormente, induziu-se melanoma em animais selvagens e knockout para o receptor B1 e verificou-se expressão de citocinas, vias de proliferação e apoptose e vascularização nesses tumores a partir técnicas de PCR, western blotting e análise histológica. Observou-se que células B16F10 estimuladas com o agonista do receptor B1, diminuem a capacidade de migração. Tumores desenvolvidos em animais knockout para o receptor B1, possuem uma menor expressão gênica desse receptor quando comparados com tumores desenvolvidos em animais selvagens e apresentam vias de proliferação celular mais ativadas, além de uma vascularização irregular. Considerando esses resultados, sugerimos que o receptor B1 de cininas contribui para o impedimento da progressão tumoral, podendo, futuramente, ser um alvo terapêutico para o tratamento de melanoma. / Malignant melanoma is between the cancer types that most have been increased in the last decades, representing a therapeutic challenge. When it is early detected, chances of cure through surgical excisions with secure margins are high. However, advanced cases of melanoma are resistant to all types of therapies; thus, one of the most challenges for research in melanoma is the identification of molecular targets to further develop new strategies of treatment. The ability to blockade the development of a tumor depends on a better understanding of cellular and molecular pathways that operate in the tumor microenvironment. A chronic and persistent inflammation contributes to cancer development, and, even tumors that are not epidemiologically linked to pathogens present inflammatory components in their microenvironment. The Kallicrein-Kinin System (KKS) is responsible for several biological effects, like, vasodilatation, modulation of pain and inflammation, contraction/relaxation of smooth muscles and cell proliferation. The kinin B1 receptor is well related to inflammatory processes, however, the involvement of the KKS in cancer development is, yet, not well described in the literature. Regarding to melanoma, studies relating the involvement of the KKS in melanoma development is still not available. This way, identification of genetic mechanisms and signaling pathways that drive melanoma formation and progression is extremely important for designing rational therapies in the future. Thus, the aim of this study was evaluate the participation of the kinin B1 receptor in melanoma progression. First, in vitro assays with murine melanoma cells, B16F10, were performed to verify the presence of the KKS components in this cell lineage, as well as the capacity of migration when these cells are stimulated with the B1 receptor agonist and antagonist. Then, melanoma was induced in wild type and B1 receptor knockout mice and the expression of cytokines, proliferation and apoptosis pathways and vascularization were studied by PCR, western blotting and histological analyses. We observed that B16F10 cells stimulated with the B1 receptor agonist had their capacity of migration decreased. Tumors developed in B1 receptor knockout mice showed a lower expression of this gene comparing to the tumors developed in wild type animals, also presenting higher activation of proliferation pathways and abnormal vessels. Considering these results, we suggest that the kinin B1 receptor contributes to blockade, at least in part, the tumor progression which can, in the future, become a therapeutic target for melanoma treatment.

Leung Man-hin. / Thesis submitted in: August 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 119-133). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgement --- p.iv / Abbreviations --- p.v / Figure List --- p.vii / Chapter Chapter 1: --- General introduction / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Pathogenesis of Alzheimer's disease / Chapter 1.2.1 --- Amyloid cascade hypothesis --- p.2 / Chapter 1.2.2 --- Tauopathy --- p.5 / Chapter 1.3 --- The amyloid precursor proteins / Chapter 1.3.1 --- Structure of amyloid precursor proteins and related peptides --- p.5 / Chapter 1.3.2 --- Amyloid precursor protein mutations --- p.6 / Chapter 1.3.3 --- Amyloid precursor protein processing --- p.10 / Chapter 1.3.4 --- Physiological roles of APP --- p.12 / Chapter 1.3.5 --- The pathophysiological role of Ap --- p.17 / Chapter 1.3.6 --- The pathophysiological role of APP-CTF --- p.20 / Chapter 1.4 --- The role of proteases in amyloid precursor protein processing / Chapter 1.4.1 --- α-secretase and p-secretase --- p.22 / Chapter 1.4.2 --- γ-secretase complex --- p.30 / Chapter 1.4.3 --- Caspases --- p.36 / Chapter 1.4.4 --- Kallikrein-like proteases --- p.37 / Chapter 1.5 --- Objective of the present study --- p.40 / Chapter Chapter 2: --- Materials and methods / Chapter 2.1 --- Experimental procedure / Chapter 2.1.1 --- Plasmid construction --- p.42 / Chapter 2.1.2 --- "DNA purification, ligation and restriction enzyme digestion" --- p.44 / Chapter 2.1.3 --- Competent cell preparation --- p.47 / Chapter 2.1.4 --- Transformation --- p.47 / Chapter 2.1.5 --- Plasmid miniprep --- p.48 / Chapter 2.1.6 --- Prokaryotic expression of neurosin --- p.49 / Chapter 2.1.7 --- SDS-PAGE --- p.51 / Chapter 2.1.8 --- Protein sample preparation --- p.51 / Chapter 2.1.9 --- Western Blot --- p.52 / Chapter 2.1.10 --- Immobilized metal affinity chromatography --- p.54 / Chapter 2.1.11 --- Enzyme assay --- p.55 / Chapter 2.1.12 --- Cell culture and transfection --- p.56 / Chapter 2.1.13 --- Live cell imaging --- p.57 / Chapter 2.2 --- Materials --- p.59 / Chapter Chapter 3 : --- Results / Chapter 3.1 --- Recombinant expression and characterization of neurosin / Chapter 3.1.1 --- Construction of neurosin prokaryotic expression vectors --- p.62 / Chapter 3.1.2 --- Prokaryotic expression of neurosin --- p.64 / Chapter 3.1.3 --- Neurosin was expressed as inclusion bodies --- p.68 / Chapter 3.1.4 --- Co-expression of molecular chaperones with neurosin --- p.70 / Chapter 3.1.5 --- Purification of recombinant neurosin by IMAC --- p.76 / Chapter 3.1.6 --- Enzyme assay --- p.81 / Chapter 3.2 --- Effect of neurosin on APP processing in neuronal cells / Chapter 3.2.1 --- Generation of APP constructs --- p.84 / Chapter 3.2.2 --- Expression of APP in mammalian cultures --- p.89 / Chapter 3.2.3 --- Cellular localization of APP and its processing products --- p.96 / Chapter 3.2.4 --- The role of over-expression of neurosin on APP processing in B103 cells --- p.101 / Chapter Chapter 4: --- Discussion / Chapter 4.1 --- Discussion of neurosin expression --- p.103 / Chapter 4.2 --- Discussion of APP cell model --- p.109 / Chapter 4.3 --- Conclusion --- p.119 / References --- p.121 / Appendix / Chapter A. --- Tables on primers used --- p.138 / Chapter B. --- Plasmid maps --- p.139 / Chapter C. --- Raw data on the DNA sequencing --- p.141

Στο πλαίσιο της παρούσης διατριβής, αξιοποιήθηκαν οι αλληλουχίες γνωστών γονιδιωμάτων, αλλά και γονιδιωμάτων που αποκρυπτογραφήθηκαν πρόσφατα, για να μελετηθεί η εξελικτική ιστορία τριών λειτουργικά σημαντικών πρωτεϊνικών οικογενειών: (α) των φυτικών DNA μεθυλομεταφορασών και (β) των ευκαρυωτικών RNA μεθυλομεταφορασών, οι οποίες είναι ένζυμα που επιφέρουν μεθυλίωση στις νουκλεοτιδικές αλληλουχίες, καθώς και (γ) των πεπτιδασών συγγενών της καλλικρεΐνης (KLKs), οι οποίες είναι γνωστές σερινοπρωτεϊνάσες με δράση τύπου θρυψίνης ή χυμοθρυψίνης. Οι εξελικτικές σχέσεις των χαρακτηρισμένων και καινοφανών πρωτεϊνών των τριών οικογενειών διερευνήθηκαν με κατασκευή φυλογενετικών δένδρων. Επίσης, αναλύθηκε η δευτεροταγής και τριτοταγής δομή των ομόλογων πρωτεϊνών, η δομή των γονιδίων που κωδικοποιούν τις πρωτεΐνες αυτές, και κατασκευάσθηκαν διαγνωστικά πρωτεϊνικά μοτίβα από τις αλληλουχίες των τριών οικογενειών ενζύμων. Τα αποτελέσματα των αναλύσεων οδήγησαν στην εξαγωγή σημαντικών συμπερασμάτων σχετικά με την πιθανή βιολογική λειτουργία των καινοφανών πρωτεϊνών. Συγκεκριμένα, ομόλογες φυτικές DNA μεθυλομεταφοράσες και καινοφανείς ευκαρυωτικές RNA μεθυλομεταφοράσες ταυτοποιήθηκαν σε δημόσιες βάσεις δεδομένων. Λεπτομερής φυλογενετική ανάλυση οδήγησε στην ταυτοποίηση των τεσσάρων ήδη γνωστών οικογενειών φυτικών DNA μεθυλομεταφορασών και μιας καινοφανούς υποοικογένειας (Pavlopoulou and Kossida, 2007). Επίσης, ταυτοποιήθηκαν πέντε υποοικογένειες ευκαρυωτικών RNA μεθυλομεταφορασών. Πέραν των τριών ήδη γνωστών υποοικογενειών (NOP2, NCL1 και YNL022C), ταυτοποιήθηκε μια καινοφανής υποοικογένεια (RCMT9), και μια υποοικογένεια, FMU, η οποία εθεωρείτο ότι απαντάται αποκλειστικά σε προκαρυωτικούς οργανισμούς (Pavlopoulou and Kossida, 2009). Επιπλέον, κατασκευάσθηκαν πρωτεϊνικά αποτυπώματα της οικογένειας (και των επιμέρους υποοικογενειών) των RNA μεθυλομεταφορασών, τα οποία καταχωρήθηκαν στη δευτερογενή πρωτεϊνική βάση δεδομένων PRINTS (http://www.bioinf.man.ac.uk/dbbrowser/PRINTS). Στα πλαίσια της διατριβής, αναπτύχθηκε το υπολογιστικό πρόγραμμα RCMTHMM, με σκοπό τη διάκριση/ταυτοποίηση των ευκαρυωτικών RNA μεθυλομεταφορασών, το οποίο διατέθηκε για δημόσια χρήση στη διεύθυνση URL: http://www.bioacademy.gr/bioinformatics/RCMTHMM. Σημαντική συνεισφορά της παρούσας μελέτης είναι η αναδιατύπωση της εξελικτικής ιστορίας των καλλικρεϊνών (Pavlopoulou et al., 2010). Οι καλλικρεΐνες είναι σημαντικά πρωτεολυτικά ένζυμα που δρουν ατομικά ή σε πρωτεολυτικούς καταρράκτες και ρυθμίζουν σημαντικές φυσιολογικές λειτουργίες, ενώ η απορρυθμισμένη δράση τους έχει συνδεθεί με σοβαρές ασθένειες (καρδιοαγγειακές, νευροεκφυλιστικές, φλεγμονώδεις, δερματικές, διάφορους τύπους καρκίνου). Για την απομόνωση νέων καλλικρεϊνών αξιοποιήθηκε το γεγονός ότι τα KLK γονίδια συνεντοπίζονται στο ίδιο χρωμόσωμα υπό μορφή μη διακοπτόμενης συστάδας γονιδίων. Σε προηγούμενες μελέτες είχε προταθεί ότι οι καλλικρεΐνες απαντώνται μόνον στα θηλαστικά και ότι εμφανίσθηκαν πριν από περίπου 150 εκατομμύρια χρόνια. Στην παρούσα μελέτη, ομόλογες καινοφανείς ακολουθίες καλλικρεϊνών ανιχνεύθηκαν in silico στα γονιδιώματα διάφορων οργανισμών. Ορθόλογα των καλλικρεϊνών ταυτοποιήθηκαν για πρώτη φορά στα ερπετά, στα πτηνά και στα αμφίβια, υποδεικνύοντας την εξελικτική καταγωγή των καλλικρεϊνών πριν από 330 εκατομμύρια χρόνια. Επιπροσθέτως, πέραν των 15 γνωστών KLKs (KLΚ1-15), ταυτοποιήθηκαν τρία καινοφανή μέλη (ορφανές Klks) και με σύγκριση των προβλεπόμενων δομών δείχθηκε ότι τα δομικά χαρακτηριστικά που σχετίζονται με την καταλυτική δράση είναι συντηρημένα στις καινοφανείς πρωτεϊνικές αλληλουχίες KLK. Σημειωτέον, δείχθηκε ότι στα γονιδιώματα όλων των υπό εξέταση οργανισμών, τα ορθόλογα γονίδια KLK χαρτογραφούνται στην ίδια χρωμοσωμική περιοχή, διευθετημένα εν σειρά με τον ίδιο προσανατολισμό και χωρίς παρεμβολή μη καλλικρεϊνικών γονιδίωνΠροτείναμε ότι η οικογένεια των καλλικρεϊνών προέκυψε από μια σειρά γονιδιακών διπλασιασμών και μεταλλάξεων, και ότι οι καλλικρεΐνες έχουν συνεξελιχθεί με τα ειδικά υποστρώματά τους (Pavlopoulou et al., 2010). / In the present thesis, the availability of an increasing number of complete or almost complete genomes, including those that were completed recently, enabled the study of the evolutionary history of three functionally important protein families: (a) the plant DNA methyltransferases and (b) the eukaryotic RNA methyltransferases, which are enzymes that catalyze the transfer of a methyl group to nucleotide sequences, as well as (c) the kallikrein-related peptidases or KLKs, which are trypsin- or chymotrypsin-like serine proteases. The evolutionary relationships of the already known and the novel proteins of the three families that were identified here were investigated using phylogenetic trees. Moreover, the secondary and tertiary structures of the homologous proteins were analyzed, as well as the structure of the protein-encoding genes, and diagnostic protein motifs were constructed based on the sequences of the three enzyme families. Our results led to suggestions pertaining to the biological function of the identified novel proteins. In particular, homologous plant DNA methyltransferases and novel eukaryotic RNA methyltransferases were identified in publicly accessible sequence databases. Detailed phylogenetic analysis of plant DNA methyltransferases identified four already known families and a novel subfamily in addition (Pavlopoulou and Kossida, 2007). Moreover, five distinct eukaryotic RNA methyltransferase subfamilies were identified; apart from the three already known subfamilies (NOP2, NCL1 and YNL022C), one novel subfamily (RCMT9) and the FMU which hitherto was considered to exist exclusively in prokaryotes were also identified (Pavlopoulou and Kossida, 2009). Furthermore, protein fingerprints were constructed from the generic family of RNA methyltransferases (and the individual subfamilies), which were deposited in the PRINTS database (http://www.bioinf.man.ac.uk/dbbrowser/PRINTS). We developed the computational program RCMTHMM, in order to discriminate/identify eukaryotic RNA methyltransferases from other proteins. The RCMTHMM program has been made publicly available in the URL: http://www.bioacademy.gr/bioinformatics/RCMTHMM. Finally, the evolutionary history of KLKs was reconstructed. Kallikreins are important proteolytic enzymes which are involved in proteolytic cascade pathways and their dysregulated expression has been associated with major human pathologies (cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, skin diseases, different cancer types). The prominent feature of the kallikrein family is that it consists of tandemly and uninterruptedly arrayed genes on a single locus at human chromosome 19q13.3-13.4. This unique feature was used in order to identify novel KLKs and KLK-like genes/proteins. Previous studies on the evolution of kallikreins were restricted to mammals and the emergence of the kallikrein genes was suggested approximately 150 million years ago. In the present study, homologous novel kallikrein protein sequences were detected in silico in the genomes of various species. For the first time, novel KLK orthologues were identified in reptiles, aves and amphibia, which allowed us to trace the evolutionary origin of kallikreins 330 million years ago. In addition, apart from the 15 already known KLK genes (KLK1-15), three novel members were identified (orphan Klks). All the defining structural features which are related to the catalytic activity of KLKs were found to be conserved in the novel KLK protein sequences. Of particular interest, the synteny of the KLK-encoding genes was analyzed and it was shown that these genes are co-localized in contiguous, uninterrupted clusters maintaining the same orientation in all species under investigation. We suggest that a series of gene duplication and mutation events gave rise to the family of KLK enzymes and KLKs have co-evolved with their specific substrates (Pavlopoulou et al., 2010).

Orientador: Prof. Dr. Luciano Puzer / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / 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. / 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.

O melanoma malígno está entre os cânceres que mais têm aumentado nas últimas décadas representando um grande desafio terapêutico. Quando diagnosticado precocemente, as chances de cura por excisão cirúrgica com margens de segurança adequadas são altas. Entretanto, casos avançados de melanoma são resistentes às formas atuais de terapia; assim, um dos maiores desafios para a pesquisa em melanoma é a identificação de alvos moleculares para o desenvolvimento de novas estratégias de tratamento. A capacidade de impedir o desenvolvimento de um tumor depende do melhor entendimento das vias celulares e moleculares que operam no microambiente tumoral. Uma inflamação crônica e persistente contribui para o desenvolvimento do câncer, e mesmo tumores que não são epidemiologicamente ligados a patógenos, são caracterizados pela presença de componentes inflamatórios em seu microambiente. O Sistema Calicreínas-Cininas (SCC) é responsável por uma série de efeitos biológicos, como vasodilatação, modulação da dor e inflamação, contração/relaxamento da musculatura lisa e efeitos sobre a proliferação celular. A participação do receptor B1 de cininas é bem relacionada a processos inflamatórios; contudo, a relação entre o SCC e câncer ainda é pouco descrita na literatura. Com relação ao melanoma, não existem na literatura, estudos que relacionam a participação do SCC e essa patologia. Portanto, a identificação de mecanismos genéticos e de vias de sinalização que levam à formação e progressão tumoral é de extrema importância para um desenho racional de terapias. Assim, o objetivo desse trabalho foi estudar a participação do receptor B1 de cininas no desenvolvimento de melanoma. Primeiramente realizou-se ensaios in vitro com células de melanoma murino, B16F10, verificando-se a presença dos componentes do SCC nesta linhagem celular, bem como a capacidade de migração das células quando estimuladas com o agonista e antagonista do receptor B1. Posteriormente, induziu-se melanoma em animais selvagens e knockout para o receptor B1 e verificou-se expressão de citocinas, vias de proliferação e apoptose e vascularização nesses tumores a partir técnicas de PCR, western blotting e análise histológica. Observou-se que células B16F10 estimuladas com o agonista do receptor B1, diminuem a capacidade de migração. Tumores desenvolvidos em animais knockout para o receptor B1, possuem uma menor expressão gênica desse receptor quando comparados com tumores desenvolvidos em animais selvagens e apresentam vias de proliferação celular mais ativadas, além de uma vascularização irregular. Considerando esses resultados, sugerimos que o receptor B1 de cininas contribui para o impedimento da progressão tumoral, podendo, futuramente, ser um alvo terapêutico para o tratamento de melanoma. / Malignant melanoma is between the cancer types that most have been increased in the last decades, representing a therapeutic challenge. When it is early detected, chances of cure through surgical excisions with secure margins are high. However, advanced cases of melanoma are resistant to all types of therapies; thus, one of the most challenges for research in melanoma is the identification of molecular targets to further develop new strategies of treatment. The ability to blockade the development of a tumor depends on a better understanding of cellular and molecular pathways that operate in the tumor microenvironment. A chronic and persistent inflammation contributes to cancer development, and, even tumors that are not epidemiologically linked to pathogens present inflammatory components in their microenvironment. The Kallicrein-Kinin System (KKS) is responsible for several biological effects, like, vasodilatation, modulation of pain and inflammation, contraction/relaxation of smooth muscles and cell proliferation. The kinin B1 receptor is well related to inflammatory processes, however, the involvement of the KKS in cancer development is, yet, not well described in the literature. Regarding to melanoma, studies relating the involvement of the KKS in melanoma development is still not available. This way, identification of genetic mechanisms and signaling pathways that drive melanoma formation and progression is extremely important for designing rational therapies in the future. Thus, the aim of this study was evaluate the participation of the kinin B1 receptor in melanoma progression. First, in vitro assays with murine melanoma cells, B16F10, were performed to verify the presence of the KKS components in this cell lineage, as well as the capacity of migration when these cells are stimulated with the B1 receptor agonist and antagonist. Then, melanoma was induced in wild type and B1 receptor knockout mice and the expression of cytokines, proliferation and apoptosis pathways and vascularization were studied by PCR, western blotting and histological analyses. We observed that B16F10 cells stimulated with the B1 receptor agonist had their capacity of migration decreased. Tumors developed in B1 receptor knockout mice showed a lower expression of this gene comparing to the tumors developed in wild type animals, also presenting higher activation of proliferation pathways and abnormal vessels. Considering these results, we suggest that the kinin B1 receptor contributes to blockade, at least in part, the tumor progression which can, in the future, become a therapeutic target for melanoma treatment.

Orientadora: Profa. Dra. Maria Cristina Carlan da Silva / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, Santo André, 2018. / Calicreínas (KLKs) são uma família de serino-proteases com especificidade semelhante à tripsina ou quimotrispsina. Pertencem às calicreínas teciduais, 15 membros denominados KLK1 a KLK15. As KLKs estão envolvidas com diversas funções fisiológicas, como mecanismos que regulam a descamação da pele, formação do esmalte dentário, função renal, liquefação seminal, plasticidade sináptica neural e funcionamento cerebral. No sistema nervoso central (SNC), a calicreína predominantemente expressa é a KLK6, sendo que a desregulação dessa enzima pode estar envolvida com desordens como mal de Alzheimer, mal de Parkinson e esclerose múltipla, além de estar envolvida em processos de desmielinização. Alterações na expressão de KLK6 também podem estar relacionadas com a diminuição da sensibilidade de células tumorais à agentes citotóxicos como tratamentos quimioterápicos e radioterápicos. A KLK8 também é expressa no SNC em áreas do sistema límbico, sendo que sua desregulação está relacionada ao desenvolvimento de esquizofrenia. O Citomegalovírus Humano é um agente viral de alta prevalência mundial. Em casos de imunosupressão, o vírus pode causar sérias patologias e até levar a morte. O HCMV é capaz de infectar células do SNC e sua presença em tumores tem sido reportada em diversos estudos. É sugerido que o vírus possui um papel oncomodulatório, aumentando a malignidade tumoral. Gliomas são os tipos mais comuns de tumores do SNC, sendo o glioblastoma multiforme (GBM) o tipo mais maligno e infiltrativo de glioma, com baixa sobrevida. A presença do HCMV em gliomas foi reportada em diversos estudos e no Brasil por nosso grupo de pesquisa e investigações recentes estudam os possíveis mecanismos relacionadas ao vírus e a progressão tumoral. O HCMV é também capaz de modular a expressão de proteases celulares. Apesar disso, até o momento não existem relatos mostrando a relação entre HCMV e a expressão de KLKs. Esse trabalho teve como objetivo avaliar a expressão e atividade de KLKs durante a infecção de células de GBM pelo HCMV, bem como suas consequências na replicação viral e oncogênese. Os dados mostram que o HCMV induz a expressão de KLK6 em linhagens de GBM U138 e U251. / Kallikreins (KLKs) belong to a family of serine-proteases with trypsin-like or chymotrypsin-like specificities. Fifteen members compose the tissue kalikreins, named KLK1 to 15. The KLKs are involved in many physiological functions related to skin dequamation, teeth enamel formation, renal function, semen liquefaction, neural synaptic plasticity and brain functioning. In the central nervous system (CNS), KLK6 is highly expressed and its deregulation appears to be involved with disorders such as Alzheimer¿s disease, Parkinson¿s disease and multiple sclerosis. In addition, KLK6 overexpression can be involved with demyelinating processes. Changes in KLK6 expression are also related to the reduced sensibility of GBM cells to chemo and radiotherapy. The KLK8 is also expressed in CNS in certain areas of the limbic system and its deregulation is linked to the development of schizophrenia. Recent studies demonstrated the involvement of human kallikreins with replication of Human Papillomavirus and Influenza virus. The Human Cytomegalovirus (HCMV) is a viral agent highly prevalent worldwide. After primary infection the virus remains latent for life in the host and can be reactivated in immunosuppressed individuals, causing serious diseases, which can lead to death. The HCMV has tropism for CNS cells and many groups reported its presence in tumors. It is suggested that the virus can increase tumor malignity in a process denominated oncomodulation. Gliomas are the most common SNC tumor types, and Glioblastoma Multiforme (GBM) is the most malignant and infiltrative tumor, with an extremely low survival rate. Our group has reported the presence of HCMV in gliomas all over the world and specially In Brazil. It is known that HCMV is capable of modulate the expression of many cellular proteases, however there are no reports regarding the possible effect of the virus in kallikreins expression. The main goal of this work was to evaluate the expression and activity of kallikreins during HCMV infection in GBM cell lines. The data obtained show that the virus induces the expression of KLK6 in U138 and U251 GBM cell lines.

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2016-10-04T14:10:11Z No. of bitstreams: 1 anacarolinabuzzostefanini_dissert.pdf: 2564521 bytes, checksum: fc9c0d25882e400f1002a6ed99657319 (MD5) / Made available in DSpace on 2016-10-04T14:10:11Z (GMT). No. of bitstreams: 1 anacarolinabuzzostefanini_dissert.pdf: 2564521 bytes, checksum: fc9c0d25882e400f1002a6ed99657319 (MD5) Previous issue date: 2014-10-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction - The head and neck squamous cell carcinomas (HNSCCs) are among the most frequent types of cancer, with more than 600,000 new cases per year worldwide. The five-year survival rate is low in this disease and one of the reasons is that patients with tumours in early stages frequently exhibit few symptoms, resulting in diagnosis delay and severe morbidity. The understanding of the molecular pathways involved in the initiation and progression of these tumors is therefore important not only for understanding their biology, but also for the development of more effective preventive and therapeutic approaches. In previous studies of our group, the kallikrein 8 gene (KLK8) was observed differentially expressed in laryngeal carcinomas and its surgical margins, which highlights its potential as a tumor marker, similar to another member of KLK family, kallikrein 3 or PSA. Objectives and Methodology - The overall objective of the present study was to investigate the participation of the kallikrein 8 in the HNSCC development. The specific objectives included (a) to investigate the phylogenetic relationships among KLKs genes using the MEGA program, (b) to analyze, by real time PCR, the expression pattern of the KLK8 gene and its most abundant isoform in HNSCC and their surgical margins, (c) to evaluate the effect of elevated expression of KLK8 in HNSCC secretome, using conditioned medium and proteomic and metabolomic approaches, (d) to develop molecular homology modeling of protein hK8 by bioinformatics tools. Results - The phylogenetic analysis of human kallikrein genes and their isoforms confirmed the idea that these genes evolved from a single common ancestor by successive tandem duplications and chromosomal rearrangements facilitated by repetitive elements. The results of gene expression analysis in tumor tissues showed that the variant 1 of KLK8 has significantly reduced levels in HNSCC, unlike the other five variables. The ectopic expression of this variant resulted in changes of cell morphology, increased proliferation, viability and migratory capacity, but no alterations in invasiveness. The data from cells with ectopic expression of KLK8 revealed small differences in their proteomes compared to control cells. Otherwise, these cells exhibited changes in their glycolytic pattern and in the effect of their secretome on the metabolism of other cells. Conclusion - This study was important to answer some questions and raise others questions about the role of KLK8 gene in carcinomas of the head and neck. For the first time, differences in expression of KLK8 isoforms were observed in HNSCC and the effects of ectopic KLK8 expression on the proteome and the secretome of HNSCC cells. / Introdução - Os carcinomas epidermóides de cabeeça e pescoco (CECPs) estão entre os mais frequentes tipos de câncer, com mais de 600 mil novos casos por ano no mundo e taxas de sobrevida reduzidas. Além de sua incidência e mortalidade altas, os CECPs são clinicamente relevantes por causa de sua morbidade, em geral decorrente do diagnóstico tardio. O entendimento das vias moleculares envolvidas na iniciação e na progressão desses tumores é, portanto, importante, não somente para o entendimento de sua biologia, mas também para o desenvolvimento de abordagens preventivas e terapêuticas mais eficazes. Em estudos prévios do nosso grupo, o gene da calicreína 8 (KLK8) foi observado com expressão diferencial entre carcinomas de laringe e suas margens cirúrgicas, o que evidencia seu potencial como marcador tumoral, como ocorre com outro membro de sua família, a calicreína 3 ou PSA. Objetivos e Metodologia – O presente projeto teve como objetivo geral investigar a participação da calicreína 8 no desenvolvimento de CECPs. Seus objetivos específicos compreenderam (a) investigar as relações filogenéticas entre os genes KLKs com o auxílio do programa MEGA, (b) analisar, por PCR em tempo real, o padrão de expressão do gene KLK8 e de sua isoforma mais abundante em CECP e em tecidos normais correspondentes, (c) avaliar o efeito da expressão elevada de KLK8 no secretoma de células de CECP, utilizando ensaios com meio condicionado e abordagens proteômicas e metabolômicas, (d) desenvolver a modelagem molecular por homologia da proteína hK8 com ferramentas de bioinformática. Resultados – A análise filogenética da família de genes das calicreínas humanas e suas isoformas confirmou a idéia de que esses genes evoluíram de um único ancestral comum por sucessivas duplicações em tandem e rearranjos cromossômicos facilitados por elementos repetitivos. Os dados de expressão gênica em tecidos tumorais mostraram que a variante 1 de KLK8 apresenta níveis significativamente reduzidos em CECP, ao contrário das outras cinco variantes. A indução permanente in vitro desta variante resultou em mudança da morfologia celular, aumento de proliferação, viabilidade e capacidade migratória, sem aumento concomitante de invasividade. Os dados obtidos sobre essas células com expressão ectópica de KLK8 revelaram pequenas diferenças em seu proteoma quando comparadas com células controle. Por outro lado, exibiram modificação no padrão glicolítico e no potencial de seu secretoma de afetar o metabolismo de outras células. Conclusão - O presente estudo foi importante para responder a alguns questionamentos e levantar outras questões sobre a função do gene KLK8 em carcinomas de cabeça e pescoço. O estudo identificou, pela primeira vez, as diferenças de expressão entre as isoformas de KLK8 em CECP e os efeitos da indução de sua expressão sobre o proteoma e o secretoma de suas células.

Abstract Human prostate-specific antigen (hPSA, KLK3) and glandular kallikrein 2 (hK2, KLK2), two members of a large human tissue kallikrein enzyme family, were produced as recombinant mature proteins for the first time and characterized. Furthermore, their association with prostate cancer was studied. Both proteins were produced with baculovirus expression vector system in pilot-scale using bioreactors. Recombinant hPSA was either active with chymotrypsin-like activity or inactive with incorrect processing of N-terminus. The molecular weight of active recombinant hPSA was 31 kD and it formed stable complexes with serine protease inhibitors, α1-antichymotrypsin (ACT) and α2-macroglobulin (2αM). Two polymorphic forms of KLK2, Arg226hK2 and Trp226hK2, were found. The recombinant Arg226hK2 had trypsin-like activity, while recombinant Trp226hK2 was inactive. The Arg226hK2 was labile with low production yields. The molecular weights of hK2 polymorphic forms were 33 kD. hPSA isoforms secreted by prostate cancer cells, LNCaP, were isolated and characterized. These proteins were N-terminally heterogeneous: 10-60% of LNCaP-PSAs were correctly processed. Molecular modeling suggested that the additions or deletions of two or four N-terminal amino acids could affect the three-dimensional structure and reduce the activity of LNCaP-PSA. Active isoforms had chymotrypsin-like activity and formed stable complexes with ACT and 2αM. The expression of hPSA and hK2 was studied with in situ hybridization and immunohistochemistry techniques in benign and cancerous prostate tissue. hK2 mRNA was expressed at a significantly higher level in prostate cancer tissue than in benign prostate tissue (P < 0.0005). The hPSA mRNA expression levels were reversed (P = 0.06). In benign tissue, the mean level of hK2 mRNA was 82% of the respective value of hPSA (P < 0.003), whereas in tumor tissue the mean hK2 expression level was 21% higher than that of hPSA (P < 0.01). The results at protein level supported the mRNA findings. There was a correlation between hPSA and hK2 mRNA levels in both benign (r = 0.735; P < 0.01) and malignant (r = 0.767; P < 0.01) prostate tissue. It was shown with competitively differential PCR that the KLK2 gene was amplified in prostate tumor tissue, while the KLK3 gene was not. These results suggest that hK2 and hPSA have a diverse value in the diagnosis of prostate cancer.