Abstract Cardiovascular diseases are the leading causes of death in the developed countries and their incidence is not expected to decrease in the future. There is a lifetime risk of one in five of developing heart failure, which still has poor prognosis and current treatments only cover part of the pathophysiology behind this syndrome. Pathological processes contributing to heart failure include cardiac hypertrophy and remodeling, which involves neurohumoral activation, reactivation of fetal genes, impaired Ca2+ cycling, increased apoptosis, and increased fibrosis. Intracellular signalling pathways and transcription factors mediating the response to various extracellular stresses have a key role in the regulation of myocardial remodeling and they are investigated in order to develop new approaches for the treatment of heart failure. The aim of this thesis was to elucidate roles of mitogen-activated protein kinases (MAPKs) and transcription factor GATA-4 in the regulation of cardiomyocyte function in cell cultures, and in hearts ex vivo and in vivo. The main findings were that (i) Inhibition of p38α MAPK enhanced function of sarco/endoplasmic reticulum Ca2+ -ATPase and thus cardiac contractility by increasing phosphorylation of protein phosphatase inhibitor-1 and phospholamban, (ii) p38 MAPK isoforms p38α and p38β regulated promoter activity of B-type natriuretic peptide via distinct pathways, (iii) p38α and p38β MAPKs also had different effects on gene expressions related to fibrosis and hypertrophy, and (iv) p38 and ERK1/2 MAPKs mediated stretch-induced activation of GATA-4 by phosphorylation at Ser 105. GATA-4 also seems to be regulated by ubiquitination. This study provides novel data of p38 MAPK and GATA-4 in the regulation of cardiomyocyte function. Inhibition of p38α MAPK could be beneficial in the treatment of heart failure. Also GATA-4 is a potential target for treatment of cardiovascular diseases. / Tiivistelmä Sydän- ja verisuonisairaudet ovat yleisin kuolinsyy länsimaissa, eikä niiden ilmaantuvuus tule vähenemään lähitulevaisuudessa. Elinikäinen riski sairastua sydämen vajaatoimintaan on 20 %, ja sydämen vajaatoiminnan ennuste on edelleen huono. Nykyisillä hoitomuodoilla voidaan puuttua vain osittain sydämen vajaatoiminnan patofysiologisiin mekanismeihin. Sydämen vajaatoiminnan kehittymiseen liittyvät sydänlihaksen liikakasvu ja uudelleenmuovautumisprosessi, johon liittyy neurohumoraalinen aktivaatio, sikiöaikaisten geenien uudelleenilmentyminen, häiriöt solunsisäisessä Ca2+-viestinnässä sekä lisääntynyt ohjelmoitu solukuolema ja sidekudoksen muodostuminen sydämeen. Solunsisäisillä viestinvälitysketjuilla sekä transkriptiotekijöillä, jotka vastaavat solunulkoisten ärsykkeiden välittämisestä solun sisällä, on keskeinen rooli edellämainittujen prosessien säätelyssä. Uusien lähestymistapojen kehittäminen sydämen vajaatoiminnan hoitoon edellyttää myös solunsisäisen viestinvälityksen ja geenien säätelyn mekanismien selvittämistä. Tämän väitöstyön tavoite oli selvittää p38 mitogeeniaktivoituvan proteiinikinaasin (p38 MAPK) ja transkriptiotekijä GATA-4:n merkitystä sydämen vajaatoiminnan patogeneesissä soluviljelymalleissa. Päälöydöksiä olivat: (i) p38α MAPK -isoformin estäminen paransi kalsiumia solulimakalvostoon pumppaavan SERCA2a:n toimintaa ja sydänlihassolun supistumiskykyä lisäämällä fosfolambaanin ja proteiinifosfataasi-inhibiittori-1:n fosforylaatiota. (ii) p38 MAPK isoformit p38α ja p38β säätelivät B-tyypin natriureettisen peptidin geenin promoottorialuetta erillisten reittien kautta. (iii) p38α ja p38β isoformit vaikuttivat myös eri tavoin sydämen sidekudoksen muodostumiseen ja hypertrofiaan liittyvien geenien ilmentymiseen. (iv) p38 ja ERK1/2 välittävät venytyksen aiheuttaman GATA-4:n aktivaation fosforyloimalla seriini-105 fosforylaatiopaikan. Lisäksi GATA-4:n toimintaa säädellään ubiquitinaation avulla. Tämä tutkimus tuo uutta tietoa p38 MAPK:n ja GATA-4:n rooleista sydämen vajaatoiminnan kehittymisessä. p38α-isoformin toiminnan estäminen voisi olla hyödyllinen hoitomuoto sydämen vajaatoiminnassa. Myös GATA-4 on potentiaalinen lääkehoidon kohde sydänsairauksien hoidossa.

Les cascades de Mitogen-Activated Protein Kinases (MAPKs) sont présentes chez tous les eucaryotes et permettent la transduction des signaux de l’extérieur vers l’intérieur de la cellule. Chez les végétaux, elles sont très abondantes et actives dans une multitude de processus, autant lors de la réponse aux stress que lors du développement. Elles fonctionnent comme un système de phosphorelais, se transférant un groupement phosphate d’une protéine à l’autre, de la MAPKKK à la MAPKK (MKK), puis de la MKK à la MAPK (MPK) et finalement, de la MPK vers des facteurs de transcription ou toute autre protéine qui permettra un changement au niveau de la réponse cellulaire. Depuis quelques années, plusieurs membres de la grande famille des MAPKs ont été étudiés pour leur rôle dans la reproduction sexuée des végétaux. Des mutants ont été caractérisés, mais jusqu’à maintenant, peu de voies complètes ont été décelées. Des précédents travaux dans le laboratoire ont démontré que deux MAPKKKs, de la sous-famille des MEKKs, ScFRK1 et ScFRK2, sont importantes pour le développement normal de l’ovule et du pollen chez Solanum chacoense, une espèce de pomme de terre sauvage diploïde. Sachant que les mutants des gènes les plus orthologues chez Arabidopsis thaliana ne possèdent pas les mêmes phénotypes, nous avons émis l’hypothèse que les Solanacées, du moins S. chacoense, possèdent une famille de MAPKKKs différente, qui n’est pas présente chez A. thaliana. Nous avons donc analysé les génomes/transcriptomes/protéomes de 15 espèces issues de différents clades du règne végétal afin d’étudier les relations phylogénétiques à l’intérieur de la sous-famille des MEKKs. Cela nous a permis d’observer que ScFRK1 et ScFRK2 ne sont pas seuls, mais sont inclus dans un groupe monophylétique que nous avons nommé la classe des FRKs (FRK pour Fertilization-Related Kinase). De plus, nous avons observé une expansion considérable de cette classe chez les Solanacées, comparativement à d’autres dicotylédones comme le peuplier, la vigne ou le coton. La classe des FRKs est absente chez les monocotylédones étudiées (riz et maïs) et ne possède qu’un seul membre (une FRK primitive) chez l’angiosperme basal Amborella trichopoda. Cette analyse phylogénétique des MEKKs nous a poussés à nous poser des questions sur l’origine de la classe des FRKs ainsi que sur son rôle au sein des Solanacées. Dans un deuxième temps, nous avons fait la caractérisation fonctionnelle de ScFRK3, un troisième membre de la classe des FRKs chez S. chacoense, aussi impliqué dans le développement des gamétophytes mâle et femelle. Du patron d’expression jusqu’à l’établissement d’une voie de signalisation potentielle, en passant par la caractérisation phénotypique des mutants, plusieurs expériences ont été réalisées dans le but de comprendre le rôle de ScFRK3 au niveau de la reproduction chez S. chacoense. Dans un contexte plus global, il est important de se questionner sur les rôles semblables, mais forcément différents, des trois membres de la famille FRKs qui ont été caractérisés jusqu’à présent. / Mitogen-Activated Protein Kinases (MAPKs) signaling cascades are found in all Eucaryotes and allow signal transduction from the outside of the cell to the inside. In plants, they are particularly numerous and play roles in several signaling processes, including stress responses and response to developmental cues. Their system involves a phosphorelay: they interact with each other to transfer a phosphate group. It starts with an activated MAPKKK, which transfers the phosphate group to a MAPKK (MKK), then this MKK transfers the signal to a MAPK (MPK), which ends this relay by phosphorylating transcription factors or any other proteins that will, in a way or an other, change the cell response according to the signal. During the last few years, many MAPKs members have been studied for their role in plants sexual reproduction. Some mutants were characterized, but until now, our knowledge of complete signaling cascades is very limited. Previous studies in our lab have shown that two MAPKKKs from the MEKK subfamily, ScFRK1 and ScFRK2, are important for male and female gametophytes development in Solanum chacoense, a wild diploid potato species. Genes that are the most orthologous to ScFRK1 and ScFRK2 in Arabidopsis thaliana, AtMAPKKK19, 20 and 21, do not seem to play the same roles in reproduction, which led us to make the hypothesis that in solanaceous species, at least in S. chacoense, there is one MAPKKK family that is different and not present in A. thaliana. At first, we did analyze the genomes/transcriptomes/proteomes of 15 species from different clads of the plant kingdom to find all the members of the MEKK subfamily of MAPKKKs in order to study their phylogenetic relationship. We then observed that ScFRK1 and ScFRK2 are included in a large monophyletic group which was called the FRK class (Fertilization Related Kinase). Moreover, we also observed that this class has considerably expanded within the solanaceous species, compared to other species like A. thaliana, poplar, cotton or grape vine. The FRK class is totally absent in the monocot species studied (rice and maize) and only one member is found in the basal angiosperm Amborella trichopoda. This phylogenetic analysis led us to ask questions about the origins of the FRK class and its role inside the Solanaceae family. Secondly, we characterized ScFRK3, a third member of the FRK class in S. chacoense, which is also involved, as its two FRK sisters, in male and female gametophytes development. From its expression pattern to the establishment of a potential signaling cascade, analysis and phenotyping of ScFRK3 mutant lines, many experiments were realized in order to understand the role of ScFRK3 in S. chacoense sexual reproduction. Overall, the appearance of this new and expanded class of MEKKs questions its specific role in comparison to other species that have much lesser members, mainly when compared to the model plant A. thaliana, which harbor only a fifth of the FRKs found in solanaceous species.

Les voies de transduction du signal cellulaire permettent aux cellules de répondre aux signaux de l'environnement et de les traiter. Les voies de transduction de kinases MAP (MAPK) sont bien conservées dans toutes les cellules eucaryotes et sont impliquées dans la régulation de nombreux processus cellulaires importants. Les régions intrinsèquement désordonnées (RID), présentes dans de nombreuses MAPK, n'étaient pas encore structurellement caractérisées. Les RID de MAPK sont particulièrement importantes car elles contiennent des motifs de liaison qui contrôlent les interactions entre les protéines MAPK elles-mêmes et aussi entre les protéines MAPK et d'autres protéines contenant les mêmes motifs. La résonance magnétique nucléaire (RMN) en combinaison avec d'autres techniques biophysiques a été utilisée pour étudier les RID de kinase des voies de transduction du signal MAPK. La spectroscopie RMN est bien adaptée pour l'étude des protéines intrinsèquement désordonnées à l'échelle atomique. Les déplacements chimiques et couplages dipolaires résiduels peuvent être utilisés conjointement avec des méthodes de sélection d'ensemble pour étudier la structure résiduelle dans les RID. La relaxation de spin nucléaire nous renseigne sur les mouvements rapides. Des titrations par RMN et des techniques de spectroscopie d'échange peuvent être utilisées pour surveiller la cinétique d'interactions protéine-protéine. Cette étude contribuera à la compréhension du rôle des RID dans les voies de transduction du signal cellulaire. / Protein signal transduction pathways allow cells respond to and process signals from the environment. A group of such pathways, called mitogen-activated protein kinase (MAPK) signal transduction pathways, is well conserved in all eukaryotic cells and is involved in regulating many important cell processes. Long intrinsically disordered region (IDRs), present in many MAPKs, have remained structurally uncharacterised. The IDRs of MAPKs are especially important as they contain docking-site motifs which control the interactions between MAPK proteins themselves and also between MAPKs and other interacting proteins containing the same motifs. Nuclear magnetic resonance (NMR) spectroscopy in combination with other biophysical techniques was used to study IDRs of MAPKs. NMR spectroscopy is well suited for studying intrinsically disordered proteins (IDPs) at atomic-level resolution. NMR observables, such as for example chemical shifts and residual dipolar couplings, can be used together with ensemble selection methods to study residual structure in IDRs. Nuclear spin relaxation informs us about fast pico-nanosecond motions. NMR titrations and exchange spectroscopy techniques can be used to monitor kinetics of protein-protein interactions. The mechanistic insight into function of IDRs and motifs will contribute to understanding of how signal transduction pathways work.

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-05-21T13:12:10Z No. of bitstreams: 1 Vers?o completa da disserta??o-stefaniemuraro.pdf: 4426933 bytes, checksum: 5733e09060e6e08135de26c11374b171 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-05-28T17:29:36Z (GMT) No. of bitstreams: 1 Vers?o completa da disserta??o-stefaniemuraro.pdf: 4426933 bytes, checksum: 5733e09060e6e08135de26c11374b171 (MD5) / Made available in DSpace on 2018-05-28T17:34:08Z (GMT). No. of bitstreams: 1 Vers?o completa da disserta??o-stefaniemuraro.pdf: 4426933 bytes, checksum: 5733e09060e6e08135de26c11374b171 (MD5) Previous issue date: 2018-03-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Respiratory syncytial virus (RSV) is a major cause of diseases of the respiratory tract in humans being mainly associated with bronchiolitis, chronic obstructive pulmonary disease (COPD) and asthma exacerbation. RSV infection occurs primarily in pulmonary epithelial cells and, once infection is established, an innate immune response is triggered and mainly neutrophil recruitment is induced. Neutrophils can extrude neutrophil extracellular traps (NETs) capable of entrapping and inactivate a multitude of microorganisms because of its composition and due to the stringy nature of DNA fibers. Recently, was demonstrated that RSV particles and its fusion (F) protein were able to induce the release NETs coated with neutrophil elastase and myeloperoxidase, both antimicrobial peptides. Also, was observed that the excessive formation of NETs can have negative consequences to the host, such as airway obstruction during RSV infection. Therefore, the aim was to evaluate the mechanisms involved in NET formation induced by RSV infection of neutrophils, alveolar epithelial cells (A549) or lung fibroblasts (MRC5). Human neutrophils were infected with RSV and were able to induce NETs release only after 3 hours of stimulation indicating classical NETosis. Next was characterized NETs formation during infection associating DNA extrusion with MPO, NE and F protein of RSV. Was also observed NADPH oxidase and PAD4 dependence and PI3K/AKT, ERK and p38 MAPK pathways during infection. The inhibition of these signaling pathways, PAD4 and ROS production abolished NET formation. Considering a possible involvement of necroptosis during NETs production, were tested MLKL and RIPK inhibitors and evaluated LDH release in the supernatant of infected neutrophils. Neutrophils released LDH and depend on necroptosis induction to produce NETs. Likewise, neutrophils were co-cultured with A549 or MRC5 cells infected with RSV. Both A549 and MRC5 cells triggered NET release by human neutrophils in a virus concentration-dependent manner, the opposite occurs when used UV-inactivated virus. Briefly, RSV induces the classical/ROS-dependent NETosis by human neutrophils, and this effect relies on specific kinases activity. Furthermore, neutrophils are able to recognize pulmonary cells infected by RSV, releasing NETs. Thus, NETs release control could be crucial for minimizing tissue inflammation caused by RSV infection. / O v?rus sincicial respirat?rio (VSR) ? uma das principais causas de doen?as do trato respirat?rio em humanos sendo associado principalmente com bronquiolite, doen?a pulmonar obstrutiva cr?nica (DPOC) e exacerba??o de asma. O VSR infecta principalmente c?lulas epiteliais pulmonares e, uma vez que a infec??o ? estabelecida, uma resposta imune inata ? desencadeada e ocorre o recrutamento de c?lulas do sistema imune, principalmente neutr?filos. Os neutr?filos podem liberar redes extracelulares de neutr?filos (NETs) capazes de capturar e inativar uma grande quantidade de microrganismos devido ? sua composi??o e natureza fibrosa das fibras de DNA. Recentemente, foi demonstrado que part?culas do VSR al?m da prote?na de fus?o (F) do v?rus foram capazes de induzir a libera??o de NETs revestidas com elastase neutrof?lica e mieloperoxidase, ambos pept?deos com atividade antimicrobiana. Al?m disso, observou-se que a forma??o excessiva de NETs pode ter consequ?ncias negativas para o hospedeiro, como a obstru??o das vias a?reas durante a infec??o por VSR. Portanto, o objetivo foi avaliar os mecanismos envolvidos na forma??o de NET induzida pela infec??o por RSV em neutr?filos humanos, c?lulas epiteliais alveolares (A549) ou fibroblastos pulmonares (MRC5). Neutr?filos humanos foram infectados com VSR e foram capazes de induzir a libera??o de NETs somente ap?s 3 horas de infec??o, indicando uma NETose cl?ssica. Em seguida, foi caracterizada a forma??o de NETs durante a infec??o associando a extrus?o de DNA com as prote?nas MPO, NE e com a prote?na F do VSR. Tamb?m se observou a depend?ncia de NADPH oxidase e PAD4 e das vias de sinaliza??o PI3K / AKT, ERK e p38 MAPK durante a infec??o. A inibi??o dessas vias de sinaliza??o, da produ??o de PAD4 e de EROs aboliu a forma??o de NET. Considerando um poss?vel envolvimento da necroptose na produ??o de NETs, foram utilizados inibidores de MLKL e RIPK1 e foi avaliada a libera??o de LDH no sobrenadante de neutr?filos infectados. Os neutr?filos liberaram LDH e dependeram da ativa??o da necroptose para produzir NETs. Do mesmo modo, os neutr?filos foram co-cultivados com c?lulas A549 ou MRC5 infectadas com VSR. Ambas as c?lulas A549 e MRC5 desencadearam a libera??o de NET por neutr?filos humanos de uma maneira dependente da concentra??o de v?rus, o oposto ocorreu quando usado um v?rus UV-inativado. Resumidamente, o VSR induz a NETose cl?ssica / dependente de EROs em neutr?filos humanos, e este efeito depende de atividade espec?fica de quinases. Al?m disso, os neutr?filos s?o capazes de reconhecer c?lulas pulmonares infectadas pelo VSR, induzindo a libera??o NETs. Assim, o controle de libera??o de NETs pode ser crucial para minimizar a inflama??o do tecido causada pela infec??o por VSR.

A subfamília p21 RAS de pequenas GTPases, incluindo KRAS, HRAS e NRAS, participa de muitas redes de sinalização, incluindo proliferação celular, organização do citoesqueleto e apoptose, e é o alvo mais freqüente de mutações ativadoras em câncer. Mutações germinativas em KRAS e HRAS causam graves anormalidades desenvolvimentais levando às síndromes de Noonan, cárdio-facial-cutânea e Costello, porem mutações ativadoras germinativas em NRAS não foram descritas até hoje. A síndrome autoimune linfoproliferativa (ALPS) é o mais comum defeito genético de apoptose linfocitária, cursando com autoimunidade e acúmulo excessivo de linfócitos, particularmente do tipo T + CD4- CD8-. As mutações causadoras de ALPS descritas até hoje afetam a apoptose mediada por Fas, uma das vias extrínsecas de apoptose. Nós demonstramos aqui que os principais achados clínicos de ALPS, bem como uma predisposição para tumores hematológicos, podem ser causados por uma mutação heterozigota ativadora G13D no oncogene NRAS, sem causar prejuízo na apoptose mediada por Fas. O aumento na quantidade intracelular de NRAS ativo, ligado a GTP, induziu a um aumento da sinalização na via RAF/MEK/ERK, o que suprimiu a expressão da proteína pró-apoptótica BIM, e atenuou a apoptose intrínseca mitocondrial. Desta forma, uma mutação germinativa ativadora em NRAS causou um fenótipo clinico diferente do visto em pacientes com mutações em outros membros da família p21 RAS, cursando com um defeito imunológico seletivo, sem distúrbios generalizados do desenvolvimento / The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4-, CD8- ab T cells. Mutations in ALPS typically affect Fas-mediated apoptosis, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair Fas-mediated apoptosis. The increase in active, GTP-bound NRAS augmented RAF/MEK/ERK signaling which markedly decreased the pro-apoptotic protein BIM and attenuated intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects

A infecção persistente por HPV-16 está fortemente associada ao risco de desenvolvimento de neoplasias do colo do útero, vagina, vulva, pênis, canal anal e orofaringe. O estudo detalhado da variabilidade nucleotídica intra-típica de HPV-16 resultou em importantes achados no que concerne à filogenia e evolução viral, e à história natural das infecções. Variantes Asiático-Americanas (AA) e E-350G de HPV-16 foram associadas com maior risco de persistência da infecção viral e desenvolvimento de câncer de colo de útero quando comparadas à variante Européia protótipo (E-P ou E-350T), embora esta ainda apresente alto risco quando comparada aos outros tipos virais. Mais recentemente, diferenças funcionais entre as proteínas E6/E7 das distintas variantes moleculares de HPV- 16 estão sendo descritas, a fim de explicar as diferenças nas associações epidemiológicas observadas. Dados do nosso grupo apontaram para a transcrição aumentada do gene MEK2 especificamente em queratinócitos humanos primários (PHKs) transduzidos com E6/E7 da variante E-350G. Pelo exposto, objetivou-se: (1) Analisar os níveis de ativação de proteínas efetoras das vias de transdução de sinal mediadas por MAPK e PI3K/AKT em queratinócitos imortalizados por E6/E7 de três variantes moleculares de HPV-16 (AA, E-P, E-350G); (2) Analisar os efeitos das proteínas E6/E7 dessas variantes sob as vias de MAPK quanto à indução de fatores de transcrição; (3) Analisar o potencial transformante de PHKs imortalizados pelas diferentes variantes, e em cooperação com a proteína celular c-MYC; (4) Analisar o potencial de migração e invasão em PHKs imortalizados pelas diferentes variantes de HPV-16, e em cooperação com a proteína celular c-MYC. Neste estudo observou-se que a variante AA de HPV-16 induziu a maior ativação das vias de sinalização estudadas (MAPK, e PI3K/AKT). Ademais, PHKs imortalizados por esta variante apresentaram maior capacidade de migração, de invasão através de uma matriz de colágeno, além de maior potencial transformante. Adicionalmente, as células imortalizadas pela variante AA apresentaram maior expressão da proteína mesenquimal vimentina e diminuição dos níveis da proteína epitelial E-caderina, sugerindo ativação parcial de Transição Epitélio Mesênquima (EMT) nestes queratinócitos. Ademais, quando o oncogene c-MYC foi co-transduzido nas diferentes linhagens infectadas por E6/E7 de HPV-16, foi observado que em PHKs imortalizados pela variante AA também houve maior ativação da via de MAPK-ERK, maior migração, e um potencial transformante semelhante, em relação às células co-transduzidas pela variante E-350G e c-MYC. Em conjunto, estes dados sugerem que a variante AA de HPV-16 possui vantagem seletiva sob as outras variantes em promover transformação celular, migração e invasão, e isto poderia explicar, ao menos em parte, a maior prevalência desta variante no câncer cervical. Os resultados gerados neste estudo são de extrema relevância para avaliar o impacto da variabilidade intra-típica de HPV-16 sobre o potencial oncogênico observado em estudos epidemiológicos / Persistent infection with HPV-16 is strongly associated with risk of developing neoplasia in the uterine cervix, vagina, vulva, penis, anal canal and oropharynx. The detailed study of HPV-16 intra-typical nucleotide variability resulted in important findings regarding phylogeny and viral evolution, and the natural history of infections. Asian-American (AA) and E-350G variants of HPV-16 were associated with increased risk of persistent viral infection and development of cervical cancer compared to the European prototype (E-P or E-350T), although this variant still presents higher risk when compared to other viral types. More recently, functional differences between the E6/E7 proteins of distinct molecular variants of HPV-16 are being described, in order to explain the differences in the epidemiological associations observed. Data from our group pointed to increased transcription of the MEK2 gene specifically in primary human keratinocytes (PHKs) transducing E6/E7 of the E-350G variant. Consequently, the aims of this study were: 1) To examine the activation levels of effector proteins of the signal transduction pathways mediated by MAPK and PI3K/AKT in PHKs immortalized by E6/E7 of three different molecular variants of HPV-16 (AA, E-P, E-350G); (2) To analyze the effects of E6/E7 of different molecular variants of HPV-16 upon MAPK pathways concerning the induction of transcription factors; (3) To analyze the transforming potential of PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC; (4) To analyze the potential of migration and invasion in PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC. In this study we observed that the AA variant of HPV-16 induced higher activation of both signaling pathways studied (MAPK, and PI3K/AKT). Furthermore, this variant presented increased migration capacity, higher invasion through a collagen matrix, and greater transforming potential. Moreover, cells immortalized by the AA variant showed higher expression of the mesenchymal protein vimentin and a decrease of the epithelial protein E-cadherin, suggesting partial activation of Epithelial Mesenchymal Transition (EMT). In addition, when the c-MYC oncogene was co-transduced in the different cells lines infected with HPV-16 E6/E7, we observed that in PHKs immortalized by the AA variant there was also an enhanced activation of the MAPK-ERK pathway, a higher ability to migrate, and similar transformation potential in comparison with cells co-transduced with the E-350G variant and c-MYC. Taken together, this data suggest that the AA molecular variant of the HPV-16 has a selective advantage over the other variants to promote cell transformation, migration and invasion, and this could partly explain the higher prevalence of this variant in cervical cancer. The results generated in this study are very important to assess the impact of intra-typical variability of HPV-16 on the oncogenic potential observed in epidemiological studies

L'action de la dopamine dans les cellules est transmise par sa fixation sur des récepteurs qui appartiennent à deux classes, D1 et D2. Quatre sous-types du récepteur D1 (D1A, D1B/D5, D1C et D1D) ont été clonés jusqu'à présent chez les vertébrés. L'analyse évolutive montre que les sous-types D1A et D1B sont les plus conservés alors que les sous-types D1C et D1D sont absents chez les mammifères. Malgré cette diversité, les fonctions de la dopamine dans l'organisme ne peuvent pas être rapportées à l'action d'un sous-type précis. C'est pourquoi au cours de ce travail nous avons identifié des caractères fonctionnels capables de distinguer chacun des sous-types et de comprendre pour quelle raison ils ont été conservés chez les vertébrés. La désensibilisation est un des paramètres fonctionnels les plus important. Le récepteur D1A se caractérise par une baisse d'activité forte et biphasique, le récepteur D1B par un profil proche avec une amplitude plus faible conséquence de son activité constitutive. Enfin, le récepteur D1C ne semble pas être capable de se désensibiliser. La construction de chimères entre chacun des sous-types du récepteur D1 et la protéine GFP ont permis la visualisation des récepteurs au cours de la désensibilisation. Elles montrent que l'internalisation ne semble pas, pour les récepteurs D1 de la dopamine, intervenir dans le processus de désensibilisation fonctionnelle. L'activation simultanée des récepteurs A1 de l'adénosine bloque l'activité des récepteurs D1. L'analyse des voies de signalisation MAPK a montré que l'activation de la voie ERK était rapide et forte, et différente selon les sous-types. La voie p38 n'est que faiblement activée et la voie JNK semble ne pas l'être du tout. Il semble donc que les mécanismes d'activation et de régulation des voies de signalisation différencient les sous-types du récepteur D1 chez les vertébrés. Ces paramètres participent de façon majeure à la transmission régulée des fonctions de la dopamine dans l'organisme.

<p>Transforming growth factor-β (TGF-β) is a member of a large superfamily of cytokines which participate in many different types of cellular processes, such as growth inhibition, cell migration, differentiation, cell adhesion, wound healing and immunosuppression. Alterations of TGF-β superfamily signalling results in several different disorders, including bone disease, vascular disease and cancer. The TGF-β signalling pathways involve several different proteins, such as the Smad proteins, which upon receptor activation are translocated to the nucleus, where they affect transcriptional responses. </p><p>The actin cytoskeleton is an organised network of filaments with a highly dynamic structure, which is under a continuous reconstruction to control the morphology, survival, growth and motility of eukaryotic cells. The members of the family of small GTP-binding proteins have been shown to be important regulators of the actin cytoskeleton.</p><p>TGF-β was found to induce short term as well as long term actin reorganisation in prostate cancer cells. The short term response included membrane ruffling, and required signalling by the small GTPases Cdc42 and Rho as well as, the involvement of the mitogen-activated protein kinases p38 (p38 MAPK). The long term response included formation of stress fibers and required a cooperation between Smad and Rho GTPase signalling pathways involving the Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). </p><p>The TGF-β-induced activation of Cdc42 was, furthermore, shown to require the inhibitory Smad7 and p38 MAP kinase, via a PI3K-dependent pathway. Mixed lineage kinase 3 (MLK3), a mediator downstream of Cdc42, was necessary for the Cdc42-dependent actin filament reorganisation.</p><p>Apoptosis is an important and carefully regulated process in human development and disease, which allows the multicellular organisms to remove cells that are in excess or potentially dangerous. TGF-β family members can induce apoptosis in many different cell types, in the presence or absence of other growth factors. Smad7 had previously been shown to be necessary for TGF-β-induced apoptosis of epithelial cells. We could show that Smad7 is required for TGF-β-induced activation of the TGF-β activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK pathway, which subsequently leads to apoptosis in prostate cancer cells.</p><p>Members of the lymphoid enhancer factor-1/T-cell factor (LEF1/TCF) family of transcription factors have, together with β-catenin, been shown to be nuclear effectors in the Wnt-signalling pathway. We investigated a possible cross-talk between the TGF-β and Wnt signalling pathways. We found that TGF-β, in a Smad7-dependent manner induced a nuclear accumulation of β-catenin and enhanced the transcriptional activity of β-catenin and the induction of the downstream target gene <i>c-myc</i>. Since β-catenin and c-Myc has been shown to promote apoptosis, our results suggests the possibility that β-catenin contributes to TGF-β-induced apoptosis</p>

<p>Diabetes is a major risk factor for ischemic stroke and is associated with increased mortality. Additionally, hyperglycemia, a common complication in acute stroke, is associated with poor outcome.</p><p>In order to identify the correlation between blood glucose and early mortality, multiple logistic regression analyses were used and odds ratios calculated in a retrospective study of 447 stroke patients. Eighty-one patients (18%) had diabetes. The odds ratios for 30-day case-fatality and blood glucose were 1.9 and 1.6 in diabetic and non-diabetic patients respectively. Optimal blood glucose concentrations in respective group were 10.3 and 6.3 mmol/L, as determined by receiver operator characteristic (ROC) curves.</p><p>Cerebral ischemia triggers different signaling pathways including mitogen-activated protein kinases (MAPK) which regulate fundamental cell functions. In an experimental rat model of combined hyperglycemia and transient middle cerebral artery occlusion (MCAO), the activation pattern of one such MAPK, extracellular signal-regulated kinase (ERK) was studied along with infarct volumes and neurological function. Hyperglycemia resulted in markedly increased ERK activation and approximately three-fold increase of infarcts compared with controls. </p><p>Based on the increased ERK activation, further experiments were conducted to limit the hyperglycemic-ischemic damage by interfering with ERK and supposedly related mechanisms. Consequently, rats were given U0126 (inhibiting ERK activation), PBN (anti-oxidative), PP2 (inhibiting src-family kinases), or vehicle. PBN reduced infarcts and improved neurological function compared with controls while no statistically significant effects were observed for U0126 or PP2. However, when the dose was doubled, U0126 significantly reduced infarcts and improved neurological function after 1 day in hyperglycemic rats. Post-ischemic ERK activation was completely inhibited by U0126 as demonstrated with Western immunoblotting. The findings suggest that ERK is an important mediator of hyperglycemic-ischemic brain injury and possible target for future interventions.</p>

Transforming growth factor-β (TGF-β) is a member of a large superfamily of cytokines which participate in many different types of cellular processes, such as growth inhibition, cell migration, differentiation, cell adhesion, wound healing and immunosuppression. Alterations of TGF-β superfamily signalling results in several different disorders, including bone disease, vascular disease and cancer. The TGF-β signalling pathways involve several different proteins, such as the Smad proteins, which upon receptor activation are translocated to the nucleus, where they affect transcriptional responses. The actin cytoskeleton is an organised network of filaments with a highly dynamic structure, which is under a continuous reconstruction to control the morphology, survival, growth and motility of eukaryotic cells. The members of the family of small GTP-binding proteins have been shown to be important regulators of the actin cytoskeleton. TGF-β was found to induce short term as well as long term actin reorganisation in prostate cancer cells. The short term response included membrane ruffling, and required signalling by the small GTPases Cdc42 and Rho as well as, the involvement of the mitogen-activated protein kinases p38 (p38 MAPK). The long term response included formation of stress fibers and required a cooperation between Smad and Rho GTPase signalling pathways involving the Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). The TGF-β-induced activation of Cdc42 was, furthermore, shown to require the inhibitory Smad7 and p38 MAP kinase, via a PI3K-dependent pathway. Mixed lineage kinase 3 (MLK3), a mediator downstream of Cdc42, was necessary for the Cdc42-dependent actin filament reorganisation. Apoptosis is an important and carefully regulated process in human development and disease, which allows the multicellular organisms to remove cells that are in excess or potentially dangerous. TGF-β family members can induce apoptosis in many different cell types, in the presence or absence of other growth factors. Smad7 had previously been shown to be necessary for TGF-β-induced apoptosis of epithelial cells. We could show that Smad7 is required for TGF-β-induced activation of the TGF-β activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK pathway, which subsequently leads to apoptosis in prostate cancer cells. Members of the lymphoid enhancer factor-1/T-cell factor (LEF1/TCF) family of transcription factors have, together with β-catenin, been shown to be nuclear effectors in the Wnt-signalling pathway. We investigated a possible cross-talk between the TGF-β and Wnt signalling pathways. We found that TGF-β, in a Smad7-dependent manner induced a nuclear accumulation of β-catenin and enhanced the transcriptional activity of β-catenin and the induction of the downstream target gene c-myc. Since β-catenin and c-Myc has been shown to promote apoptosis, our results suggests the possibility that β-catenin contributes to TGF-β-induced apoptosis