The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

Abstract α-Methylacyl-CoA racemase (Amacr) is an enzyme at the merging point of two important pathways of lipid metabolism: elimination of methyl-branched fatty acids and synthesis of bile acids. Amacr is regarded as obligatory for these processes. Patients with Amacr-deficiency suffer from adult onset sensory motor neuropathy and/or severe neonatal cholestasis with coagulopathy and fat-soluble vitamin malabsorption. Amacr is also linked to cancer and so far has been proposed as a new marker for diagnosis of at least prostate and colon cancers. Common sources of phytol derived branched-chain fatty acids for man are ruminant fats, meat and dairy products. The bile acid synthesis is the main pathway for cholesterol catabolism. Amacr is considered to be a member of family III of the CoA transferases (L-carnitine dehydratase - bile acid inducible protein F (CaiB-BaiF) family) and localized to two subcellular compartments, mitochondria and peroxisomes. In this work the mouse gene encoding Amacr was characterized, the gene was inactivated and mutational and structural studies were used to determine the loop and the active site structure of the enzyme. It was shown that mouse Amacr which locates both to mitochondria and peroxisomes, is an identical product of a single gene, which is located at chromosome 15, region 15B1. Neither alternative replication, splicing, or any post-translational modifications of the enzyme occur. The mouse model for Amacr-deficiency indicated a role of Amacr in detoxification of methyl-branched fatty acids, and suggested that a diet free from these phytol metabolites may function as a treatment for the deficiency. Furthermore, major changes were observed in the bile acid pool of the knock-out mice compared to wild type mice. However, the study suggests that there is an Amacr-independent pathway for synthesis of bile acids albeit of low capacity, which provides a way for Amacr-deficient individuals to survive. The mutational and structural studies confirmed Amacr as a member of family III of the CoA transferases. Furthermore, according to comparisons of the structural data of Amacr and other members of the family (FRC, YfdW), the superfamily can be divided into two subgroups, racemases and transferases. Proteins in the subfamilies share the CoA-binding mode, but the substrate specificities as well as the catalysed reaction differ greatly.

Submitted by JESSICA KIRSCH MICHELETTI null (jessicamicheletti@hotmail.com) on 2016-01-07T18:04:37Z No. of bitstreams: 1 JÉSSICA KIRSCH MICHELETTI.pdf: 1986588 bytes, checksum: dd0a0d26b8a3217f5d310580899619c8 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-01-08T15:53:32Z (GMT) No. of bitstreams: 1 micheletti_jk_me_prud.pdf: 1986588 bytes, checksum: dd0a0d26b8a3217f5d310580899619c8 (MD5) / Made available in DSpace on 2016-01-08T15:53:32Z (GMT). No. of bitstreams: 1 micheletti_jk_me_prud.pdf: 1986588 bytes, checksum: dd0a0d26b8a3217f5d310580899619c8 (MD5) Previous issue date: 2015-12-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A imersão em água fria (IAF) aparece no cenário atual como uma técnica recuperativa eficaz no meio esportivo, atuando sobre diferentes desfechos. Entretanto, o conceito de recuperação pós-exercício merece destaque, uma vez que análise de parâmetros isolados parece ferir esse conceito. Para tanto, a construção de um desenho a partir de um único mecanismo de estresse, com a mesma característica de aplicação de técnica e envolvendo elementos de dimensões diversas parece pertinente. Objetivo: analisar a recuperação após um treino imediato e verificar o comportamento isolados e em conjunto dos parâmetros clínicos, funcionais, metabólico e autonômico a partir do uso da IAF como técnica recuperativa. Métodos: Amostra composta por 64 jogadores de futebol do sexo masculino, randomizados em dois grupos, grupo controle (GC) e grupo experimental (GE). Os procedimentos foram realizados em duas etapas. 1ª: Participantes foram submetidos a testes basais de função muscular. 2ª: Após uma semana de descanso, foram submetidos a um treino chave (50 minutos) e imediatamente após realizaram a intervenção por 15 minutos, GE recebeu a IAF (13º±1ºC) e GC permaneceu sentado. As variáveis investigadas foram percepção subjetiva de dor, percepção de recuperação, alteração de sensibilidade, concentração de lactato sanguíneo, modulação autonômica cardíaca e os testes funcionais, estas foram mensuradas durante momentos específicos da recuperação até ao máximo 2 horas pós-treino chave. Para análise estatística utilizou-se o pacote estatístico SPSS Statistics 22.0. Para os dados numéricos, nos marcadores funcionais, a distribuição dos dados foi testada (Komolgorov-Smirnov) e como normal utilizou-se o teste t de Student para amostras independentes. Para os demais marcadores foram testados a esfericidade dos dados (teste de Mauchly). Os dados foram analisados utilizando a Análise de Variância para Medidas Repetidas (pós-teste de Bonferroni). Para os dados dicotomizados foi realizado para os dados de frequências relativa (%) da pontuação total (todos os desfechos) o teste Mann-Whitney. E para a frequência absoluta (de casos) o teste de qui-quadrado. Para calcular a probabilidade de recuperação foi utilizada regressão logística. Resultados: Dados numéricos: Para os desfechos clínicos, não foram observadas diferenças estatisticamente significantes entre os grupos, e ambos se recuperam no mesmo momento (15 minutos pós-treino chave). Para o desfecho metabólico, ambos os grupos se recuperaram no mesmo momento (2 horas pós-exercício). No desfecho função, não houve diferenças significantes entre momentos e entre grupos. Para análise da variabilidade da frequência cardíaca observou-se antecipação da recuperação de todos os índices no GE comparado ao GC. Dados dicotomizados: A análise em conjunto das categorias de variáveis não demonstrou melhor eficácia da técnica de IAF, em que o GC se recuperou 63,4% e o GE: 69,17. Quando avaliado os sistemas isolados há uma melhor probabilidade de chance da técnica em ser melhor para desfechos metabólico e autonômico. Conclusões: Gerais: Quando analisado em conjunto as categorias dos desfechos, as técnicas apresentam recuperações próximas, sem diferenças estatísticas. Específicos: Quando analisado as categorias de desfechos isoladas há elevada probabilidade de recuperação em até 2 horas pós-treino chave para participantes dos dois grupos nas variáveis clínicas, metabólica, autonômica e funcionais, exceção para o desfecho contração isométrica voluntária máxima (na análise dos dados dicotomizados). Para os conjuntos de variáveis metabólicas e autonômicas a probabilidade de recuperação mostrou-se maior para o GE (na análise dos dados dicotomizados). Por fim, o sistema autonômico é beneficiado com a técnica em ambas as análises. / Cold water immersion (CWI) appears in the current scenario as an effective recuperative technique in sports, working on different outcomes. However, the concept of post-exercise recovery deserves attention, since analysis of individual parameters seem to hurt this concept. Therefore, the construction of a drawing from a unique mechanism of stress, with the same technique application feature and involving elements of various dimensions seems pertinent. Objective: To analyze the immediately recovery after a training and to verify the isolated and combined behavior of clinical, functional, metabolic and autonomic parameters from the use of immersion in cold water as recuperative technique. Methods: A sample of 64 football players, randomized into two groups, control group (CG) and experimental group (EG). The procedures were performed in two stages. 1st: Participants underwent basic tests of muscle function. 2nd: After a week of rest, underwent a key workout (50 minutes) and immediately after the intervention performed for 15 minutes, GE received the IAF (13 ± 1 ° C) and GC stay sitting. The investigated variables were subjective perception of pain, perceived recovery, abnormal sensitivity, blood lactate concentration, cardiac autonomic modulation and functional tests, these were measured during specific recovery times up to a maximum two hours post training. Statistical analysis was performed using the statistical package SPSS Statistics 22.0. For numeric data, the functional markers, the distribution of the data was tested (Komolgorov-Smirnov) and as normal we used the t student test for independent samples. For the other markers were tested sphericity data (Mauchly test). Data were analyzed using analysis of variance for repeated measures (Bonferroni post-test). For dichotomized data was performed for data on frequency (%) of total score (all outcomes) Mann-Whitney test. And the absolute frequency (cases) chi-square test. To calculate the probability of recovery was used logistic regression. Results: numerical data: For clinical outcomes, statistically significant differences were observed between the groups, and both recover at the same time (15 minutes post training). For metabolic outcomes, both groups recovered at the same time (2 hours after exercise). The function outcome, there were no significant differences between moments and between groups. For the analysis of heart rate variability was observed anticipation of recovery of all indices in the EG compared to the CG. Dichotomized data: Analysis together variable categories did not show better efficacy CWI technique where the GC has recovered 63.4% and GE: 69,17. When assessed isolated systems there is a better chance of technique likely to be better for metabolic and autonomic outcomes. Conclusions: General: When analyzed together the categories of outcomes, the techniques presented close recoveries, with no statistical differences. Specific: When analyzed the categories of isolated outcomes there is high probability of recovery within 2 after training hours for participants of the two groups in clinical, metabolic, autonomic and functional, except for the outcome of maximal voluntary isometric contraction (in the analysis of dichotomized data). For groups of metabolic and autonomic variables the probability of recovery was higher for the GE (in the analysis of dichotomized data). Finally, the autonomic system is benefit from the technique in both analyses.

Made available in DSpace on 2016-06-02T19:48:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-12-16 / The postoperative period requires the nursing team to be prepared to deal with possible specific complications. The precision of the nursing diagnosis during that period is essential for ensuring an effective and decisive care plan. The postoperative recovery may be changed, causing it to be delayed and, therefore, to require specific nursing intervention. The human response is represented by NANDA-I, through the Delayed Surgical Recovery (DSR) diagnosis. Based on the analysis of the elements of the DSR diagnosis, the possibility of gaps may be considered, especially gaps that are related to the defining characteristics (DC) and the related factors (RF). This study aimed at analyzing the concept and validation (by experts) of the Delayed Surgical Recovery nursing diagnosis, which is proposed by NANDA-I. That is a descriptive validation study of a nursing diagnosis, based on the model that was proposed by Hoskins. A bibliographical review was accomplished on LILACS, PubMed, CINAHL and Cochrane Library databases, in order to select studies that are related to the theme, for to ground the concept analysis, performed according to the Walker and Avant model, which describe an investigation process on basic elements of an eight-step concept. Fifty-seven studies were elected to conduct the concept analysis. Posteriorly, a content validation of the nursing diagnosis was made according to the Fehring model, in which the opinion of experts is surveyed, regarding the adequacy of the DSR nursing diagnosis. The DSR concept is found to be used in medicine, followed by nursing, psychology, social service, speech therapy, nutrition and physical therapy. DSR is usually discussed in relation to the deterioration of physical, psychological and social functioning interrelationship of patients and their relatives. The identified main defining attributes were: impaired ability to walk, need for help to perform daily activities, difficulty in performing selfcare, difficulty in reassuming social roles, persistent fatigue, necessity of a longer time for recovery, and others. Among the identified background items, there were: pain, anxiety, negative experiences on previous surgeries, infection, and others. The main consequences were: rehospitalization, prolonged hospitalization, depression, and others. Given the concept analysis data, some modifications on the enunciation and definition of DSR diagnosis were suggested, as well as some modifications on the Defining Characteristics (among them: delays the return to social activities and persistent pain ), and some suggestions for including DC (such as need for help to perform daily activities , cardiovascular and pulmonary problems ). The inclusion of Related Factors as advanced age , problems arising from anesthesia and presence of comorbidities were also suggested. The elements of the nursing diagnosis and the proposed modifications after the concept analysis were subjected to the review of 45 experts. As a result, changes were made on the diagnosis enunciation text which was modified to Postoperative Delayed Recovery. The nursing diagnosis definition text was changed to extension of the number of days of mediate or delayed postoperative to resume activities that sustain life, health and welfare , validation of the DC proposed by NANDA-I, modification of the DC text, such as Delays the return to social work, job, familiar, study, religious activities, etc. , Loss of appetite , Postoperative persistent pain and Persistent fatigue . Yet, it was possible to add six DCs for suggestion to be included in the Nursing Diagnosis of NANDA-I need for help to perform daily activities , Cardiovascular problems and Pulmonary problems were the ones that received the highest scores. As for the RF, all the ones that were presented by NANDA-I were validated and seven new RF were suggested to be included. Among the ones that received the highest scores by the experts are: Problems arising from anesthesia , Presence of comorbidities , Compromised nutritional conditions and Advanced age . The identified results offer the possibility of reviewing the DSR diagnosis by NANDA-I, contributing to knowledge about postoperative period and, consequently providing a better quality of nursing care to surgical patients who experience delayed recovery. / O período pós-operatório requer preparo por parte da equipe de enfermagem para atender possíveis complicações específicas deste período. A precisão dos diagnósticos de enfermagem durante este período é fundamental para garantir um efetivo e resolutivo planejamento de cuidados. A recuperação pós-operatória pode sofrer interferências resultando em um tempo maior para que ocorra e, portanto, requerer intervenções de enfermagem específicas. Esta resposta humana está representada pela NANDA-I através do diagnóstico de enfermagem Recuperação Cirúrgica Retardada (RCR). A partir da análise dos elementos diagnóstico RCR considera-se a possibilidade de existência de lacunas, especialmente em relação às características definidoras (CD) e fatores relacionados (FR). Este estudo teve como objetivo realizar a análise de conceito e validação por peritos do diagnóstico de enfermagem Recuperação Cirúrgica Retardada proposto pela NANDA-I. Trata-se de um estudo descritivo de validação de diagnóstico de enfermagem, com base no modelo proposto por Hoskins. Foi realizada uma revisão bibliográfica nas bases de dados LILACS, PubMed, CINAHL e Cochrane Library com o intuito de selecionar estudos envolvendo o tema estudado para fundamentar a análise de conceito, realizada segundo o modelo de Walker e Avant, que descrevem um processo de investigação de elementos básicos de um conceito desenvolvido em oito passos. Foram elegíveis para a condução da análise de conceito 57 estudos. Posteriormente, realizou-se a validação de conteúdo do diagnóstico de enfermagem conforme modelo de Fehring, em que se obtém a opinião de peritos quanto à adequação do diagnóstico de enfermagem RCR. Identifica-se que o conceito RCR foi utilizado principalmente pela medicina, seguido da enfermagem, psicologia, serviço social, fonoaudiologia, nutrição e fisioterapia. Em geral, a RCR é abordada quanto ao comprometimento da inter-relação do funcionamento físico, psicológico e social de pacientes e seus familiares. Os principais atributos definidores identificados foram: habilidade para caminhar comprometida, necessidade de auxílio para desempenhar atividades de vida diária, dificuldade para desempenhar o autocuidado, dificuldade em reassumir papéis sociais, persistência de fadiga, necessidade de mais tempo para recuperação, entre outros. Entre os antecedentes identificados apresentam-se: dor, ansiedade, experiências negativas de cirurgias prévias, infecção, entre outros. As principais conseqüências foram: reinternações hospitalares, internações prolongadas, depressão, entre outros. Diante dos dados da análise de conceito foram sugeridas alterações no enunciado e definição do DE RCR, alterações na redação de CD entre elas, adia o retorno para as atividades sociais e persistência de dor , ainda, sugestões de inclusão de CD com destaque para necessidade de auxílio para desempenhar atividades de vida diária , problemas cardiovasculares e pulmonares . Foi ainda sugerida a inclusão de FR como idade avançada , problemas decorrentes da anestesia e presença de comorbidades . Os elementos do DE e as alterações propostas após a análise de conceito foram submetidas à análise de 45 peritos. Como resultado foram realizadas adequações quanto a redação do enunciado diagnóstico que foi alterad para Recuperação Pós-operatória Retardada, adequações quanto a redação da definição do DE para extensão do número de dias de pós-operatório mediato e/ou tardio para reiniciar atividades que mantêm a vida, a saúde e o bem estar , validação das CD propostas pela NANDA-I, alteração da redação de CD como Adia o retorno às atividades sociais de trabalho, emprego, familiares, estudo, religiosas, etc , Perda de apetite , Persistência de dor pós-operatória e Persistência de fadiga . Ainda, foi possível acrescentar 6 CD para sugestão de inclusão ao DE pela NANDA-I Necessidade de auxílio para desempenhar atividades de vida diária , Problemas cardiovasculares e Problemas pulmonares foram as que receberam maiores escores. Quanto aos FR, todos os apresentados pela NANDA-I foram validados e 7 novos FR foram sugeridos para inclusão. Entre os que receberam maiores escores pelos peritos estão: Problemas decorrentes da anestesia , Presença de comorbidades , Condições nutricionais comprometidas e Idade avançada . Os resultados identificados oferecem possibilidade de revisão do diagnóstico RCR pela NANDA-I, contribuem com o conhecimento sobre o período pós-operatório e consequentemente, com uma melhor qualidade na assistência de enfermagem ao paciente cirúrgico que vivencia o prolongamento da recuperação.

Les MAP kinases sont des enzymes essentielles impliquées dans 7 voies de signalisation distinctes qui permettent à la cellule de répondre de manière adéquate aux stimuli extra-cellulaires. Chez les mammifères, les MAP kinases les mieux caractérisées sont Erk1/2, Jnk, p38 et Erk5. Ces enzymes jouent un rôle important dans l’embryogenèse, la prolifération et la différenciation cellulaire ainsi que dans la réponse au stress. Erk4 est un membre atypique de la famille MAP kinase. D’une part, la boucle d’activation de Erk4 possède un motif SEG au lieu du motif TXY, très conservé chez les MAP kinases. D’autre part, Erk4 possède une extension en C-terminal du domaine kinase qui n’est pas présente chez les MAP kinases classiques. Jusqu’à présent aucune fonction n’a été attribuée à Erk4. De plus, la voie de signalisation ainsi que le mode de régulation conduisant à l’activation de Erk4 ne sont pas connus. Le seul substrat de Erk4 identifié jusqu’à maintenant est la MAPKAP kinase MK5. L’impact fonctionnel de cette interaction n’est également pas connu. Afin d’en apprendre davantage sur la MAP kinase atypique Erk4, nous avons étudié le mécanisme d’activation de cette kinase ainsi que sa fonction physiologique par une approche de délétion génique chez la souris. En ce qui concerne l’activation de Erk4, nous avons montré que la boucle d’activation de Erk4 (S186EG) est constitutivement phosphorylée in vivo et que cette phosphorylation n’est pas modulée par les stimuli classiques des MAP kinases dont le sérum et le sorbitol. Cependant, nous avons observé que la phosphorylation de la S186 augmente en présence de MK5 et que cette augmentation est indépendante de l’activité kinase de l’une ou l’autre de ces kinases. De plus, nous avons établi que la phosphorylation de la boucle d’activation de Erk4 est requise pour l’interaction stable entre Erk4 et MK5 ainsi que pour l’activation, et la relocalisation cytoplasmique de MK5. Ainsi, notre étude a permis de révéler que Erk4 est régulée de manière différente des MAP kinases classiques et que la phosphorylation de la boucle d’activation de Erk4 joue un rôle essentiel dans la régulation de l’activité de MK5. Parallèlement, nos résultats mettent en évidence l’existence d’une “Erk4 kinase”, dont le recrutement et/ou l’activation semble être facilité par MK5. Afin identifier la fonction physiologique de Erk4, nous avons généré des souris Erk4-déficientes. L’inactivation génique de Erk4 est viable et les souris ne présentent aucune anomalie apparente. Dans le but d’expliquer l’absence de phénotype, nous avons regardé si l’expression de Erk3, le paralogue de Erk4, pouvait compenser la perte de Erk4. Notre analyse a révélé que l’expression de Erk3 dans les souris Erk4-/- n’augmente pas au cours du développement embryonnaire ou dans les tissus adultes afin de compenser pour la perte de Erk4. Par la suite, nous avons adressé la question de redondance entre Erk4 et Erk3. Dans notre laboratoire, les souris Erk3-déficientes ont également été générées et le phénotype de ces souris a récemment été analysé. Cette étude a révélé que l’inactivation génique de Erk3 entraîne un retard de croissance intra-utérin, un défaut de maturation pulmonaire et la mort néo-natale des souriceaux. Nous avons donc regardé la contribution de Erk4 dans ces phénotypes. L’analyse des souris Erk4-/- a révélé que l’inactivation de Erk4 n’entraîne pas de retard de croissance ou de maturation du poumon. De plus, nous avons montré que l’inactivation additionnelle de Erk4 dans les souris Erk3-/- n’accentue pas le phénotype des souris Erk3-déficientes. Ainsi, notre étude a révélé que contrairement à Erk3, Erk4 n’est pas essentielle au développement murin dans des conditions physiologiques. Parallèlement, nous avons montré que Erk4 et Erk3 possèdent des fonctions non-redondantes in vivo. / MAP kinases are essential enzymes implicated in 7 distinct signaling pathways that allow cells to respond appropriately to extracellular stimuli. In mammals, Erk1/2, Jnk, p38 and Erk5 are the best characterized MAP kinases. These enzymes play important roles in embryogenesis, cell proliferation and differentiation and in response to cellular stresses. Erk4 is an atypical member of the MAP kinase family. First, its activation loop is composed of an SEG motif instead of the well conserved TXY motif found in MAP kinases. Second, Erk4 has a C-terminal extension following the kinase domain that is not present in classical MAP kinases. Despite its identification more than a decade ago, the function of Erk4 remains elusive. Moreover, the signaling pathway as well as the regulatory mechanism leading to Erk4 activation in still uncharacterized. The only identified substrate of Erk4 is the MAPKAP kinase MK5, but the functional relevance of this interaction is still unknown. To gain information about the atypical MAP kinase Erk4, we decided to study the activation mechanism of Erk4 and its physiological function using a gene targeted deletion approach in mice. Regarding the activation of Erk4, we showed that the activation loop of Erk4 (S186EG) is constitutively phosphosphorylated in vivo and that this phosphorylation is not modulated by classical MAP kinase stimuli such as serum and sorbitol. However, we observed that phosphorylation of S186 increases in the presence of MK5 and we showed that this increase is independent of the kinase activity of either kinases. Moreover, we established that phosphorylation of Erk4 activation loop is required for the stable interaction between Erk4 and MK5 as well as for the activation and cytoplasmic relocalisation of MK5. Thus, our study reveals that Erk4 is differently regulated than classical MAP kinases and that Erk4 activation loop phosphorylation is important for its role in the regulation of MK5 activity. In parallel, our results revealed the existence of an “Erk4 kinase” whose recruitment and/or activation seems to be facilitated by MK5. To gain information about the physiological function of Erk4 we generated Erk4 deficient mice. Gene-targeted inactivation of Erk4 is viable and these mice present no gross abnormality. To explain the absence of phenotype, we analyzed the expression of Erk3, the paralog of Erk4, to determine if it could compensate for the loss of Erk4. Our analysis revealed that Erk3 expression in Erk4-/- mice is not up-regulated during embryogenesis nor in adult mice tissues in order to compensate for the loss of Erk4. We next addressed the question of redundancy between Erk4 and Erk3. In our laboratory, Erk3-/- deficient mice were also generated and the phenotype of these mice was recently analyzed. This study revealed that gene inactivation of Erk3 leads to intra-uterine growth retardation, lung maturation defect and neo-natal lethality. We then investigated the contribution of Erk4 in these phenotypes. The analysis of Erk4-/- mice revealed that inactivation of Erk4 did not delay intra-uterine growth nor cause pulmonary maturation defect. Moreover, we showed that additional loss of Erk4 in Erk3-/-mice does not accentuate Erk3-deficient mice phenotype. Thus, this study reveals that, contrary to Erk3, Erk4 is dispensable for mice development under normal condition and that Erk4 and Erk3 have non-redundant functions in vivo.

Les MAP kinases sont des enzymes essentielles impliquées dans 7 voies de signalisation distinctes qui permettent à la cellule de répondre de manière adéquate aux stimuli extra-cellulaires. Chez les mammifères, les MAP kinases les mieux caractérisées sont Erk1/2, Jnk, p38 et Erk5. Ces enzymes jouent un rôle important dans l’embryogenèse, la prolifération et la différenciation cellulaire ainsi que dans la réponse au stress. Erk4 est un membre atypique de la famille MAP kinase. D’une part, la boucle d’activation de Erk4 possède un motif SEG au lieu du motif TXY, très conservé chez les MAP kinases. D’autre part, Erk4 possède une extension en C-terminal du domaine kinase qui n’est pas présente chez les MAP kinases classiques. Jusqu’à présent aucune fonction n’a été attribuée à Erk4. De plus, la voie de signalisation ainsi que le mode de régulation conduisant à l’activation de Erk4 ne sont pas connus. Le seul substrat de Erk4 identifié jusqu’à maintenant est la MAPKAP kinase MK5. L’impact fonctionnel de cette interaction n’est également pas connu. Afin d’en apprendre davantage sur la MAP kinase atypique Erk4, nous avons étudié le mécanisme d’activation de cette kinase ainsi que sa fonction physiologique par une approche de délétion génique chez la souris. En ce qui concerne l’activation de Erk4, nous avons montré que la boucle d’activation de Erk4 (S186EG) est constitutivement phosphorylée in vivo et que cette phosphorylation n’est pas modulée par les stimuli classiques des MAP kinases dont le sérum et le sorbitol. Cependant, nous avons observé que la phosphorylation de la S186 augmente en présence de MK5 et que cette augmentation est indépendante de l’activité kinase de l’une ou l’autre de ces kinases. De plus, nous avons établi que la phosphorylation de la boucle d’activation de Erk4 est requise pour l’interaction stable entre Erk4 et MK5 ainsi que pour l’activation, et la relocalisation cytoplasmique de MK5. Ainsi, notre étude a permis de révéler que Erk4 est régulée de manière différente des MAP kinases classiques et que la phosphorylation de la boucle d’activation de Erk4 joue un rôle essentiel dans la régulation de l’activité de MK5. Parallèlement, nos résultats mettent en évidence l’existence d’une “Erk4 kinase”, dont le recrutement et/ou l’activation semble être facilité par MK5. Afin identifier la fonction physiologique de Erk4, nous avons généré des souris Erk4-déficientes. L’inactivation génique de Erk4 est viable et les souris ne présentent aucune anomalie apparente. Dans le but d’expliquer l’absence de phénotype, nous avons regardé si l’expression de Erk3, le paralogue de Erk4, pouvait compenser la perte de Erk4. Notre analyse a révélé que l’expression de Erk3 dans les souris Erk4-/- n’augmente pas au cours du développement embryonnaire ou dans les tissus adultes afin de compenser pour la perte de Erk4. Par la suite, nous avons adressé la question de redondance entre Erk4 et Erk3. Dans notre laboratoire, les souris Erk3-déficientes ont également été générées et le phénotype de ces souris a récemment été analysé. Cette étude a révélé que l’inactivation génique de Erk3 entraîne un retard de croissance intra-utérin, un défaut de maturation pulmonaire et la mort néo-natale des souriceaux. Nous avons donc regardé la contribution de Erk4 dans ces phénotypes. L’analyse des souris Erk4-/- a révélé que l’inactivation de Erk4 n’entraîne pas de retard de croissance ou de maturation du poumon. De plus, nous avons montré que l’inactivation additionnelle de Erk4 dans les souris Erk3-/- n’accentue pas le phénotype des souris Erk3-déficientes. Ainsi, notre étude a révélé que contrairement à Erk3, Erk4 n’est pas essentielle au développement murin dans des conditions physiologiques. Parallèlement, nous avons montré que Erk4 et Erk3 possèdent des fonctions non-redondantes in vivo. / MAP kinases are essential enzymes implicated in 7 distinct signaling pathways that allow cells to respond appropriately to extracellular stimuli. In mammals, Erk1/2, Jnk, p38 and Erk5 are the best characterized MAP kinases. These enzymes play important roles in embryogenesis, cell proliferation and differentiation and in response to cellular stresses. Erk4 is an atypical member of the MAP kinase family. First, its activation loop is composed of an SEG motif instead of the well conserved TXY motif found in MAP kinases. Second, Erk4 has a C-terminal extension following the kinase domain that is not present in classical MAP kinases. Despite its identification more than a decade ago, the function of Erk4 remains elusive. Moreover, the signaling pathway as well as the regulatory mechanism leading to Erk4 activation in still uncharacterized. The only identified substrate of Erk4 is the MAPKAP kinase MK5, but the functional relevance of this interaction is still unknown. To gain information about the atypical MAP kinase Erk4, we decided to study the activation mechanism of Erk4 and its physiological function using a gene targeted deletion approach in mice. Regarding the activation of Erk4, we showed that the activation loop of Erk4 (S186EG) is constitutively phosphosphorylated in vivo and that this phosphorylation is not modulated by classical MAP kinase stimuli such as serum and sorbitol. However, we observed that phosphorylation of S186 increases in the presence of MK5 and we showed that this increase is independent of the kinase activity of either kinases. Moreover, we established that phosphorylation of Erk4 activation loop is required for the stable interaction between Erk4 and MK5 as well as for the activation and cytoplasmic relocalisation of MK5. Thus, our study reveals that Erk4 is differently regulated than classical MAP kinases and that Erk4 activation loop phosphorylation is important for its role in the regulation of MK5 activity. In parallel, our results revealed the existence of an “Erk4 kinase” whose recruitment and/or activation seems to be facilitated by MK5. To gain information about the physiological function of Erk4 we generated Erk4 deficient mice. Gene-targeted inactivation of Erk4 is viable and these mice present no gross abnormality. To explain the absence of phenotype, we analyzed the expression of Erk3, the paralog of Erk4, to determine if it could compensate for the loss of Erk4. Our analysis revealed that Erk3 expression in Erk4-/- mice is not up-regulated during embryogenesis nor in adult mice tissues in order to compensate for the loss of Erk4. We next addressed the question of redundancy between Erk4 and Erk3. In our laboratory, Erk3-/- deficient mice were also generated and the phenotype of these mice was recently analyzed. This study revealed that gene inactivation of Erk3 leads to intra-uterine growth retardation, lung maturation defect and neo-natal lethality. We then investigated the contribution of Erk4 in these phenotypes. The analysis of Erk4-/- mice revealed that inactivation of Erk4 did not delay intra-uterine growth nor cause pulmonary maturation defect. Moreover, we showed that additional loss of Erk4 in Erk3-/-mice does not accentuate Erk3-deficient mice phenotype. Thus, this study reveals that, contrary to Erk3, Erk4 is dispensable for mice development under normal condition and that Erk4 and Erk3 have non-redundant functions in vivo.