Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Non-Latin script record Includes bibliographical references (leaves 112-128). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 199-250.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-04-27T15:00:50Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-05-16T19:24:00Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) / Made available in DSpace on 2017-05-16T19:24:00Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) Previous issue date: 2016 / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O excesso de gordura corporal caracter?stico da obesidade est? relacionado a diversas altera??es metab?licas, que incluem a resist?ncia ? insulina. Dentre as medidas n?o farmacol?gicas empregadas para a melhora da sensibilidade ? insulina est? o treinamento f?sico aer?bio, como o treinamento intervalado de alta intensidade (HIIT, do ingl?s high intensity interval training). Sendo assim, esse estudo avaliou os efeitos do HIIT em componentes bioqu?micos, celulares e moleculares relacionados ? resist?ncia ? insulina em obesos. Indiv?duos obesos sens?veis (n=9) e resistentes ? insulina (n=8) foram submetidos a 8 semanas de HIIT, em cicloerg?metro, realizado 3 vezes por semana, com intensidade e volume progressivos (8 a 12 est?mulos; 80 a 110% da pot?ncia m?xima). Amostras de sangue venoso e do m?sculo vasto lateral foram obtidas antes e ap?s o programa de HIIT. Ap?s o programa de treinamento houve aumento da sensibilidade ? insulina nos obesos resistentes ? insulina, mas n?o houve redu??o da massa de gordura. A concentra??o de citocinas no soro, o estresse oxidativo sist?mico e frequ?ncia das c?lulas imunes n?o foram modificadas ap?s o treinamento. No m?sculo esquel?tico, o HIIT promoveu aumento da fosforila??o do substrato do receptor de insulina (IRS) (Tyr612), da Akt (Ser473) e da prote?na quinase dependente de c?lcio/calmodulina (CAMKII) (Thr286), e aumento do conte?do da ?-hidroxiacil-CoA desidrogenase (?-HAD) e citocromo C oxidase (COX-IV). Houve ainda, redu??o da fosforila??o da quinase regulada por sinal extracelular (ERK1/2) nos obesos resistentes ? insulina. Conclu?mos que 8 semanas de HIIT promoveram melhora da sensibilidade ? insulina, modificou componentes da via de sinaliza??o da insulina e do metabolismo oxidativo no m?sculo esquel?tico. Essas altera??es ocorreram independentes de mudan?as na gordura corporal total e de par?metros inflamat?rios sist?micos. / Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / Obesity is characterized by excess of body fat, and its development can lead to a variety of metabolic disorders, including insulin resistance. Exercise is recognized as a non-pharmacological approach to increasing skeletal muscle insulin sensitivity, although the mechanisms are not elucidated. Additionally, the understanding of high intensity interval training (HIIT, high intensity interval training) treat insulin resistance is less understood. Therefore, this study evaluated the effects of HIIT on biochemical, molecular, and cellular markers related to insulin resistance in sedentary obese individuals. Sensitive (n=9) and insulin resistant (n=8) obese individuals (body mass index ? 30 kg/m-2) were engaged in 8 weeks of HIIT using a cycle ergometer. The HIIT was performed 3 times a week, and its intensity and volume progressively increased throughout the training period (from 8 to 12 stimuli; from 80 to 110% of the maximum power). Venous blood and the vastus lateralis muscle samples were obtained before and after the HIIT. HIIT enhanced insulin sensitivity in insulin-resistant obese individuals without changing body fat mass. Cytokine concentration in serum, blood oxidative stress, and frequency of some immune cells were not altered by HIIT. In skeletal muscle, HIIT increased the phosphorylation of insulin receptor substrate (IRS) (Tyr612), Akt (Ser473), and protein kinase dependent calcium/calmodulin (CaMKII) (Thr286). HIIT also increased the expression of ?-hydroxyacyl-CoA dehydrogenase (?-HAD) and cytochrome C oxidase (COX-IV). A reduction of the kinase phosphorylation of extracellular signal-regulated (ERK1/2) was only seen in obese insulin resistant individuals. The results show that 8 weeks of HIIT enhanced insulin sensitivity, modified components of the insulin-signaling pathway, and improved skeletal muscle oxidative metabolism. These changes were independent of alterations in body fat and inflammatory parameters.

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

Abstract Intracellular signalling cascades regulate cardiomyocyte hypertrophic response. Initially hypertrophy of individual myocytes occurs as an adaptive response to increased demands for cardiac work, e.g. during hypertension or after myocardial infarction, but a prolonged hypertrophic response, accompanied by accelerated fibrosis and apoptosis, predisposes the heart to impaired performance and the syndrome of heart failure. The goal of this work was to elucidate some of the main signalling pathways in experimental models of the cardiac hypertrophic response. Mechanical stretching of cultured neonatal rat cardiomyocytes in vitro activates the B-type natriuretic peptide (BNP) gene, a well-established marker of the hypertrophic response, through intracellular signalling cascades mitogen-activated protein kinases (MAPKs) and protein kinase A (PKA) -pathway. Further, transcription factors transcriptional enhancer factor-1 (TEF-1) and activating transcription factor 3 (ATF3) were induced during stretch, and TEF-1 activation was shown to be regulated by extracellular signal-regulated kinase (ERK), while ATF3 activation was modulated by PKA. The BNP gene was also activated by the adenoviral overexpression of the p38 MAPK isoforms p38α and p38β in vitro. Importantly, p38α–induced activation was mediated through activator protein-1 (AP-1) while p38β mediated BNP transcription through GATA-4, which suggests distinct physiological roles for different p38 isoforms. This was further confirmed by quantitative PCR, which demonstrated pro-fibrotic role for the p38α isoform and a pro-hypertrophic role for the p38β isoform. Finally, adenoviral overexpression of ATF3 in vitro and in vivo resulted in activation of cardiac survival factors nuclear factor-κВ and Nkx-2.5, and attenuation of central pro-inflammatory and pro-fibrotic mediators. Together these data suggest a protective role for ATF3 in the heart. Overall this study provides new insights into the role of several signalling molecules involved in cardiac hypertrophic process and suggests potential therapeutic strategies for the diagnosis and treatment of heart failure. / Tiivistelmä Sydämen kammioiden seinämät paksuuntuvat kuormituksen lisääntyessä mm. verenpainetaudissa tai sydäninfarktin jälkeen. Lisääntynyt kuormitus aiheuttaa sydänlihassolujen koon kasvun (hypertrofioitumisen) ohella sidekudoksen kertymistä (fibroosia) ja solukuolemaa. Nämä solutason muutokset lopulta vioittavat sydämen rakennetta niin, että sen toiminta pettää, ja sydän ajautuu vajaatoimintaan. Tätä taudin etenemistä säätelevät molekyylitasolla lukuisat solunsisäiset signaalinvälitysjärjestelmät, joita tässä väitöskirjatyössä tutkittiin eri koemalleissa. Sydämen täyttöpaineen nousun aiheuttama sydänlihassolujen mekaaninen venytys aktivoi natriureettisten peptidien (eteispeptidi, ANP ja B-tyypin natriureettinen peptidi, BNP) synteesiä ja vapautumista verenkiertoon. BNP geenin säätelyä mekaanisen venytyksen aikana tutkittiin rotan sydänlihassoluviljelmissä. Mitogeeni-aktivoituvat proteiinikinaasit (MAPK) sekä proteiinikinaasi A (PKA) säätelivät mekaanisen ärsykkeen aiheuttamaa BNP geenin ekspressiota. Venytys aktivoi myös transkriptiotekijöitä TEF-1 (transcriptional enhancer factor-1) ja ATF3 (activating transcription factor 3). TEF-1 sääteli venytyksen aiheuttamaa BNP:n aktivaatiota ERK:n (extracellular signal-regulated kinase) välityksellä BNP geenin säätelyalueella olevan sitoutumispaikkansa (M-CAT elementti) kautta. ATF3:n säätelyssä PKA:lla oli keskeinen merkitys. Tutkimus osoitti myös, että p38 MAPK:n alatyypeistä p38α lisäsi fibroosiin liittyvien geenien aktiivisuutta, kun taas p38β aiheutti solujen hypertrofioitumista lisäävien geenien ekspressiota. Molemmat alatyypit aktivoivat BNP geenin ekspressiota, mutta aktivaatio tapahtui eri transkriptiotekijöiden kautta. Tutkimuksessa havaittiin myös, että ATF3:n yliekspressio adenovirusvälitteisellä geeninsiirrolla lisäsi kahden sydäntä suojaavan transkriptiotekijän (nuclear factor-κВ ja Nkx-2.5) aktiivisuutta, sekä vähensi sydämen tulehdusvastetta ja fibroosia lisäävien tekijöiden (interleukiini-6 ja plasminogeeniaktivaattorin inhibiittori-1) ekspressiota. Väitöskirjatutkimus antaa uutta tietoa solunsisäisistä signaalinvälitys-järjestelmistä, jotka säätelevät sydänlihaksen kuormitusvastetta sydän- ja verenkiertoelimistön sairauksissa. Näiden solutason mekanismien tunteminen osaltaan edesauttaa jatkossa uusien menetelmien kehittämistä sydämen vajaatoiminnan ehkäisyyn ja hoitoon.

La mycotoxine déoxynivalénol (DON) et son métabolite déépoxy-déoxynivalenol (DOM-1) ont des effets significatifs sur la modification de la fonction des cellules thècales de l’ovaire bovin. L'objectif de cette étude était d'identifier les différentes voies de signalisation impliquées dans le mécanisme d'action de DON et DOM-1 par la spectrométrie de masse. Méthodes: Les cellules thécales de l'ovaire bovin ont été récoltées à partir des vaches adultes, indépendamment du stade du cycle œstral, et ont été cultivées à une densité de 500000 cellules viables dans 1 ml de milieu de McCoy pendant 5 jours. Les cellules ont ensuite été traitées au jour 5 de la culture avec 1 ng/ml de DON ou DOM-1 pendant 30 minutes et des échantillons cellulaires de protéins totales ont été préparés pour spectrométrie de masse. Résultats: la spectrométrie de masse a montré que DON et DOM-1 induisent une surexpression simultanée de ERK1/2, MAPK14 (p38alpha) et MAPK13 (p38delta). La spectrométrie de masse a également indiqué que 94 peptides ont été surexprimés tels que GNGT1, EDN1 et YWHAB. Ils régulent la plupart des voies de prolifération des cellules et sont impliqués dans la biosynthèse des lipides et des glucides. Néanmoins, 255 peptides ont été régulés à la baisse, tels que CALR3, PTGES3, RAD21, ACVR2B et TGFBR1 dont leurs activités sont principalement l'activation ou la désactivation des processus apoptotiques, et le métabolisme du glucose et de la choline. Nos résultats montrent que DON et DOM-1, à une dose de 1 ng/ml, ont le potentiel de stimuler la surexpression de MAPK distinctes et réguler négativement les voies de signalisation spécifiques qui stimulent la prolifération les cellules de la thèque de l’ovaire de bovin. / The mycotoxin deoxynivalenol (DON) and its metabolite deepoxy-DOM-1 have significant effects on bovine ovarian theca cell function. The objective of this study was to identify different signaling pathways involved in the mechanism of action of DON and DOM-1 by mass spectrometry. Methods: bovine ovarian theca cells were harvested from adult cows independently of the stage of the estrous cycle, and were cultured at a density of 500000 viable cells in 1 ml McCoy’s medium for 5 days. The cells were then treated on day 5 of culture with 1 ng/mL DON or DOM-1 for 30 minutes and total cell protein was collected for mass spectrometry. Results from mass spectrometry showed that both DON and DOM-1 induce simultaneous upregulation of ERK1/2 , MAPK14 (p38alpha) and MAPK13 (p38delta). Mass spectrometry also indicated that 94 peptides such as GNGT1, EDN1 and YWHAB were upregulated. They mostly regulate cell proliferation pathways and are involved in biosynthesis of lipid and carbohydrates. Nevertheless, 255 peptides such as CALR3, PTGES3, RAD21, ACVR2B and TGFBR1 were downregulated whose activities are mainly activation or deactivation of apoptotic processes, and glucose and choline metabolism. Our findings show that both DON and DOM-1 at least at a low dose (1 ng/ml) have the potential to stimulate upregulation of distinct MAPKs and downregulate specific signaling pathways that stimulate bovine ovarian theca cell proliferation.

La reproduction est un événement crucial pour la vie des plantes. Ce processus nécessite la formation du pollen et des ovules. Les cellules germinales vont subir la méiose puis une succession de mitoses, deux pour le pollen et trois pour l’ovule, ce qui va leur permettre d’acquérir leurs structures finales. Une fois formés, ces deux gamètes doivent se rencontrer. Pour cela, le grain de pollen va germer sur le stigmate puis former le tube pollinique. Le tube pollinique en croissance va traverser les différents tissus femelles et ainsi tracter les deux cellules spermatiques jusqu’à l’ovule, permettant la reproduction. Un important réseau de signalisation cellulaire est nécessaire pour permettre ces événements. Les cascades des Mitogen-Activated Protein Kinases (MAPKs) sont l’un des réseaux de signalisation les plus étudiés chez les plantes. Ces kinases sont impliquées dans de très nombreux processus développementaux tels que la formation de l’embryon ou des stomates. Pour autant, leurs rôles restent encore peu caractérisés pendant de la fécondation. Ce projet a pour objectif de mieux comprendre le rôle que jouent les MAPK lors de la formation des gamètes mâles et femelles ainsi que lors de la croissance des tubes polliniques. Plusieurs membres de la superfamille des MAPKs ont été caractérisés pour leurs rôles dans la reproduction sexuée des végétaux. De précédents travaux dans le laboratoire de Daniel P. Matton, ont démontré l’implication de deux MAPK Kinases Kinases (MAP3K), la Solanum chacoense Fertilization-Related Kinase 1 (ScFRK1) et ScFRK2. Ces deux kinases sont nécessaires pour le développement de l’ovule et du pollen chez S. chacoense, une espèce de pomme de terre sauvage diploïde. Dans un premier temps, nous avons étudié la fonction d’une nouvelle ScFRK, la ScFRK3. Ce troisième membre de la classe des FRKs chez S. chacoense, est, elle aussi, impliquée dans le développement des gamétophytes mâles et femelles. 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 lors de la formation des gamètes chez Solanum chacoense. Nous montrons que la ScFRK3 est impliquée dans la formation du pollen ainsi que celui des ovules. Nous avons ensuite poursuivi nos recherches en affinant le phénotypage du mutant de surexpression ScFRK2. En effet, les précédentes études ont permis de montré que la surexpression de ScFRK2 conduit le primordium ovulaire à la formation de structures carpéloïdes. Pour autant, les ensembles des primordius ovulaires ne sont pas devenu des strutures capéloïde. Nous montrons ici que seulement 10 % des ovules dans l'ovaire sont devenu 4 des carpéloïde. Notre étude montre qu’en plus des structures carpéloïdes, un grand nombre d'ovule n'ont pas de sac embryonnaire à l'anthèse ce qui explique le faible nombre de graines par fruit. L’analyse du développement des sacs embryonnaires monte que la surexpression de ScFRK2 entraine l’arrêt au stade mégaspore fonctionnelle. Ce phénotype est similaire à ce qui a pu être observé dans les lignées ARN interférant pour la ScFRK1 et la ScFRK3. De précédentes études faites chez Arabidopsis thaliana semblent montrer que les membres de la superfamille des MAPK ne sont pas essentiels pour la croissance du tube pollinique. Pour comprendre le rôle que jouent les MAPK dans l’élongation du tube pollinique, nous avons utilisé un inhibiteur des MAP Kinase Kinase (MKK), appelé U0126. La présence de cette drogue dans le milieu de croissance des grains de pollen provoque une diminution de la germination et de l’élongation du tube pollinique. L’utilisation de la méthode semi-in vivo montre une perte de la polarisation de la croissance des tubes polliniques causée par l’inhibition des MKK. La présence de l’inhibiteur conduit à la diminution de la quantité de filaments d’actine ainsi qu’à leur désorganisation à l’apex du tube. L’exocytose est aussi affectée par l’inhibition des MKK. Les cascades MAPK sont nécessaires à la croissance polarisée du tube pollinique chez Arabidopsis thaliana. Pour finir, nous avons voulu identifier certains membres de la superfamille des MAPK impliqués dans la croissance du tube pollinique. Nous nous sommes intéressés en premier lieu aux orthologues de la famille ScFRK chez Arabidopsis thaliana. Les AtMAP3K19-20-21 sont les orthologues les plus proches de ScFRK3. Ces AtMAP3K sont exprimées lors du développement des grains de pollen et lors de l’élongation de tube pollinique. L’analyse du pollen des différentes lignées mutantes montre qu’en leur absence, le pollen ne présente aucun problème de développement contrairement à ScFRK3. Par contre, les doubles mutants et le triple mutant pour les AtMAP3K19-20-21 montrent une diminution de la capacité de germination. L’élongation du tube pollinique est affectée lors de la mutation d’au moins une des AtMAP3K. Ces deux études démontrent que les MAPK sont essentielles dans la formation et l’élongation du tube pollinique. / Reproduction is a crucial event for plant life. This process requires the formation of pollen and ovules. The germ cells will undergo meiosis and then a succession of mitosis, two for the pollen and three for the ovule, which will allow them to acquire their final structures. Once formed, these two gametes must meet each other. For this, the pollen grain will germinate on the stigmas to form the pollen tube. The growth of the pollen tube will pass through the different female tissues and thus pull the two sperm cells to the ovum for reproduction. An important cellular signaling network is necessary to allow these events to occur. The Mitogen-Activated Protein Kinase (MAPKs) cascades are one of the most studied signaling networks in plants. These kinases are involved in a wide range of developmental processes such as embryo formation and stomata. However, their roles remain poorly characterized during fertilization. The aim of this project is to better understand the role played by MAPKs during the formation of male and female gametes as well as during the growth of pollen tubes. Several members of the MAPK superfamily have been characterized for their role in the sexual reproduction of plants. Previous work in Daniel P. Matton's laboratory has demonstrated the involvement of two MAPK Kinases (MAP3K), Solanum chacoense Fertilization-Related Kinase 1 (ScFRK1) and ScFRK2. These two kinases are necessary for egg and pollen development in S. chacoense, a diploid wild potato species. In a first step, we studied the functionality of a new ScFRK, ScFRK3. This third member of the class of FRKs in S. chacoense, is also involved in the development of male and female gametophytes. From the expression pattern to the establishment of a potential signaling pathway, through the phenotypic characterization of mutants, several experiments have been performed in order to understand the role of ScFRK3 in the formation of gametes in S. chacoense. We show that ScFRK3 is involved in the formation of pollen as well as that of the embryonic sac. We then continued our research by refining the phenotyping of the overexpression mutant ScFRK2. Indeed, previous studies have shown that ScFRK2 overexpression leads the ovular primordium to the formation of carpeloid structures. However, the sets of ovular primordia have not become capeloid structures. We show here that only 10% of the eggs in the ovary have become carpeloid. Our study shows that in addition to the carpeloid structures, a large number of ova do not have an embryonic sac in the anthesis, which explains the low number of seeds per fruit. The analysis of the development of the embryonic sacs shows that 7 overexpression of ScFRK2 leads to the cessation of the functional megaspore stage. This phenotype is similar to what has been observed in interfering RNA lines reducing expression of ScFRK1 and ScFRK3. Previous studies in Arabidopsis thaliana suggest that members of the MAPK superfamily are not essential for pollen tube growth. To understand the role that MAPKs play in pollen tube elongation, we used a MAP Kinase Kinase (MKK) inhibitor called U0126. The presence of this drug in the growth medium of pollen grains causes a decrease in germination and elongation of the pollen tube. The use of the semi in vivo method shows a loss of polarization of the pollen tube growth caused by the inhibition of MKK. The presence of the inhibitor leads to a decrease in the number of actin filaments and their disorganization at the apex of the tube. Exocytosis is also affected by MKK inhibition. We show in this chapter that MAPK cascades are necessary for polarized pollen tube growth in Arabidopsis thaliana. Finally, we wanted to identify some members of the MAPK superfamily involved in pollen tube growth. We were first interested in the ScFRK family orthologs in Arabidopsis thaliana. AtMAP3K19-20-21 are the closest orthologs to ScFRK3. These AtMAP3K are expressed during the development of pollen grains and during the elongation of the pollen tube. Pollen analysis of the different mutant lines shows that in their absence the pollen does not present any development problems unlike ScFRK3. On the other hand, double mutants and triple mutant for AtMAP3K19-20-21 show a decrease in germination capacity. Pollen tube elongation is affected when at least one of the AtMAP3Ks is mutated. These two studies demonstrate that MAPKs are essential for the formation and elongation of the pollen tube and that AtMAP3K19-20-21 participate in these biological processes.

No description available.

The cytokine tumor necrosis factor α (TNFα) stimulates the NF-кB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting Rip1 and Traf2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that Rip1 links the TNFR1 to the IкB kinase (IKK) complex, whereas Traf2 couples the TNFR1 to the SAPK/JNK cascade. We found TNFα-induced p38 MAP kinase activation and interleukin-6 (IL-6) production is impaired in rip1-/- murine embryonic fibroblasts (MEF) but unaffected in traj2-/- MEF, demonstrating that Rip1 is also a specific mediator of the p38 MAP kinase response to TNFα. Moreover, we demonstrate that endogenous Rip1 associates with the MAP3K, Mekk3 in response to TNFα and that TNFα-induced p38 MAP kinase activation is impaired in mekk3-/- cells, indicating that Rip1 may mediate the p38 MAP kinase response to TNFα by recruiting Mekk3. We also demonstrate that Rip1 is phosphorylated and ubiquitinated in response to Tnfα and that Rip1 phosphorylation is not required for ubiquitination of Rip1. Furthermore, TNFα-induced ubiquitination of Rip1 is impaired in Traf2-/- cells, suggesting that Traf2 is the E3 ubiquitin ligase responsible for the TNFα-dependent ubiquitination of Rip1. Finally, recruitment of the ubiquitinated Tak1 complex is dependent on the presence of Rip1, suggesting that Rip1 ubiquitination rather than its phosphorylation is critical in TNFR1 signaling.

Arterial occlusive diseases are major causes of morbidity and mortality in industrialized countries and represent a huge economic burden. The extent of the native collateral circulation is an important determinant of blood perfusion restoration and therefore the severity of tissue damage and functional impairment that ensues following arterial occlusion. Understanding the mechanisms responsible for collateral artery development may provide avenues for therapeutic intervention. Here, we identify a critical requirement for mixed lineage kinase (MLK) – cJun-NH2-terminal kinase (JNK) signaling in vascular morphogenesis and native collateral artery development. We demonstrate that Mlk2-/-Mlk3-/- mice or mice with compound JNK-deficiency in the vascular endothelium display abnormal collateral arteries, which are unable to restore blood perfusion following arterial occlusion, leading to severe tissue necrosis in animal models of femoral and coronary artery occlusion. Employing constitutive and inducible conditional deletion strategies, we demonstrate that endothelial JNK acts during the embryonic development of collateral arteries to ensure proper patterning and maturation, but is dispensable for angiogenic and arteriogenic responses in adult mice. During developmental vascular morphogenesis, MLK – JNK signaling is required for suppression of excessive sprouting angiogenesis likely via JNK-dependent regulation of Dll4 expression and Notch signaling. This function of JNK may underlie its critical requirement for native collateral artery formation. Thus, this study introduces MLK – JNK signaling as a major regulator of vascular development. In contrast, we find that JNK in hematopoietic cells, which are thought to share a common mesodermally-derived precursor with endothelial cells, is cellautonomously dispensable for normal hematopoietic development and hematopoietic stem cell self-renewal, illustrating the highly context dependent function of JNK.