Period 2 (Per2) is a core circadian factor responsible for its own negative regulation. It operates in the circadian clock, which affects multiple biological functions such as metabolic rate, hormone release, and core body temperature. The Per2 protein functions directly with factors in other biological functions such as tumor suppression, immune system, and metabolism. In many cases, the Per2 deficiency caused by disrupted expression is sufficient to create severe abnormalities in many of the mentioned functions. The sequence contains several domains and motifs in Per2 that are traditionally involved in protein interactions which suggests that Per2 serving a regulatory role by effecting downstream biological roles dependent on Per2 stability. In this work, we perform a two-hybrid screening assay using the C-terminal region of human Per2 and identified an extensive number of interactors. Utilizing a genetic ontology program, we assorted the list of clones into groups of proteins that are biologically relevant or operated in similar function. Through this program, we validated the two-hybrid screening by the clusters of biological function already attributed to hPer2 and identified new putative biological functions. We use the new putative interactors to gain further insight on the regulatory roles that hPer2 performs, in conjunction with operating as a core factor in circadian rhythmicity. We also show that Translationally Controlled Tumor Protein (TCTP) is capable of binding to hPer2 and is a novel interaction. When a sufficient amount of TCTP (1:1 molar stoichiometric ratio) is present in a system, a cleavage of hPer2 is observed in vitro. This cleavage occurs in reactions independent of ATP, ubiquitin, and the proteasome. The data points towards a method of cleavage similar to that of the archael lon-tk (Thermococcus kodakaraensis) that preferentially cleaved unstructured substrates in ATP-independent reactions. / Master of Science

La croissance d’un organisme multicellulaire pour atteindre une taille bien définie, nécessite une coordination de la prolifération cellulaire, de l’expansion et de la différentiation cellulaire ainsi que de la mort cellulaire. Ces processus sont sous l’influence de l’état nutritionnel de l’organisme, les conditions de son environnement et des signaux hormonaux. Translationally controlled tumor protein (TCTP) est un facteur essentiel à la croissance des plantes et des animaux. La protéine TCTP de plante contrôle la croissance mitotique, tandis que la protéine TCTP animale contrôle la croissance mitotique et post-mitotique. Une voie importante dans la régulation de la croissance en réponse aux nutriments est la voie Target of Rapamycin (TOR). Chez la Drosophile, il a été montré que dTCTP serait un régulateur positif en amont de TOR. Au cours de ma thèse, j’ai étudié le lien entre TCTP et la voie TOR, afin de savoir si, comme chez les animaux, AtTCTP agit en amont de la voie TOR pour contrôler la croissance des organes. Afin de savoir si la voie TCTP était liée à l’état nutritionnel, j’ai recherché l’impact du milieu de culture sur la létalité de la mutation tctp. J’ai ensuite caractérisé l’impact de la mutation tctp sur le transport et l’homéostasie de l’hormone auxine. J’ai enfin analysé pourquoi TCTP de plante ne contrôle pas la croissance post-mitotique par expansion cellulaire, contrairement à TCTP animale. Les données de la littérature montrent que chez les animaux TCTP est un activateur positif en amont de la voie TOR. Chez la plante Arabidopsis thaliana, mes données d’interactions génétiques sont en faveur d’un modèle dans lequel AtTCTP agit indépendamment de la voie TOR, contrairement de ce qu’il a été proposé chez les animaux. Chez les plantes, la perte de fonction de TCTP est associée à un retard du développement embryonnaire et à la mort. Cette létalité peut être complémentée par sauvetage des embryons sur du milieu riche en nutriments. J’ai montré que l’ajout de sucrose ou de glutamine dans le milieu de sauvetage des embryons tctp est nécessaire à leur développement. Ces données suggèrent qu’in vitro, AtTCTP n’est pas nécessaire à l’approvisionnement et à l’utilisation des nutriments sucrose, glucose ou glutamine. Dans leur ensemble, ces résultats réévaluent le rôle du régulateur de croissance TCTP en montrant que le gène AtTCTP régule la croissance mitotique indépendamment de la voie TOR et des voies de signalisation liées aux nutriments. L’observation des flux d’auxine en suivant la localisation de PIN1-GFP dans les embryons et les inflorescences du mutant tctp ne montre aucune altération par rapport au phénotype sauvage. De même, l’homeostasie de l’auxine, suivie à l’aide du rapporteur DR5::GFP n’est pas altérée dans les embryons tctp. Ceci suggère que le défaut de croissance du mutant tctp n’est pas lié à une altération du flux ou de l’homéostasie de l’auxine. La protéine TCTP de plante ne contrôle pas la croissance post-mitotique, contrairement à la protéine TCTP animale. J’ai réalisé un échange de domaines protéiques entre AtTCTP et Drosophila dTCTP. Le but était d’identifier les domaines protéiques de la protéine TCTP animale qui permettent la croissance post-mitotique. La plupart des protéines chimères étaient instables dans la Drosophile. Afin de comprendre pourquoi, j’ai réalisé du modelage par homologie et j’ai discuté la structure des chimères dans ma thèse.L’ensemble de mes résultats permet de mieux comprendre la fonction de TCTP chez les végétaux, en montrant que cette fonction s’exerce indépendamment de la voie TOR. / The growth of a multicellular organism and its size determination require the tight regulation of cell proliferation, cell differentiation, cell growth and apoptosis. These processes are influenced by the nutritional state of the organism, its environmental conditions and hormonal signals. Translationally controlled tumor protein (TCTP) is an essential regulator of growth in plants and animals. In plants it controls mitotic growth, whereas in animals, it controls mitotic and post-mitotic growth. One of the important pathways involved in the control of growth in response to nutrients is the Target of Rapamycin (TOR) pathway. In Drosophila, dTCTP was proposed to act a positive regulator upstream of TOR, although this role remains a matter of debate in the animal field.During the past 3 years of my PhD. thesis, I addressed the question whether plant TCTP acts upstream of TOR to control organ growth. I studied the impact of nutrient availability and hormones on TCTP role to control growth in plants and vice versa. Finally, I examined why plant TCTP does not control post-mitotic cell expansion growth, conversely to animal TCTP using a structure-function approach.In animals, TCTP was proposed to act as a positive activator upstream of the TOR pathway. In plants, my data support a model in which AtTCTP acts independently from the plant TOR pathway, thus in contrast to what has been proposed in animals. TCTP loss of function leads to delay of embryo development and death. Nutrient supplement rescues this embryos lethality. First, I demonstrate that embryos grown on nutrients lacking sucrose or glutamine fail to develop correctly. My data demonstrate that in vitro AtTCTP is not essential to the uptake, the use of and the response to the nutrients glucose, sucrose or glutamine. Taken together, these results reevaluate the role of AtTCTP as a growth regulator controlling mitotic growth independently from the TOR pathway and likely from nutrient related signaling pathways. Interestingly, my data also show that AtTCTP controls growth independently from auxin flux or homeostasis and that auxin-induced growth can occur without TCTP. To address why plant TCTP do not control post-mitotic growth conversely to animal counterpart, I performed protein domain swaps and created chimera proteins between Arabidopsis AtTCTP and Drosophila dTCTP. The rational was to identify protein domains that differentiate plant and animal TCTPs with regard to post-mitotic growth control. Most of chimera proteins were instable and I was unable to complement tctp loss of function in Drosophila. I performed a structure based modeling to understand this phenotype and the outcome is discussed in my PhD thesis.Altogether my results improve the understanding of plant morphogenesis by reevaluating the role of the central growth regulator TCTP.

Le cancer de la prostate (CaP) représente la deuxième cause de mortalité par cancer chez l'homme. La suppression androgénique (castration-thérapie) demeure la seule thérapie efficace du CaP avancé du fait de son caractère castration-sensible. Cependant, elle n'empêche pas la progression castration-résistante (CR) de la maladie dans les 1 à 3 ans après le début du traitement hormonal. Récemment, l'implication d'Hsp27 (Heat shock protein 27) dans l'échappement thérapeutique des CaPs a été montrée et un oligonucléotide antisens inhibiteur d'Hsp27, OGX-427, est en cours d'évaluation clinique II/III pour le traitement des CaPs CR. Afin de comprendre le rôle d'Hsp27 dans le mécanisme de résistance à la castration, nous avons réalisé le criblage de l'ensemble des protéines partenaires d'Hsp27 par double hybride. Mes travaux de thèse ont permis d'identifier une nouvelle protéine cliente d'Hsp27, TCTP (translationally controlled tumor protein) dont l'expression est indétectable dans les cellules normales. J'ai également montré que la progression CR des CaPs corrélait avec une surexpression de TCTP, une perte de P53 et que l'inhibition de TCTP par un oligonucléotide antisens restaurait l'expression de P53. Cette étude suggère, pour la première fois, un lien direct entre P53 et la sensibilité à la castration des CaPs. De plus, l'étude de l'interactome d'Hsp27 a mis en évidence son implication dans de nouvelles fonctions telles que la réparation de l'ADN ou l'épissage alternatif des ARNm. L'ensemble de ces travaux ont permis de mieux comprendre les mécanismes d'action d'Hsp27 dans la progression CR des CaPs et de développer de nouvelles approches thérapeutiques. / Prostate cancer (PC) is the second most common cause of cancer-related mortality in men in the Western world. Androgen ablation (castration-therapy) is usually the initial therapy in patients with advanced or metastatic disease. Unfortunately, the disease gradually progresses to a metastatic castration-resistant (CR) state, which remains incurable. Recently, the involvement of Hsp27 (Heat Shock Protein 27) in CR progression has been identified and an oligonucleotide antisense (OGX-427), inhibitor of Hsp27 is currently in phase II/III clinical trials to treat CRPC. In order to understand Hsp27 mechanisms of action in CR progression, we started to screen for Hsp27 partner proteins by using two-hybrid system. My PhD work has reported that Translationally Controlled Tumor Protein (TCTP) was a new Hsp27 protein partner that mediated Hsp27 cytoprotection in CRPC and that TCTP expression was absent in normal prostate tissues. We have further found that CR progression correlated with TCTP overexpression, the loss of P53 and that TCTP silencing using an antisense was able to restore P53 expression and function. This study suggests for the first time that castration-sensitivity is directly linked to P53 expression. In addition, we revealed exciting new aspects of the Hsp27 involvement in essential metabolic and cellular processes such as DNA repair and mRNA splicing. In summary, my PhD results have provided an enriched understanding of Hsp27 mechanisms of cytoprotection contributing to CRPC progression and opened a new promising field of research for multi-target therapeutic approaches.

Made available in DSpace on 2015-03-26T13:51:57Z (GMT). No. of bitstreams: 1 texto completo .pdf: 1092167 bytes, checksum: 8586b9a4bcc728e0319bbbdb012ca136 (MD5) Previous issue date: 2012-07-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Viruses are organisms with small genomes of simple organization, which coding about 3 to encode 10 viral proteins. The success of the infection depends on the manipulation of the cell by the virus, by means of complex interactions occurring between viral factors and host factors. The induced changes by virus infection include cell morphology changes, cell cycle changes and alterations in gene expression, among others. Understanding the processes that favor viral infection necessarily involves the study of virus-host interactions. In order to better understand the processes related to infection by tomato potyvirus Pepper yellow mosaic virus (PepYMV) a subtractive library was built 72 hours after infection. Several genes were identified as induced or repressed by viral infection. Among the induced genes, is the gene encoding the Translationally controlled tumor protein (TCTP). TCTP protein is highly conserved in all eukaryotes. Its functions are related to growth control and cell cycle, anti-apoptotic activity, and response to different biotic and abiotic stresses. The involvement of this protein in infection PepYMV has not been established, but studies in a strain of transgenic tomato plants silenced for TCTP showed that the silenced plants have a lower accumulation of PepYMV, indicating that TCTP promotes viral infection. In this study, we sought to advance the understanding of mechanisms involving TCTP in the process of infection by PepYMV. N. benthamiana plants silenced by VIGS TCTP were used to study the effect of silencing in viral infection, and the silenced plants accumulate fewer viruses in early stages of virus infection. Individual expression of viral proteins in N. benthamiana identified P3 and CP as capable of inducing TCTP expression at similar levels to those induced during PepYMV infection, and expression of NIb reduced expression of TCTP. The verification of direct interactions occurrence between viral proteins and TCTP by double-hybrid assay showed that TCTP not interact separately with any of the proteins of viral origin. Purification of proteins of health and infected N. benthamiana plants by affinity with TCTP identified several proteins that putativaly interacts with TCTP. As in two hybrid assay, interactions involving PepYMV proteins were not detected. These results sugests that TCTP actuation must involve the formation of protein complexes involving viral and plant proteins or contribute indirectly to PepYMV infection, without involving direct interactions between TCTP and viral proteins. / Os vírus são organismos com genomas pequenos, de organização simples, que codificam em média 3 a 10 proteínas. O sucesso da infecção depende da manipulação da célula pelo vírus, por meio de interações complexas que ocorrem entre fatores virais e fatores do hospedeiro. As modificações induzidas na célula incluem alterações morfológicas, alteração do ciclo celular e na expressão gênica, entre outras. A compreensão dos processos que favorecem a infecção viral passa necessariamente pelo estudo de interações vírus-hospedeiro. No intuito de compreender melhor os processos relacionados à infecção de tomateiros pelo potyvírus Pepper yellow mosaic virus (PepYMV) uma biblioteca subtrativa foi construída 72 horas após a infecção. Diversos genes cuja expressão foi alterada pela infecção foram identificados. Dentre os genes induzidos, se encontra o gene que codifica a Translationally controlled tumor protein (TCTP). A proteína TCTP é altamente conservada em todos os eucariotos. Suas funções estão relacionadas a controle do crescimento e ciclo celular, atividade anti-apoptótica, e resposta a diferentes tipos de estresses abióticos e bióticos. O envolvimento desta proteína na infecção pelo PepYMV ainda não foi estabelecido, porém estudos em uma linhagem de tomateiro transgênica silenciadas para a TCTP, mostraram que as plantas silenciadas apresentam um menor acúmulo de PepYMV, indicando que a TCTP favorece a infecção por este vírus. Neste trabalho, buscou-se avançar na compreensão dos mecanismos que envolvem a TCTP no processo de infecção pelo PepYMV. Plantas de Nicotiana benthamiana silenciadas para TCTP por VIGS (Virus Induced Gene Silence) foram utilizadas para estudar o efeito do silenciamento na infecção viral, sendo que as plantas silenciadas acumularam menos vírus no início da infecção. A expressão individual das proteínas de origem viral em N. benthamiana identificou a P3 e a CP como capazes de induzir a expressão de TCTP em níveis semelhantes aos observados durante a infecção pelo PepYMV, sendo que a expressão da proteína NIb reduziu a expressão de TCTP. A verificação da ocorrência de interações diretas entre a TCTP e as proteínas virais, por ensaio de duplo híbrido, mostrou que a TCTP não interage separadamente com as proteínas de origem viral. A purificação de proteínas de plantas de N. benthamiana, sadias e infectadas, por afinidade com a TCTP identificou diversas proteínas que possivelmente 7 interagem com a TCTP. Assim como no ensaio de duplo híbrido, a interação com proteínas virais não foi detectada. Estes resultados sugerem que o papel da TCTP deve envolver a formação de complexos proteicos entre proteínas virais e da planta, ou favorecer a infecção de forma indireta.

Das translationell kotrollierte Tumorprotein (TCTP) ist ein bei Eukaryonten vorkommendes hochkonserviertes Protein, das eine Rolle bei der Pathogenese allergischer Erkrankungen spielt. Bei atopischen Kindern vermittelt es eine IgE abhängige Histaminfreisetzung aus basophilen Granulozyten. Die zugrundeliegenden Mechanismen sind jedoch unklar. TCTP hat die Eigenschaft, an das Tubulin des Zytoskeletts der Zelle zu binden und besitzt eine hohe Affinität für Kalzium. Seine Synthese wird auf dem transkriptionellen und translationellen Niveau reguliert. Eine früher angenommene spezifische Funktion in Tumorzellen konnte nicht bestätigt werden. Das für TCTP kodierende Gen wird als TPT1 bezeichnet. Um die molekulare Basis für die Kontrolle der Synthese des TCTP zu verstehen, wurden in dieser Arbeit Struktur und Funktion des TPT1-Gens bei Mensch und Kaninchen untersucht. Erstmalig wurde die vollständige Struktur eines Säuger-TPT1-Gens durch Klonierung und Sequenzierung aufgeklärt und die funktionelle Rolle des Promotors analysiert. Das 3,8 kb große Kaninchengen wird durch fünf Introns unterbrochen, und kodiert für zwei mRNAs von 843 und 1163 nt, die sich in der Länge der 3' untranslatierten Region unterscheiden. Sie entstehen durch alternative Polyadenylierung. Vom Human-Gen wurden genomische Rekombinanten isoliert und seine vorläufige Struktur ermittelt. Es besitzt eine identische Intron/Exon Architektur und unterscheidet sich nur geringfügig in der Länge der Introns. Auch bei der Expression des Human-Gens entstehen zwei mRNAs. Hybridisierungsexperimente mit RNA aus 10 Kaninchen- und 50 Human-Geweben zeigten, daß beide TCTP mRNAs in allen untersuchten Geweben in ähnlichem Verhältnis zueinander exprimiert werden. Die Gesamtkonzentrationen der TCTP- mRNAs unterschied sich jedoch in verschiedenen Gewebegruppen bis zum Faktor 100. Dies deutet auf eine ausgeprägte Regulation der gewebsspezifischen Transkription hin. Die Promotorstrukturen von 1,2 kb 5'-flankierender Sequenzen des Kaninchen- Gens wurden mit Computerprogrammen auf Bindungsstellen für Transkriptionsfaktoren analysiert. Für funktionelle Aussagen wurden Promotorfragmente mit dem Chloramphenicol-Acetyltransferase-Gen (cat) gekoppelt und die Promotoraktivität durch Bestimmung der CAT-Enzymaktivität nach Zelltransfektionen ermittelt. Ein minimaler Promotor von 66 bp Länge, der eine TATA-Box enthält, konnte eingegrenzt werden. Die maximale Promotoraktivität, die 90% im Vergleich zum starken Thymidinkinase-Promotor betrug, war mit einem 290 bp langem Fragment assoziiert und enthielt eine SP-1, zwei AP-1/CREB und zwei ETS Bindungsstellen. Diese Konstellation ist ein häufiges Merkmal von Genen, die wie das TPT1-Gen durch Phorbolester und Lipopolysaccharide induzierbar sind. Im Sequenzbereich bis -160 sind die Promotoren des Human- und des Kaninchen-Gens sehr ähnlich (89% Homologie), alle Bindungsorte für Transkriptionsfaktoren sind hier konserviert. Weiterhin wurde im Kaninchengenom eine Vielzahl von prozessierten TPT1- Pseudogenen.gefunden. Sechs von ihnen und ihre genomisch-flankierenden Integrationsorte wurden sequenziert. Sie repräsentierten beide mRNA Typen und waren zu über 99% zu den korrespondierenden mRNAs homolog. Die Leserahmen aller Pseudogene waren intakt, bei zwei Pseudogenen war die Aminosäuresequenz sogar unverändert erhalten. Die durch CAT-Assays getestete Transkriptionsaktivität der 5'flankierenden Region eines Pseudogens zeigte eine Aktivität von über 15% gegenüber dem authentischen TPT1-Promotor. Dies ist ein Indiz für eine mögliche Expression von TPT1 Pseudogenen in vivo. / The translationally controlled tumor protein (TCTP) is a conserved eukaryotic protein, which is involved in the pathogenesis of allergic diseases. In atopic children it has been reported to mediate histamine release from basophilic leukocytes in an IgE dependent way. The underlying mechanism, however, is unknown. TCTP is characterized by an efficient binding to tubulin of cytoskeletal structures and by a high calcium affinity. Its synthesis is regulated at the transcriptional and translational level. A specific function in tumor cells, which was assumed initially, could not be confirmed. The gene coding for TCTP is called TPT1. To understand the molecular basis for the control of TCTP expression structure and function of the human and rabbit TPT1 genes were investigated including their promoter regions. The first mammalian TPT1 gene (rabbit) was cloned and sequenced. It consists of 3.8 kb and is interrupted by five introns. Two mRNAs of 843 and 1163 nt length are transcribed differing in their 3'untranslated regions. They are generated by alternative polyadenylation. Furthermore genomic recombinants were isolated containing the human TPT1 gene and a preliminary structure of the gene was established. The human gene has the same intron/exon architecture as the rabbit gene just differing in the length of its introns. Human multi-tissue dotblots revealed an identical transcription pattern for both mRNAs. The concentration of the TCTP mRNAs differed up to the factor 100 between different tissues, indicating distinct tissue specificity in transcriptional control. 1.2 kb 5'flanking promoter structures were analyzed for transcription factor binding sites. For functional studies TPT1 promoter fragments were fused to the chloramphenicol acetyltransferase (CAT) reportergene and assayed by cell transfection and CAT enzyme activity. A basic promoter of 66 bp length containing a TATA box could be defined. Maximal promoter activity of 90% compared to the strong thymidine kinase promoter was associated with a fragment of 290 bp containing a SP-1, two AP-1/CREB and two ETS binding sites. This is a common feature of genes like TPT1, which are inducible by phorbolesters and lipopolysaccharides. Furthermore, numerous processed TPT1 pseudogenes were found spread through the rabbit genome. Six pseudogenes and their flanking genomic integration sites were sequenced. They represented both mRNA types and were at least 99% homologous to the corresponding mRNAs. In all pseudogenes the open reading frames were retained and in two of them the original amino acid sequence was even conserved completely. The 5'flanking region of one pseudogene was tested for transcriptional activity by CAT assays and revealed an activity of about 15% of the authentical TPT1 promoter. This could suggest a possible expression of TPT1 pseudogenes in vivo.