Ribosomal transcription produces RNAs essential for the structure and function of the ribosomes. The up-regulation of this transcription during growth is known to address the increased needs of the cell for proteins and thus for ribosomes. However, the mechanisms involved in this regulation are not well characterized. Our laboratory was the first to demonstrate a direct link between growth and ribosomal transcription. Stimulation of mammalian cells with serum or EGF causes an immediate increase in ribosomal RNA synthesis. This activation is dependant on the ERK pathway and on the phosphorylation of the architectural factor UBF by ERK. However, the increase of transcription resulting from this stimulation is not correlated with an increase in RNA polymerase I loading on the ribosomal genes but is rather explained by a higher elongation rate of the polymerase. We also showed that UBF blocks elongation when it is not phosphorylated and is permissive to it when it is phosphorylated by ERK. These results argue against the actual model suggesting that regulation occurs only at initiation level and demonstrate for the first time the existence of a regulation at the elongation level. Theoretically, ribosomal transcription could also be regulated at the level of gene activation since more than half of the ribosomal genes are silenced at ail times. According to the current view, DNA methylation is involved in the silencing process, but we have found that, at least in human cells, it is only important to silence a certain portion of the ribosomal genes. Loss of methylation following genetic inactivation of both DNA methyltransferase 1 and 3b in human colorectal carcinoma cell line results in a decrease in both ribosomal transcription and proliferation rates and in defects in ribosomal RNA processing and nucleolar structure. Moreover, loss of DNA methylation leads to an ingression of RNA polymerase II in the ribosomal locus thus probably causing the problems observed in these cells. We suggest that both DNA methylation and regulation of the elongation rates are essential mechanisms to maintain a high density of RNA polymerase I elongating complexes on the ribosomal genes in order to exclude RNA polymerase II and ensure an efficient ribosome biogenesis. / La transcription ribosomale synthétise les ARN qui sont au coeur de la fonction et de la structure des ribosomes. L'augmentation de cette transcription en condition de croissance est connue et répond aux besoins accrus de la cellule en protéines et donc en ribosomes. Toutefois, les mécanismes impliqués dans cette régulation étaient inconnus. Notre laboratoire a montré l'existence d'un lien direct entre la croissance et la transcription ribosomale. Nous avons montré que la stimulation de cellules de mammifères par le facteur EGF ou l'ajout de sérum cause une augmentation rapide de la transcription ribosomale. Cette activation est dépendante de la voie ERK qui cible le facteur architectural UBF. Nous avons aussi montré que cette stimulation n'entraîne pas de recrutement massif d'ARN polymérases 1 aux gènes ribosomaux, et ce malgré l'augmentation de la synthèse d'ARN ribosomaux. Par contre, la vitesse d'élongation de la polymérase explique quantitativement cette augmentation et celle-ci est régulée par l'état de phosphorylation d'UBF par ERK. Ces résultats remettent en question le modèle actuel de régulation unique au niveau de l'initiation des transcrits et démontrent pour la première fois l'existence d'une régulation au niveau de l'élongation. La proportion élevée de gènes ribosomaux silencieux suggère que ceux-ci puissent être activés et constituer un troisième niveau de régulation de la transcription ribosomale. La méthylation de l'ADN est suggérée comme étant importante pour l'établissement et le maintien de ces gènes dans un état silencieux. Or, nous avons montré que la méthylation de l'ADN explique l'état silencieux d'une partie et non de la totalité de ces gènes dans des cellules humaines en culture. De plus, nous avons montré que la perte de méthylation, suite à l'inactivation génique de DNMTl et 3b, entraîne une diminution de la transcription ribosomale et de la vitesse de prolifération ainsi que des défauts dans la transformation de TARN précurseur et dans la structure nucléolaire. De plus, la perte de méthylation entraîne une incursion de TARN polymérase II dans le locus ribosomal ce qui cause vraisemblablement les problèmes observés. Nous suggérons que la méthylation de l'ADN et la régulation de l'élongation des transcrits sont des mécanismes essentiels pour le maintien d'une forte densité de complexes en élongation sur les gènes ribosomaux de façon à exclure TARN polymérase II pour ainsi assurer une biogenèse efficace des ribosomes.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 29, 2010). Vita. Thesis advisor: Grace Y. Sun. "May 2008" Includes bibliographical references.

L'apoptose lymphocytaire joue un rôle essentiel dans le contrôle de la réponse immunitaire et de la prolifération cellulaire. De nombreuses voies interviennent dans sa régulation, dont certaines dépendantes de l’oncogène RAS. Un défaut d'apoptose lymphocytaire induit l'apparition de maladies auto-immunes et lympho-prolifératives comme l'Autoimmune LymphoProliferative Syndrome (ALPS). L'ALPS fait suite à des anomalies du principal récepteur membranaire de mort FAS, pivot de l'apoptose lymphocytaire. Le RAS-Associated Lymphoproliferative Disease (RALD) est une entité décrite récemment, se rapprochant de l'ALPS par la symptomatologie et la physiopathologie sous-jacente. Cependant, dans le RALD, le défaut d'apoptose lymphocytaire n’est pas lié à des mutations de FAS mais à une hyperactivation de la voie RAS, mettant ainsi en lumière le rôle essentiel de cette voie dans la régulation du processus en question. Dans les Leucémies Myélo-Monocytaires Juvéniles Chroniques (JMML), les mêmes mutations que celles observées dans les RALD sont trouvées, sur les mêmes populations cellulaires. Il existe une hétérogénéité clinique et biologique au sein des JMML, certaines étant indolentes (LS-JMML) et d'autres sévères (S-JMML). A cette hétérogénéité au sein même des JMML s'ajoute celle observée entre JMML et RALD. L'objectif de ce travail a été de comprendre les tenants des différences phénotypiques observées, au travers du scope de l'apoptose des lymphocytes T activés, en comparant les trois entités résultant de mutations activatrices de RAS dans des cellules pluripotentes hématopoïétiques : RALD, LS-JMML et S-JMML. Nous rapportons des conséquences distinctes pour des mutations identiques ou équivalentes, avec différentes voies de l'apoptose touchées, différenciant les phénotypes induits. Ce travail a permis de démontrer que l’hyperactivation de la voie RAS seule n’entraîne pas nécessairement une dérégulation de la réponse immunitaire T. Des événements additionnels aux mutations présentes sont nécessaires au développement des symptômes. Ces événements ont bien des conséquences sur l'apoptose lymphocytaire, au niveau post-traductionnel, qu’ils concernent la voie RAS ou non. Les différences observées entre les trois phénotypes sur le plan expérimental pourraient être une aide au pronostic. De plus, ce travail ouvre la voie à l'identification en détails des facteurs additionnels et voies défaillantes et permettrait ainsi d'obtenir des thérapeutiques spécifiques actuellement inexistantes. / Lymphocytes apoptosis is essential in maintaining homeostasis and avoiding abnormal proliferation. When defective, autoimmune diseases as the Autoimmune LymphoProliferative Syndrome (ALPS), due to mutations of the death receptor FAS, can occur. Several pathways are important actors influencing the apoptosis cascade, including the RAS proto-oncogene signaling. The RAS Associated Lymphoproliferative Disease (RALD) is a newly described entity, similar to ALPS but with RAS mutations instead of FAS mutations, enlightening the primary role of RAS in apoptosis regulation. Interestingly, the same RAS mutations as observed in RALD are also the cause of a malignant proliferation, the Juvenile Myelo Monocytic Leukemia (JMML). In the case of JMML, RAS mutations can lead either to a mild (LS-JMML) or a severe (S-JMML) phenotype. Thus, three different phenotypes can be caused by the same oncogenic RAS mutations. In order to better understand and characterize the influence of oncogenic RAS mutations in lymphocytes’ apoptosis we studied it in patients presenting with RALD, LS-JMML and JMML. We showed that isolated RAS hyperactivity is not sufficient to induce an immune deregulation. Additional factors are required to do so. These factors influence both mitochondrial and extrinsic apoptosis pathways at a post-transcriptional level. They are due to probable genetic events, and their identification can lead to new therapeutic strategies. Furthermore, activated lymphocytes’ in vitro apoptosis assessment can help differentiating the three phenotypes and thus facilitate prognosis prediction.

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.

Epidermolysis bullosa simplex (EBS) and epidermolytic ichthyosis (EI) are rare skin fragility diseases characterized by intra-epidermal blistering due to autosomal dominant-negative mutations in basal (KRT5 or KRT14) and suprabasal (KRT1 or KRT10) keratin genes,  respectively. Despite vast knowledge in the disease pathogenesis, the pathomechanisms are not fully understood, and no effective remedies exist. The purpose of this work was to search for keratin gene mutations in EBS patients, to develop in vitro models for studying EBS and EI, and to investigate novel pharmacological approaches for both diseases. We identified both novel and recurrent KRT5 mutations in all studied EBS patients but one which did not show any pathogenic keratin mutations. Using cultured primary keratinocytes from EBS patients, we reproduced a correlation between clinical severity and cytoskeletal instability in vitro. Immortalized keratinocyte cell lines were established from three EBS and three EI patients with different phenotypes using HPV16-E6E7. Only cell lines derived from severely affected patients exhibited spontaneous keratin aggregates under normal culture conditions. However, heat stress significantly induced keratin aggregates in all patient cell lines. This effect was more dramatic in cells from patients with a severe phenotype. In organotypic cultures, the immortalized cells were able to differentiate and form a multilayered epidermis reminiscent of those observed in vivo. Addition of two molecular chaperones, trimethylamine N-oxide dihydrate (TMAO) and sodium 4-phenylbutyrate (4-PBA), reduced the keratin aggregates in both stressed and unstressed EBS and EI keratinocytes, respectively. The mechanism of action of TMAO and 4-PBA was shown to involve the endogenous chaperone system (Heat shock proteins e.g. Hsp70). Besides, MAPK signaling pathways also seemed to be incriminated in the pathogenesis of EBS. Furthermore, depending on which type of keratin is mutated, 4-PBA up-regulated Hsp70 and KRT4 (possibly compensating for mutated KRT1/5), and down-regulated KRT1 and KRT10, which could further assist in protecting EBS and EI cells against stress. In conclusion, novel and recurrent pathogenic keratin mutations have been identified in EBS. Immortalized EBS and EI cell lines that functionally reflect the disease phenotype were established. Two pharmacologic agents, TMAO and 4-PBA, were shown to be promising candidates as novel treatment of heritable keratinopathies in this in vitro model.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 97-109.

Made available in DSpace on 2015-07-22T20:50:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-30. Added 1 bitstream(s) on 2015-08-11T03:26:36Z : No. of bitstreams: 1 Publico-Tese%20Paloma%20Korehiza%20Maza%20versao%20final.pdf: 1568579 bytes, checksum: 8a94eab5c79046acd8162d9a949d2c36 (MD5) / Muitos patógenos são capazes de manipular a sinalização de células do hospedeiro para facilitar sua infecção. Nesta tese, demonstramos que o fungo Paracoccidioides brasiliensis promove um aumento na ativação de ERK1/2 e SFKs em células epiteliais A549 (de pulmão humano), de aproximadamente 6 e 7 vezes, respectivamente, em relação aos níveis basais. Utilizando PP2, inibidor da atividade de SFKs, e PD98059, inibidor da ativação da via ERK1/2, verificamos que a ativação de ERK1/2 é parcialmente dependente de SFKs ativadas e que provavelmente outras quinases também estão envolvidas neste evento. Além da modulação da sinalização de células do hospedeiro, diversos estudos têm demonstrado que patógenos seqüestram domínios presentes nas membranas da célula hospedeira denominados “membrane rafts”, os quais são enriquecidos em esfingolipídeos e colesterol e estão envolvidos em diversos eventos celulares, incluindo a sinalização celular. Por diferentes metodologias, como a desorganização de “membrane rafts” por drogas que extraem (metil-β- ciclodextrina, MβCD) ou que se ligam (nistatina) ao colesterol, e a localização do gangliosídeo GM1, um marcador de “membrane rafts”, utilizando a subunidade B da toxina da cólera (CTB), mostramos o envolvimento destes domínios de membranas de células epiteliais na adesão de P. brasiliensis. A partir do isolamento de membranas resistentes a detergente (DRMs) de células epiteliais após incubação com o fungo, mostramos a ativação e o deslocamento de SFKs para as frações que contêm os “membrane rafts”. Além disso, verificamos que a depleção do colesterol com MβCD inibe completamente a ativação de SFKs, corroborando a importância dos domínios de membranas para a ativação destas quinases. Os resultados apresentados nesta tese demonstram, pela primeira vez, que fungos patogênicos modulam a organização e a atividade de “membrane rafts” de células hospedeiras para o estabelecimento da infecção. / Many pathogens are able to manipulate host cell signaling in order to facilitate infection. In this thesis, we demonstrated that the fungus Paracoccidioides brasiliensis promotes an increase of ERK12 and SFK activation in A549 epithelial cells (human lung cells), by approximately 6- and 7-fold over basal levels, respectively. By using PP2, inhibitor for SFK activity, and PD98059, inhibitor for ERK1/2 activation, we verified that ERK1/2 activation is partially dependent of activated SFKs and probably other kinases are involved in this event. Besides modulation of host cell signaling, several studies have been demonstrated that pathogens hijack domains that are present in host cell membranes called membrane rafts, which are cholesterol- and sphingolipidenriched domains, that are involved in several cell events, including cell signaling. By using several approaches, such as membrane rafts disruption with cholesterolextractor (methyl-β-cyclodextrin, MβCD) or –binding (nystatin) drugs, and the localization of ganglioside GM1, a membrane raft marker, by using cholera toxin subunit B (CTB), we showed the involvement of these epithelial cell membrane domains in P. brasiliensis adhesion. By isolating detergent-resistant membrane (DRM) from epithelial cells after incubation with this fungus, we showed the activation and the dislodgment of SFKs to fractions which contain membrane rafts. Moreover, we verified that cholesteroldepletion with MβCD completely inhibits SFK activation, corroborating the importance of membrane domains in activation of these kinases.The results presented in this thesis demonstrate for the first time that pathogenic fungi may modulate the organization and activity of host cell membrane rafts for infection establishment. / TEDE / BV UNIFESP: Teses e dissertações

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Orientador: William Dias Belangero / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-28T23:43:39Z (GMT). No. of bitstreams: 1 Faria_LidiaDornelasde_M.pdf: 3066212 bytes, checksum: 6cb5506682740e2fcb2de4b1694998a3 (MD5) Previous issue date: 2015 / Resumo: A Terapia por ondas de choque (TOC) é uma alternativa não invasiva utilizada como método de indução a formação óssea que consiste em pulsos sonoros de alta energia transmitidas de modo focal a um tecido específico. Artigos demonstram aumento de vascularização, que ativação de proteínas como BMP (bone morphogenic protein) e Erk (extracellular signal-regulated kinase) induzindo diferenciação osteogênica após sua utilização no tecido ósseo. O presente estudo visou avaliar os níveis das proteínas Erk e Akt (akutely transforming), envolvidas na cascata protéica responsiva a deformação celular gerada por estímulo mecânico e consequente transformação em estímulo bioquímico induzindo a osteogênese. Os animais selecionados para o estudo foram anestesiados e divididos em dois diferentes grupos, onde no dia 1, o primeiro grupo foi submetido a TOC em sessão única de 500 impulsos gerados por aparelho eletrohidráulico a 0,12mJ/mm²na tíbia intacta e o segundo não recebeu TOC. Na sequência, os animais foram divididos em 3 subgrupos para cada tempo de segmento de 7, 14 e 21 dias A determinação dos níveis das proteínas propostas foi realizada por meio de immunoblotting. A fosforilação das proteínas Erk e Akt dos tecidos ósseos das tíbias extraídas dos ratos aumentou nos grupos submetidos a TOC após 7, 14 e se manteve elevado até o 21° dia quando comparado ao controle / Abstract: Extracorporeal shock wave therapy (ESWT) is a non-invasive alternative used as a method for inducing bone formation that consists of high-energy acoustic pulses transmitted in a focal way to a specific tissue. Studies show increase in vascularization which activate proteins such as BMP and Erk inducing osteogenic differentiation after its use in the bone tissue. The present study aimed at evaluating the levels of Erk and AKT proteins involved in the protein cascade responsive to cell deformation in biochemical stimulus inducing osteogenesis. The animals selected for the study were under anesthesia and divided in two different groups where on day 1 the first group was submitted to ESWT in one 500 pulse-session generated by an electrohydraulic device at 0,12mJ/mm² in intact tibia and fibula and the second did not receive ESWT. Then the animals were divided into 3 sub-groups, one for each segment times of 7, 14 and 21 days. Immunoblotting analysis was performed to determine the levels of the proposed proteins. The Erk and Akt protein phosphorylation of the bone tissues of extracted tibia from the animals increased in the groups submitted to ESWT and kept elevated until the 21st day when compared to the control group / Mestrado / Fisiopatologia Cirúrgica / Mestra em Ciências

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