The JAK/STAT pathway is but one of the signal transduction cascades responsible for proper development and homeostasis. Gain-of-function mutations of pathway components are causative agents of several leukemias, highlighting the necessity for proper regulation of signal transduction. Drosophila presents an attractive model to study JAK/STAT signaling because mutations in the pathway behave in an analogous manner. Furthermore, the Drosophila cascade is much simpler as only one of each component required for activation has been characterized; whereas in mammals, there are many ligands, receptors, 4 JAKs and 7 STATs.Suppressors of Cytokine Signaling (SOCS) are one family of molecules which regulate JAK/STAT signaling via a negative feedback loop. All SOCS share a distinct modular domain architecture, which we exploited to locate three putative SOCS homologues within the Drosophila genome. I present the identification and initial characterization of one of these homologues, Socs44A. I show that Socs44A is not responsive to or dependent on JAK activity. However, I demonstrate that Socs44A is capable of downregulating JAK/STAT signaling in the developing wing but not inoogenesis, indicating that its ability to regulate the pathway is tissue specific, a phenomenon observed in the mammalian model.Signal transduction pathways are integrated at multiple levels. This interplay allows for combinatorial signaling, resulting in a higher order of complexity in the signals that can be received and interpreted by a cell. Well documented are the interactions between the JAK/STAT and the EGFR/MAPK pathways. In this work, I show that Socs44A can genetically interact with, and upregulate, the EGFR/MAPK pathway, analogous to a recent report involving SOCS-3.Starting with the Drosophila genome sequence, I initiated a reverse genetic approach to studying the function of the Socs44A locus. During the course of this investigation, I designed and implemented a novel post-processor of the BLAST algorithm, called Multi-BLAST, which facilitates retrieval of multiple domain sequences from public databases. In what would have been the ultimate achievement of this study, I attempted two mutagenesis screens designed to isolate Socs44A loss-of-function alleles. Progress on these screens is reported.

RNA silencing is a cellular mechanism of post-transcriptional gene regulation which is highly conserved among the plant and animal kingdoms of life, and plays a critical part of developmental biology, maintenance of homeostasis, and host-pathogen interactions. The pathway is engaged by small double-stranded (ds)RNA molecules (small RNAs), which effect sequence specific gene silencing by targeting complementary RNA sequences. There are several classes of small RNAs which engage the pathway. MicroRNAs (miRNAs) are expressed in the genome as endogenous regulators of gene expression. Short-interfering RNAs (siRNAs) are usually from exogenous sources such as viral-derived short-interfering RNAs, or synthetic siRNAs which are applied to cells or organisms to inhibit expression of specific genes. The p19 protein is a viral suppressor of RNA silencing (VSRS) endogenous to tombusviruses, which binds small RNA duplexes of any sequence with extremely high affinity. Because of its unique binding properties, recombinant p19 proteins are an excellent platform for tool development surrounding the RNA silencing pathway and are used extensively in novel applications for modulating the activity of small RNAs in living systems and for detecting small RNAs in biological samples. Herein we present work that has increased the breadth of p19’s utility as a biotechnology tool in three distinct realms. First, we present a chemical biology approach which combines p19 and small molecules for potent inhibition of the RNA silencing pathway in human cells. Secondly, we present the development of a novel fusion protein between p19 and a cell penetrating peptide (CPP), which functions as an siRNA delivery agent to allow gene knockdown in human cells. Thirdly, we have improved the utility of p19 for detecting and sequestering human miRNAs through rationally designing the binding surface; we describe mutations which dramatically enhance p19's affinity for human miRNA-122. The work presented here adds to the growing repertoire of engineered RNA binding proteins (RBPs) as tools for studying small RNA molecules and modulating their activity for applications in human therapeutics.

No description available.

AIDS-related human cytomegalovirus (HCMV) retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. Understanding the pathogenesis of this disease is essential for developing new, safe, and effective treatments for its prevention or management, yet much remains unknown about the virologic and immunologic mechanisms contributing to its pathology. To study such mechanisms, we use a well-established, reproducible, and clinically relevant animal model with retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS) that mimics in mice the symptoms and progression of AIDS in humans. Over 8 to 12 weeks, MAIDS mice become susceptible to experimental murine cytomegalovirus (MCMV) retinitis. We have found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling (SOCS)1 and SOCS3, host proteins which dampen immune-related signaling by cytokines, including antiviral interferons. Herein we investigated virologic and/or immunologic mechanisms involved in this stimulation and how virally-modulated SOCS1 and/or SOCS3 proteins may contribute to MCMV infection or experimental MAIDS-related MCMV retinitis. Through pursuit of two specific aims, we tested the central hypothesis that MCMV stimulates and employs SOCS1 and/or SOCS3 to induce the onset and development of MCMV retinal disease. MCMV-related SOCS1 and SOCS3 stimulation in vivo occurred with intraocular infection, was dependent on method and stage of immune suppression and severity of ocular pathology, was associated with stimulation of SOCS-inducing cytokines, and SOCS1 and SOCS3 were differentially sensitive to antiviral treatment. In vitro studies further demonstrated that SOCS1 and SOCS3 stimulation during MCMV infection occurred with expected immediate early kinetics, required viral gene expression in cell-type-dependent and virus origin-dependent patterns of expression, and displayed differential sensitivity to antiviral treatment. These data suggest that SOCS1 and SOCS3 are stimulated by divergent virologic, immunologic, and/or pathologic mechanisms during MCMV infection, and that they contribute to the pathogenesis of retinal disease, revealing new insights into the pathophysiology of AIDS-related HCMV retinitis.

Les protéines de type SMC (pour « Structural Maintenance of Chromosomes ») sont impliquées dans différents aspects de la dynamique du chromosome tels que la condensation, la ségrégation et la réparation de l’ADN. En effet, une souche de Bacillus subtilis dépourvue de SMC présente des phénotypes sévères tels qu’un défaut dans la compaction et le partitionnement du chromosome, une sensibilité accrue à certaines drogues endommageant l’ADN ainsi qu’à des inhibiteurs de gyrase. Une telle souche est incapable de croître en condition de croissance rapide. Pour comprendre l’étendue des phénotypes associés à la perte de ce gène, une identification génétique de nouveaux partenaires a été entreprise : des suppresseurs spontanés de la délétion de smc ont été isolés en condition de croissance rapide. Différentes classes de suppresseurs ont été mises en évidence, suggérant que différentes mutations pouvaient restaurer la viabilité d’une souche dépourvue de SMC. Leur caractérisation a révélé qu'ils permettaient de restaurer une partie des défauts que présente le mutant Δsmc, en particulier la résistance aux inhibiteurs de gyrase, et semblaient limiter la formation de cassures de l'ADN. Par séquençage du génome complet des suppresseurs, certaines de ces mutations ont pu être identifiées, et semblent causer une perturbation de la voie de biosynthèse des ARN de transfert. Cette perturbation permet de restaurer le défaut de croissance, et ce plus efficacement qu’une inhibition de la traduction par des drogues comme le chloramphénicol, ou par la réduction du pool de nucléotides par l’hydroxyurée. L’ensemble de ces résultats suggère que la réponse stringente pourrait être en partie responsable du phénotype suppresseur. Il est proposé qu’en dehors de la compaction du chromosome, le complexe SMC soit directement impliqué dans le maintien de l’intégrité des fourches de réplication. / SMC proteins (for "Structural Maintenance of Chromosomes") are involved in different aspects of chromosome dynamic such as condensation, segregation and DNA repair. Indeed, a Bacillus subtilis mutant lacking the SMC complex shows severe phenotypes such as defects in condensation and chromosome partitioning, an increase in sensitivity DNA damaging drugs or gyrase inhibitors. The viability of such strain is limited to conditions of slow growth. To understand the range of phenotypes associated with loss of this gene, a genetic identification of new partners was undertaken: spontaneous suppressors of smc deletion were isolated in rapid growth conditions. Different classes of suppressors have been identified, suggesting that different mutations could restore the viability of a strain lacking SMC complex. Characterization of suppressors revealed they can restore some of the defects shown in Δsmc mutant, particularly resistance to gyrase inhibitors, and seemed to limit the formation of DNA breaks. By sequencing the complete genome of suppressors, some of these mutations have been identified and cause an alteration of the biosynthetic pathway of transfer RNA. This disruption can restore the growth defect more efficiently than inhibition of translation by drugs such as chloramphenicol, or by reducing the pool of nucleotides by hydroxyurea. Taken together, these results suggest that the stringent response could be partly responsible for the suppressor phenotype. It is proposed that apart from the compaction of the chromosome, the SMC complex is directly involved in maintaining the integrity of replication forks.

Adult T-cell leukemia (ATL) is an aggressive lymphoproliferative disorder associated with human T-cell leukemia virus type 1 (HTLV-1) infection. Reactivation of telomerase, a critical event in tumor progression observed in late phases of ATL development, has been shown to be caused by HBZ (HTLV-1 bZIP factor), a regulatory protein encoded by the negative strand of the HTLV-1 genome. The HBZ-mediated up-regulation of the telomerase catalytic subunit is dependent on JunD, which in the cellular context occurs in the complex with menin, the product of the MEN-1 tumor suppressor gene. Interaction with menin represses JunD-dependent transcription and converts JunD into a growth suppressor, whereas it acts as a growth promoter in the absence of menin. My results demonstrate that the viral protein HBZ abrogates tumor suppressor function of menin, resulting in the activation of JunD transcriptional activity and finally in the up-regulation of its target gene, the human telomerase reverse transcriptase (hTERT). I showed that HBZ, JunD and menin can coexist in the same protein complex and that HBZ and menin exert opposite effects on JunD transcriptional activity. Moreover menin inhibits the JunD-mediated activation of the hTERT proximal promoter and HBZ is able to counteract this effect. Finally, I proposed that HBZ, by recruiting p300 histone acetyltransferase, reverses the histone deacetylation conducted by menin-recruited HDACs and therefore up-regulates the expression of the hTERT gene. Altogether, my work led to the identification of the molecular mechanism leading to the functional impairment of the menin tumor suppressor, which results in the deregulation of AP-1 signaling in HTLV-1 infected cells. Finally this work gave new insights into the mechanism of the transcriptional up-regulation of the hTERT gene upon HTLV-1 infection, being a key event during the development of Adult T-cell leukemia and a necessary step towards the progression into more aggressive courses.

No description available.

La leucémie T de l’adulte (ATL) est une pathologie lympho-proliférative aiguë associée à l’infection par le virus HTLV-1 (human T-cell leukemia virus type 1). La réactivation de la télomérase observée lors de la phase tardive du développement de l’ATL est un évènement crucial dans la progression tumorale. Elle est induite au niveau transcriptionnel par la protéine HBZ (HTLV-1 bZIP factor) et est dépendante du facteur de transcription JunD. Ce dernier est normalement associé en complexe avec le produit du gène suppresseur de tumeur MEN-1, la ménine, dont l’interaction avec JunD réprime la transcription JunD-dépendante et convertit JunD en inhibiteur de croissance.Mes résultats démontrent que la protéine virale HBZ inhibe la fonction suppresseur de tumeur de la ménine, induisant l’activité transcriptionnelle de JunD et donc l’activation de la transcription de son gène cible : la transcriptase inverse télomérase humaine (hTERT). J’ai démontré que HBZ, JunD et la ménine peuvent coexister dans un même complexe protéique et que HBZ et la ménine ont des effets opposés sur l’activité transcriptionnelle de JunD. En effet la ménine inhibe l’activation du promoteur proximal d’hTERT par JunD, alors que HBZ est capable de contre balancer cet effet. Finalement, je propose qu’en recrutant l’histone acétyltransférase p300, HBZ réverse la déacétylation des histones induite par le recrutement des HDACs par la ménine et par conséquent active le promoteur d’hTERT. L’ensemble de ces résultats a permis d’identifier les mécanismes moléculaires aboutissant à l’inhibition fonctionnelle de la protéine suppresseur de tumeur ménine, résultant en la dérégulation de la voie AP-1 dans les cellules infectées par HTLV-1. Finalement, ce travail apporte de nouvelles précisions sur le mécanisme de la surexpression transcriptionnelle de la télomérase lors de l’infection par HTLV-1, une étape importante de la mise en place et du développement de la leucémie T de l’adulte vers des stades plus agressifs. / Adult T-cell leukemia (ATL) is an aggressive lymphoproliferative disorder associated with human T-cell leukemia virus type 1 (HTLV-1) infection. Reactivation of telomerase, a critical event in tumor progression observed in late phases of ATL development, has been shown to be caused by HBZ (HTLV-1 bZIP factor), a regulatory protein encoded by the negative strand of the HTLV-1 genome. The HBZ-mediated up-regulation of the telomerase catalytic subunit is dependent on JunD, which in the cellular context occurs in the complex with menin, the product of the MEN-1 tumor suppressor gene. Interaction with menin represses JunD-dependent transcription and converts JunD into a growth suppressor, whereas it acts as a growth promoter in the absence of menin. My results demonstrate that the viral protein HBZ abrogates tumor suppressor function of menin, resulting in the activation of JunD transcriptional activity and finally in the up-regulation of its target gene, the human telomerase reverse transcriptase (hTERT). I showed that HBZ, JunD and menin can coexist in the same protein complex and that HBZ and menin exert opposite effects on JunD transcriptional activity. Moreover menin inhibits the JunD-mediated activation of the hTERT proximal promoter and HBZ is able to counteract this effect. Finally, I proposed that HBZ, by recruiting p300 histone acetyltransferase, reverses the histone deacetylation conducted by menin-recruited HDACs and therefore up-regulates the expression of the hTERT gene. Altogether, my work led to the identification of the molecular mechanism leading to the functional impairment of the menin tumor suppressor, which results in the deregulation of AP-1 signaling in HTLV-1 infected cells. Finally this work gave new insights into the mechanism of the transcriptional up-regulation of the hTERT gene upon HTLV-1 infection, being a key event during the development of Adult T-cell leukemia and a necessary step towards the progression into more aggressive courses.

The offshore oil industry is engaged in the development of new floating platforms, such as Spar, semi-submersible, tension-leg, FPSO and monocolumn for the exploration of deep and ultra-deep waters. Some of these floating systems have circular cross sections (or cross sections of other bluff geometries) being susceptible to vortex-induced vibrations (VIV). Vortex shedding behind a bluff body can be altered, suppressed or controlled over a limited range of Reynolds numbers. Various flow-control techniques, which result in the reduction of drag and unsteady forces, have been suggested and tested in simple geometries. One such method is the moving-surface boundary layer control (MSBC), in which smaller control rotating cylinders are placed close to the bluff body. This method is considered as an inspiration for the present experimental investigation of VIV suppression for omni-directional flows. In this context, three different configurations have been assembled to compare the effect of suppression on a plain cylinder surrounded by two, four and eight control cylinders distributed symmetrically around it. Experiments were carried out with static models and models free to oscillate in one-degree-of-freedom with fixed and rotating control cylinders. Experiments with a plain cylinder were performed to serve as reference. Displacements, drag and lift forces were measured. The position of the control cylinders proved to be an important parameter to VIV suppression. Configurations with two control cylinders increased lift and drag forces. In contrast, configurations of four and eight control cylinders showed to be more effective to suppress VIV. Furthermore the results for all the cases of the configuration of eight fixed control cylinders presented a reduction of displacement amplitude, lift and drag forces when compared to a plain cylinder. However, when the control cylinders were actuated, the two cases with rotating control cylinders increased drag force when compared to fixed control cylinders. / A indústria offshore está envolvida no desenvolvimento de novas plataformas flutuantes como Spar, semi-submersível, TLP, FPSO e monocoluna para a exploração de águas profundas e ultra-profundas. Alguns destes sistemas flutuantes têm seções transversais circulares (ou de outras seções rombudas) sendo susceptíveis à vibrações induzidas por vórtices (VIV). A esteira de vórtices desprendida de um corpo rombudo pode ser alterada ou suprimida ao longo de uma faixa de número de Reynolds. Várias técnicas de controle do escoamento foram sugeridas e testadas em geometrias simples, resultando na redução de forças de sustentação e arrasto. Um desses métodos é o controle de camada limite por superfícies móveis (CCLSM), no qual cilindrinhos rotativos de controle são colocados próximos ao corpo rombudo. Neste trabalho, este método foi abordado através de uma investigação experimental como um supressor de VIV para o escoamento omnidirecional. Neste escopo três diferentes configurações foram montadas para comparar o efeito de supressão sobre um cilindro liso rodeado por dois, quatro e oito cilindros de controle, distribuídos simetricamente em torno dele. Foram realizados ensaios com o modelo estático, ensaios de VIV em um grau de liberdade com cilindros de controle fixos e rotativos. Foram medidos deslocamento e forças de sustentação e arrasto. Os resultados mostraram que a posição dos cilindros de controle é um parâmetro importante para a supressão de VIV. A configuração com dois cilindros de controle aumentou as forças de sustentação e arrasto. Diferentemente, as configurações de quatro e oito cilindros de controle mostraram-se mais eficazes para suprimir VIV. Além disso, todos os casos da configuração de oito cilindros de controle fixos apresentaram redução nas amplitudes de vibração e nas forças de sustentação e arrasto, quando comparados com um cilindro liso. No entanto, quando os cilindros de controle foram acionados para rotacionar, mostrou-se um aumento na força de arrasto em relação aos cilindros de controle fixos.

O fenômeno de vibração induzida por vórtices (VIV) é particularmente danoso para estruturas submarinas como risers de exploração de petróleo. A maneira mais usual de se atenuarem os efeitos de VIV é instalar um supressor, como por exemplo strakes ou fairings. Dentre esses, foi desenvolvido por All Brow Universal Components um supressor chamado Ventilated Trousers (VT), que consiste em uma malha permeável feita de uma rede flexível e dezenas de bobbins. Através de um estudo experimental, procurou-se investigar os mecanismos hidrodinâmicos pelos quais o supressor V T funciona. Foram construídos três modelos diferentes de supressores: um modelo idêntico ao V T e duas malhas dele derivadas, alterando-se a geometria dos bobbins e a distribuição destes ao redor da malha. Foram realizados ensaios com o modelo xo e ensaios de VIV em um grau de liberdade alterando-se o amortecimento estrutural. Foram medidos deslocamento e forças de sustentação e arrasto. Os resultados mostraram que o supressor do tipo V T reduz as amplitudes de vibração, força de sustentação e arrasto quando comparados com um cilindro oscilando. No entanto, aumenta a força de arrasto quando comparado com o cilindro xo. A geometria da malha mostrou-se de grande importância para a supressão de VIV. Modelos que possuem o disco externo no bobbin impedem o surgimento de folga entre o modelo e o cilindro, além de aumentar o amortecimento hidrodinâmico. Três hipóteses foram levantadas para explicar o funcionamento do supressor V T. A primeira diz que a supressão é provocada pelo aumento do amortecimento hidrodinâmico. Os ensaios mostraram que, de fato, o supressor V T aumenta o amortecimento e, consequentemente, diminui as VIV. No entanto, somente esse efeito não explica toda a supressão obtida. As outras hipóteses, relacionadas à alterações bi e tridimensionais da esteira, foram avaliadas, porém não se pode afirmar que alguma delas seja isoladamente responsável por produzir o mecanismo hidrodinâmico de supressão. / The phenomenon of vortex-induced vibration (VIV) is particularly harmful to submarine structures such as risers used for oil extraction. The most usual way to attenuate the effects of VIV is the installation of suppressors, like strakes or fairings. Among them, All Brow Universal Components developed a VIV suppressor called Ventilated Trousers (VT), which consist of a permeable mesh made of a flexible net and tens of bobbins. Three different models of suppressors based on permeable meshes have been assembled with the objective to understand the hydrodynamic mechanism behind the suppression: one model identical to the VT and two meshes with different bobbin geometries and distribution. Tests were carried out with xed models and models free to oscillate in one degree of freedom varying the structural damping. Displacements, drag and lift forces were measured. Results showed that the VT suppressor reduced vibration amplitudes, lift and drag forces when compared to an oscillating circular cylinder. However, it increased drag force when compared to a fixed circular cylinder. The mesh geometry proved to be important to VIV suppression. Models that had an external disc on the bobbins avoided the appearance of a gap between the model and the cylinder. Three hypotheses were formulated to explain how the VT suppressor works. The first one says that the increase on hydrodynamic damping is responsible for suppression. In fact, tests showed that the VT increased hydrodynamic damping and, consequently, reduced the VIV response. However, this effect alone does not explain the suppression as a whole. The other two hypotheses related to two-dimensional and three-dimensional wake changes were evaluated, but it cannot be stated that any of them, on its own, is responsible for the whole of the suppression mechanism.