This thesis investigates the effect of viscous damping on rogue wave formation and permanent downshift using the higher-order nonlinear Schrödinger equation (HONLS). The strength of viscous damping is varied and compared to experiments with only linear damped HONLS. Stability analysis of the linear damped HONLS equation shows that instability stabilizes over time. This analysis also provides an instability criterion in the case of HONLS with viscous damping. Numerical experiments are conducted in the two unstable mode regime using perturbations of the Stokes wave as initial data. With only linear damping permanent downshift is not observed and rogue wave formation is decreased. The addition of viscous damping leads to permanent downshift and a slight increase in rogue wave activity. Analysis of the energy and momentum gives a possible explanation for this behavior.

Maspina (SERPINB5) é um potencial gene supressor de tumor com atividades biológicas pleiotrópicas, incluindo a regulação da proliferação, morte, adesão e migração celular e a expressão gênica e a resposta ao estresse oxidativo. O mecanismo molecular subjacente a sua função é pouco conhecido. Vários estudos sugerem que a localização subcelular de maspina tem um papel essencial na sua função biológica e sua atividade supressora de tumor. A maspina nuclear tem sido associada a um bom prognóstico, enquanto que a sua localização núcleocitoplasmática tem sido correlacionada à progressão tumoral. Portanto, este trabalho teve como objetivo investigar o mecanismo molecular da translocação nuclear de maspina. Assim, identificamos um sinal de localização nuclear (NLS) bipartido na sequência de maspina usando um software de predição de NLS. Deleção do NLS resulta em diminuição dos níveis de maspina nuclear. No entanto, observamos que maspina também é capaz de se difundir ao núcleo em células HeLa permeabilizadas com digitonina. Porém, estudos prévios indicaram que a localização subcelular de maspina é regulada na célula intacta. Assim, para poder distinguir o transporte regulado do passivo, fusionamos a sequência inteira de maspina e do seu NLS a 5GFPs (MaspinFL e 5GFPs-MaspinNLS, respectivamente). Foi observado que o NLS de maspina, mas não a sequência inteira da proteína, foi capaz de translocar a proteína quimérica ao núcleo, o que sugere que a disponibilidade do NLS de maspina pode ser regulada na sua estrutura nativa. Além disso, foi observado que a translocação nuclear de 5GFPs-MaspinNLS foi inibida pela co-transfecção com mutantes de Ran-GTPase, indicando que o processo depende de Ran-GTPase e portanto ocorre ativamente. Não observamos uma interação do NLS de maspina com carioferina alfa 2 (KPNA2) nem uma inibição do transporte nuclear de maspina ao tratar células com importazole, um inibidor da via clássica, o que sugere que maspina transloca para o núcleo de uma forma não convencional. / Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration, gene expression and oxidative stress response. The molecular mechanism underlying maspin function is poorly understood. Several studies suggest that subcellular localization plays an essential role on maspin biological function and tumor suppression activity. Nuclear maspin has been associated with a good prognostic, whereas nucleocytoplasmic localization correlates with tumor progression. The objective of this project was to investigate the mechanism underlying maspin nuclear translocation. We identified a bipartite Nuclear Localization Signal (NLS) in maspin protein sequence using an NLS prediction software. Deletion of maspin NLS leads to decrease in maspin nuclear levels. However, we also observed that maspin diffuses into the nucleus of digitoninpermeabilized cells. Still, previous studies indicated that maspin subcellular localization is regulated in the intact cell. Considering this, in order to distinguish between regulated and passive nuclear transport, maspin NLS sequence and full-length protein sequence were fused to 5GFPs (5GFPs-MaspinNLS and MaspinFL, respectively). We observed that MaspinNLS, but not MaspinFL, was able to drive 5GFPs nuclear translocation, suggesting that the availability of maspin NLS may be regulated in the native maspin structure. Furthermore, 5GFPs-MaspinNLS nuclear translocation was abrogated by mutant Ran-GTPase co-transfection, indicating that this process depends on Ran-GTPase and it occurs actively. An interaction between Maspin NLS and karyopherin alpha 2 (KPNA2) was not detected neither an inhibition of maspin nuclear transport when cells were treated with importazole, an inhibitor of the classic nuclear import pathway. These data suggest that maspin may be translocating to the nucleus in a nonconventional manner.

Ime4 is the catalytic subunit of a conserved methyltransferase (MTase) complex found in yeast, S. cerevisiae. This complex is responsible for creating the RNA modification N6- methyladenosine (m6A), the most common post-transcriptional modification in higher eukaryotes. There is evidence to suggest that m6A is an important mediator of gene expression control within the cell and has been associated with a diverse array of phenotypic effects, notably as a conserved determinant of cell fate. The MTase complex is known to be a nuclear protein, the compartment where it is believed to carry out most of its methylation activity. Recently, the nuclear localization signals (NLS) of the subunits of the human MTase complex were experimentally identified, whereas the NLSs of the yeast MTase complex remain unknown. Here, we have experimentally identified the amino acid sequence 517RKYQEFMKSKTGTSHTGTKKIDKK540, located within the C-terminal region, as a putative bipartite NLS for Ime4.

The small dispersion limit of the focusing nonlinear Schroödinger equation (fNLS) exhibits a rich structure with rapid oscillations at microscopic scales. Due to the non self-adjoint scattering problem associated to fNLS, very few rigorous results exist in the semiclassical limit. The asymptotics for reectionless WKB-like initial data was worked out in [KMM03] and for the family q(x, 0) = sech^(1+(i/∈)μ in [TVZ04]. In both studies the authors observed sharp breaking curves in the space-time separating regions with disparate asymptotic behaviors. In this paper we consider another exactly solvable family of initial data, specifically the family of centered square pulses, q(x; 0) = qx[-L,L] for real amplitudes q. Using Riemann- Hilbert techniques we obtain rigorous pointwise asymptotics for the semiclassical limit of fNLS globally in space and up to an O(1) maximal time. In particular, we find breaking curves emerging in accord with the previous studies. Finally, we show that the discontinuities in our initial data regularize by the immediate generation of genus one oscillations emitted into the support of the initial data. This is the first case in which the genus structure of the semiclassical asymptotics for fNLS have been calculated for non-analytic initial data.

Orientador: Marcos Roberto de Mattos Fontes / Resumo: A comunicação entre o núcleo celular e o citoplasma acontece através de mecanismos de transporte que permitem a passagem de moléculas por poros presentes no envoltório nuclear. Na Via Clássica de Importação Nuclear, a proteína Importina-α (Impα) atua na identificação das proteínas a serem transportadas ao núcleo a partir do reconhecimento de sequências de localização nuclear (NLS). Os primeiros estudos de caracterização estrutural da Impα de Neurospora crassa (NcImpα) e a sua estrutura cristalográfica mostraram a presença de regiões que podem estar relacionadas a especificidades da proteína NcImpα no reconhecimento de NLSs de proteínas de fungos. Além disso, foram reconhecidos prováveis NLSs em proteínas reguladoras do metabolismo de carbono e nitrogênio em fungos. O objetivo desse trabalho é identificar e caracterizar o modo de interação de NLSs de proteínas fúngicas com a NcImpα e verificar possíveis especificidades da proteína NcImpα no reconhecimento de NLSs. Os potenciais peptídeos NLS de proteínas dos fungos filamentosos N. crassa e Aspergillus nidulans foram submetidos a experimentos de calorimetria de titulação isotérmica (ITC) com a proteína NcImpα, para calcular a afinidade entre as moléculas. Dentre os peptídeos testados, as sequências correspondentes ao potenciais NLS dos fatores de transcrição PAC-3, NIT-2, FLB-3, VOSA e VEA, apresentaram elevada afinidade com a NcImpα, conforme indicado pelos valores de Kd obtidos. Quando submetidos a experimentos de importação ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The communication between the cell nucleus and the cytoplasm happens through transport mechanisms that allow the passage of molecules through pores present in the nuclear envelope. In the classical nuclear import pathway, the protein Importin-α (Impα) acts in the identification of the proteins to be transported to the nucleus from the recognition of nuclear localization sequences (NLS). The first structural characterization studies of N. crassa Impα (NcImpα) and its crystallographic structure showed the presence of regions that may be related to protein specificities in the recognition of fungal NLSs. In addition, NLSs were recognized in proteins related to fungal metabolism. The objective of this work is to identify NLSs in fungal proteins and observe the specificities of the NcImpα protein. Potential NLS peptides of N. crassa were subjected to isothermal titration calorimetry (ITC) experiments with NcImpα to verify and calculate the affinity of the complexes. Among the peptides tested, the sequences corresponding to the potentials NLS of the transcription factors PAC-3, NIT-2, FLB-3, VOSA and VEA, showed high affinity with NcImpα, as indicated by the Kd values obtained. When subjected to functional experiments with HeLa cells, the peptide NIT2-NLS were efficiently transported into the cell nucleus. Crystallization tests were performed to elucidate the structure of the complexes Impα/NLS peptides. A set of data from the NcImpα/ NIT2NLS complex was collected, with 99.71% comp... (Complete abstract click electronic access below) / Doutor

Dans cette étude, nous avons démontré par des essais de type Pull-Down la capacité de la queue C-terminale du CysLT1 d'interagir in vitro avec les importines [alpha]1, [alpha]4 et [alpha]5. Cette interaction est comparable à celle du NLS de l'antigène grand T du SV40 et du NLS de la nucléoplasmine de Xenopus laevis avec les mêmes importines. Nos études démontrent aussi que les déterminants structuraux de l'interaction ne sont pas limités seulment au NLS: des mutations dirigées contre les résidus-clé qui forment le NLS n'ont pas empêché l'interaction entre la queue C-terminale et les importines. Nos résultats suggèrent que d'autres résidus que ceux qui forment le NLS potentiel sont impliqués dans la liaison avec les importines. Nous avons démontré pour la première fois la phosphorylation in vitro de la queue C-terminale du CysLT1 par la PKA, et que cette phosphorylation peut moduler la capacité d'interaction avec l'importine [alpha]4. Nos travaux visaient aussi l'étude de l'expression cellulaire du récepteur CysLT1 et de son comportement suite à une stimulation au LTD[indice inférieur 4] .--Résumé abrégé par UMI.

Das RS1 Protein (Gen RSC1A1) beteiligt sich an der Regulation des Na+-D-Glukose-kotransporters SGLT1 und einiger anderer Transporter. In subkonfluenten LLC-PK1 Zellen hemmt RS1 die Freisetzung von SGLT1 aus dem trans-Golgi-Netzwerk und die Transkription von SGLT1. Während es sich in konfluenten Zellen hauptsächlich im Zytoplasma befindet, ist RS1 in subkonfluenten Zellen im Kern und im Zytoplasma lokalisiert. In der vorliegenden Arbeit wurden Mechanismus und Regulation der konfluenzabhängigen Kernlokalisation von RS1 untersucht. Dabel konnte gezeigt werden, dass die von Konfluenz abhängige Kernlokalisation von RS1 durch den Zellzyklus reguliert wird. In RS1 aus Sus scrofa (pRS1) wurde eine Sequenz identifiziert („nuclear shuttling signal“, NS), die für die konfluenzabhängige Verteilung von RS1 verantwortlich ist und sowohl das Signal für die Kernlokalisation (NLS) als auch das Signal für den Export aus dem Kern (NES) beinhaltet. Die NLS und NES Signale von RS1 vermitteln die Translokation des Proteins in den Kern und aus dem Kern mit Hilfe von Importin β1 bzw. CRM1, wobei die Verteilung von RS1 zwischen Kern und Zytoplasma durch die Aktivität des Exportsystems bestimmt wird. Es wurde gezeigt, dass die benachbarte Proteinkinase C (PKC) Phosphorylierungsstelle an Serin 370 von pRS1 die NS-gesteuerte Kernlokalisierung kontrolliert und für die vom Zellzyklus abhängige Kernlokalisation notwendig ist. Aufgrund der Ergebnisse der ortsgerichteten Mutagenese, PKC-Aktivierungsexperimenten und Massenspektrometrie-Analyse des Phosphorylierungsmusters von RS1 wurde ein Modell vorgeschlagen, das die Regulation der Kernlokalisation des RS1 Proteins in LLC-PK1 Zellen beschreibt. Dem Modell zufolge wird RS1 in subkonfluenten Zellen stark in den Kern befördert, während der Export von RS1 aus dem Kern nicht stattfindet. Das führt zur Anreicherung von RS1 im Kern. Nach Konfluenz wird Serin 370 durch PKC phosphoryliert, was die Steigerung des RS1-Exports aus dem Kern begünstigt und die überwiegend zytoplasmatische Lokalisation des Proteins in konfluenten Zellen hervorruft. Die konfluenzabhängige Regulation der Lokalisation von RS1 kann die Expression von SGLT1 während der Regeneration von Enterozyten im Dünndarm und der Regeneration von Zellen der Nierentubuli nach hypoxämischem Stress kontrollieren. Außerdem deutet die Analyse der Genexpression in embryonalen Fibroblasten der RS-/- Mäuse deutet darauf hin, dass die transkriptionale Regulation durch RS1 im Zellzyklus und bei der Zellteilung eine wichtige Rolle spielen kann. Da die Lokalisation von RS1 zellzyklusabhängig ist, kann RS1 für die Regulation der Transporter in spezifischen Phasen des Zellzyklus wichtig sein. / The RS1 protein (gene RSC1A1) participates in regulation of Na+-D-glucose cotransporter SGLT1 and some other solute carriers. In subconfluent LLC-PK1 cells, RS1 inhibits release of SGLT1 from the trans-Golgi network and transcription of SGLT1. In subconfluent cells, RS1 is localized in the nucleus and the cytoplasm whereas confluent cells contain predominantly cytoplasmic RS1. In the present study, the mechanism and regulation of confluence-dependent nuclear location of RS1 was investigated. Confluence dependent nuclear location of RS1 was shown to be regulated by the cell cycle. A nuclear shuttling signal (NS) in pRS1 was identified that ensures confluence-dependent distribution of pRS1 and comprises nuclear localization signal (NLS) and nuclear export signal (NES). The NLS and NES of RS1 mediate translocation into and out of the nucleus via importin ß1 and CRM1, respectively, and the nuclear/cytoplasmic distribution of the RS1 protein is determined by the nuclear export activity. The adjacent protein kinase C (PKC) phosphorylation site at serine 370 of pRS1 was shown to control nuclear localization driven by NS and is necessary for the differential localization of RS1 in quiescent versus proliferating cells. Basing on the data of site-directed mutagenesis, PKC activation experiments and mass spectrometry analysis of RS1 phosphorylation, the following model of the regulation of RS1 nuclear location in LLC-PK1 cells was proposed. In subconfluent cells, RS1 is actively imported into the nucleus whereas nuclear export of RS1 is not active leading to accumulation of RS1 in the nucleus. After confluence, phosphorylation of serine 370 of pRS1 by PKC takes place leading to enhancement of RS1 nuclear export and predominantly cytoplasmic distribution of the protein in the confluent cells. The confluence-dependent regulation of RS1 localization may control SGLT1 expression during regeneration of enterocytes in small intestine and during regeneration of renal tubular cells after hypoxemic stress. Moreover, the gene expression profiling of mouse embryonic fibroblasts with RS1-/- genotype suggests that transcriptional regulation by RS1 might be important for the cell cycle and cell division. Since RS1 localization depends on the cell cycle, RS1 might play a role in the regulation of the solute carriers during specific phases of the cell cycle.

The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase complex (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also regulates vRNP activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but also interacts with host RNA helicases to enhance viral RNA replication in the nucleus. NP contains at least one additional NLS sequence, with bioinformatics revealing a third NLS in some NP proteins. Published yeast-two hybrid results indicate that the first 20 amino acids of NP can sufficiently bind with cellular protein UAP56. Suggesting the interaction of NP-UAP56 can be a major mechanism of how NP involve in viral replication. Thus, to examine the role of the N-terminus of NP aside from its vRNP nuclear localization activity N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV-40 T-antigen were constructed, termed del20NLS-NP and del20-NP. Nuclear localization of vRNPs with these constructs was assessed by GFP expression and western blotting. All these constructs exhibit nuclear localization, consistent with NLS1 being utilized for vRNP localization but not NP localization and vRNP formation in the nucleus. Furthermore, qPCR results demonstrated decreased vRNA synthesis activity, exacerbated as the vRNA template is lengthened in both plasmids, consistent with a lack of interaction with host RNA helicases. Interestingly, del20-NP vRNP activity is less severe than del20NLS-NP, suggesting perturbations of the N-terminus disrupt vRNP activity. To narrow down the region responsible for vRNA expression defect, del10-NP was constructed. GFP expression displayed similar activity between del10-NP and WT-NP with del20-NP showing a severe defection, suggesting NP amino acids 11-20 might be the major region responsible for the vRNA synthesis defect. However, sucrose density gradient results do not support the published interaction between NP and UAP56 in 293T cells. These results support the N-terminal region, potentially amino acids 11-20 of NP, is playing the important role in efficient viral gene expression during virus replication especially as vRNA template lengthen, and that the NLS1 of NP is not essential for NP/vRNP nuclear localization in our reconstituted vRNP assay.

Corticosteroid ligands activate the glucocorticoid receptor (GR). GR plays a role in glucose homeostasis, adipogenesis, inflammation, and mood and cognitive functions. Understanding the interplay of diverse forms of receptor regulation (including post-translational modification, cofactor interactions, ligand binding, and receptor localization) and their effects is important for understanding and developing more effective treatment for a variety of conditions. Prior to ligand binding, the naïve GR is primarily cytoplasmic, residing in a chaperone complex containing heat-shock proteins and immunophilins. Upon ligand-binding, alterations to the complex allow the receptor to dimerize and import into the nucleus. Nuclear GR interacts with transcriptional regulatory sequences and recruits cofactors to regulate specific gene expression. Upon hormone withdrawal, the original chaperone complex is reassembled and the receptor is exported to the cytoplasm. Interestingly, while the import of GR into the nucleus occurs very rapidly (t ½ = 5 min), the re-export is significantly slower (t ½ = 12-24h). Previous work by our lab and others has indicated the existence of a nuclear retention signal (NRS) within the GR. The NRS sequence of the GR, its interaction partners, and the role it might play in the activity of the receptor have not yet been fully defined. Work in the Hache lab indicates that mutation of the GR nuclear localization signal 1 (NL1) increases the export rate of nuclear GR to the cytoplasm, as well as compromising receptor import, suggesting that the NL1 overlapped an NRS sequence. In this work, I made a series of GR mutants, based on sequence from the SV40 large T antigen NLS, which lacks nuclear retention activity. Using these mutants, I found that GR nuclear retention is influenced by both specific residues within the hinge region and the location of the sequence within the receptor, as reintroduction of the NLS sequence at the N-terminus of the receptor retention mutant failed to reconstitute the retention activity. Agonist liganded and hormone-withdrawn receptor mutants showed a similar decrease in retention. By contrast, antagonist-withdrawn GR mutants were retained in the nucleus, possibly due to altered receptor configuration and interactions. Assays of GR-responsive promoter activation by receptor retention mutants showed that while no difference in the ability of retention mutants to activate transcription was seen at a simple promoter, activation of a complex promoter was compromised. This impaired transactivation for the SV506-523 mutant correlated with decreased histone H4 acetylation and PolII recruitment, while GR DNA-binding at the target promoter appeared to be unaffected. These results suggested that promoter-specific cofactor interactions might be implicated in GR nuclear retention. Loss of GR hinge interaction with Oct cofactors produced an incomplete loss of retention, suggesting overlapping signals, but not supporting Oct as a primary factor in GR retention. The overlap between important residues in GR nuclear retention and localization signals and the lack of retention shown by the SV40 NLS suggested that retention might be intrinsic to the sequence of particular NLS. Preliminary results suggest that the KT511-512 residues of GR may be of general importance in protein nuclear retention, while the role of proline is likely more variable. My research has focused on increasing our understanding of glucocorticoid receptor nuclear retention and its possible implications. I have determined that the KT511-512 residues of GR play an important role in its retention, and possibly also figure in nuclear retention of other proteins. These residues are involved in interactions which affect promoter-specific histone acetylation and transcriptional activation in GR, suggesting a reason for the existence of nuclear retention.

Corticosteroid ligands activate the glucocorticoid receptor (GR). GR plays a role in glucose homeostasis, adipogenesis, inflammation, and mood and cognitive functions. Understanding the interplay of diverse forms of receptor regulation (including post-translational modification, cofactor interactions, ligand binding, and receptor localization) and their effects is important for understanding and developing more effective treatment for a variety of conditions. Prior to ligand binding, the naïve GR is primarily cytoplasmic, residing in a chaperone complex containing heat-shock proteins and immunophilins. Upon ligand-binding, alterations to the complex allow the receptor to dimerize and import into the nucleus. Nuclear GR interacts with transcriptional regulatory sequences and recruits cofactors to regulate specific gene expression. Upon hormone withdrawal, the original chaperone complex is reassembled and the receptor is exported to the cytoplasm. Interestingly, while the import of GR into the nucleus occurs very rapidly (t ½ = 5 min), the re-export is significantly slower (t ½ = 12-24h). Previous work by our lab and others has indicated the existence of a nuclear retention signal (NRS) within the GR. The NRS sequence of the GR, its interaction partners, and the role it might play in the activity of the receptor have not yet been fully defined. Work in the Hache lab indicates that mutation of the GR nuclear localization signal 1 (NL1) increases the export rate of nuclear GR to the cytoplasm, as well as compromising receptor import, suggesting that the NL1 overlapped an NRS sequence. In this work, I made a series of GR mutants, based on sequence from the SV40 large T antigen NLS, which lacks nuclear retention activity. Using these mutants, I found that GR nuclear retention is influenced by both specific residues within the hinge region and the location of the sequence within the receptor, as reintroduction of the NLS sequence at the N-terminus of the receptor retention mutant failed to reconstitute the retention activity. Agonist liganded and hormone-withdrawn receptor mutants showed a similar decrease in retention. By contrast, antagonist-withdrawn GR mutants were retained in the nucleus, possibly due to altered receptor configuration and interactions. Assays of GR-responsive promoter activation by receptor retention mutants showed that while no difference in the ability of retention mutants to activate transcription was seen at a simple promoter, activation of a complex promoter was compromised. This impaired transactivation for the SV506-523 mutant correlated with decreased histone H4 acetylation and PolII recruitment, while GR DNA-binding at the target promoter appeared to be unaffected. These results suggested that promoter-specific cofactor interactions might be implicated in GR nuclear retention. Loss of GR hinge interaction with Oct cofactors produced an incomplete loss of retention, suggesting overlapping signals, but not supporting Oct as a primary factor in GR retention. The overlap between important residues in GR nuclear retention and localization signals and the lack of retention shown by the SV40 NLS suggested that retention might be intrinsic to the sequence of particular NLS. Preliminary results suggest that the KT511-512 residues of GR may be of general importance in protein nuclear retention, while the role of proline is likely more variable. My research has focused on increasing our understanding of glucocorticoid receptor nuclear retention and its possible implications. I have determined that the KT511-512 residues of GR play an important role in its retention, and possibly also figure in nuclear retention of other proteins. These residues are involved in interactions which affect promoter-specific histone acetylation and transcriptional activation in GR, suggesting a reason for the existence of nuclear retention.