Dissertations / Theses on the topic 'Signal regulatory proteins'

Non-claret disjunctional (Ncd) is a kinesin-14 microtubule motor protein involved in the assembly and stability of meiotic and mitotic spindles in Drosophila oocytes and early embryos, respectively. Ncd functions by cross-linking microtubules through the tail and motor domains. It was originally believed that the role of the Ncd tail domain was to only statically bind microtubules. However, the Ncd tail domain has recently been shown to have properties that stabilize and bundle microtubules, and contribute to the overall motility of the Ncd protein. Continued characterization of the Ncd tail domain is essential to understanding the complete role of Ncd in cell division. This work explored the regulatory function and microtubule binding properties of the Ncd tail domain. Ncd activity is regulated during interphase by nuclear sequestration. GFP-Ncd fusion proteins, containing full length Ncd, individual Ncd domains, or combinations of Ncd domains, were used to identify the presence of a nuclear localization signal (NLS) in the Ncd polypeptide. The nuclear localization of only the GFP fusion proteins containing the Ncd tail sequence indicates that the NLS is contained within the tail domain. Subsequent, experiments performed with GFP fusion proteins containing segments of the tail domain indicate that essential NLS amino acid segments may span the length of the tail domain. Attempts to characterize the microtubule binding properties of the Ncd tail domain, using bacterially expressed MBP-Ncd tail-stalk, were unsuccessful. MBP-Ncd tail-stalk proteins aggregated under binding assay conditions, preventing an accurate determination of the stoichiometric binding relationship between Ncd and the tubulin dimer.<br>Master of Science

Beclin 1 is a haplo-insufficient tumor suppressor that is decreased in many human tumors. The function of Beclin 1 in cancer has been attributed primarily to its role in the degradative process of autophagy. However, the role of autophagy itself in tumorigenesis is context-dependent and can be both preventive and promoting. Due to its dual function in cancer a better understanding of this process is necessary to develop potential novel cancer therapies. To gain insight into the role of autophagy in breast carcinoma, I analyzed the autophagydependency of different subtypes of breast cancer. My results implicate that triple-negative breast carcinoma cells are more dependent on autophagy than luminal breast carcinoma cells. Chemical inhibition of autophagy decreased the tumorigenicity of triple-negative breast carcinoma cells with regard to proliferation and anchorage-independent growth. However, RNAi-mediated suppression of two autophagy genes, ATG5 and Beclin 1, revealed different outcomes. While suppression of ATG5 decreased glycolysis, Beclin 1 depletion did not affect the glycolytic rates. These results suggest autophagy-independent pro-tumorigenic effects of loss of Beclin 1 in cancer. Beclin 1 is a core component of the Vps34/Class III PI3K (PI3KC3) and Vps15/p150 complex that regulates multiple membrane trafficking events. I describe a novel mechanism of action for Beclin 1 in breast cancer involving its control of growth factor receptor signaling. I identify a specific stage of early endosome maturation that is regulated by Beclin 1, the transition of APPL1- containing phosphatidyIinositol 3-phosphate-negative (PI3P-) endosomes to PI3P+ endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P-/APPL+ signaling competent compartment. As a result, suppression of BECN1 sustains growth factor stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, Beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Taken together my data identify a novel role for Beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of Beclin 1 expression would enhance breast cancer progression independent of its impact on autophagy.

The 21st century brought about a dramatic increase in knowledge about genetic and molecular profiles of cancer. This information has validated the complexity of tumor cells and increased awareness of “nodal proteins”, but has yet to advance the development of rational targeted cancer therapeutics. Nodal proteins are critical cellular proteins that collect biological inputs and distribute the information across diverse biological processes. Survivin acts as a nodal protein by interfacing the multiple signals involved in mitosis and apoptosis and functionally integrate proliferation, cell death, and cellular homeostasis. By characterizing survivin as a target of both Type 1 Insulin-like Growth Factor (IGF-1) and Notch developmental signaling, we contribute to the paradigm of survivin as a nodal protein. The two signaling systems, Notch and IGF-1, regulate survivin by two independent mechanisms. Notch activation induces survivin transcription preferentially in basal breast cancer, a breast cancer subtype with poor prognosis and lack of molecular therapies. Activated Notch binds the transcription factor RBP-Jк and drives transcription from the survivin promoter. Notch mediated survivin expression increases cell cycle kinetics promoting tumor proliferation. Inhibition of Notch in a breast xenograft model reduced tumor growth and systemic metastasis. On the other hand, IGF-1 signaling drives survivin protein translation in prostate cancer cells. Binding of IGF-1 to its receptor activates downstream kinases, mammalian target of rapamycin (mTOR) and p70 S6 protein kinase (p70S6K), which modulates survivin mRNA translation to increase the apoptotic threshold. The multiple roles of survivin in tumorigenesis implicate survivin as a rational target for the “next generation” of cancer therapeutics.

Adaptive immunity requires T cell responses to foreign pathogens to be counterbalanced with the need to limit collateral destruction of the host’s own tissues. Further, the presence of a substantial pool of lymphocytes capable of recognizing selfantigen in the periphery poses a threat to the maintenance of peripheral tolerance and prevention of autoimmunity. Regulatory T cells (Treg) that can suppress potentially self-reactive T cells are critical regulators of peripheral tolerance as well as initiation of immune responses. Treg cells employ several context-dependent mechanisms to establish regulation. In this thesis, we describe two distinct pathways of regulation used by Treg cells involving negative costimulation by CTLA-4 and immunomodulation by the morphogen, TGFβ. CTLA-4 is a co-inhibitory receptor on T cells essential for maintaining T cell homeostasis and tolerance to self. CTLA-4 expression is induced in conventional T cells following activation, whereas it is constitutively expressed in regulatory FOXP3+CD4+ regulatory T cells. Mice lacking CTLA-4 develop an early onset, fatal breakdown in T cell tolerance. Whether this autoimmune disease occurs because of the loss of CTLA-4 function in regulatory T cells, conventional T cells, or both, is not known. We present evidence here that in addition to a critical CTLA-4 function in regulatory T cells, CTLA-4 in conventional T cells is also necessary for controlling the consequences of abnormal T cell activation. CTLA-4 expression in activated conventional T cells only in vivois unable to compensate for the impaired function of CTLA-4-less regulatory T cells that results in systemic lymphoproliferation, but it can prevent the aberrantly activated T cells from infiltrating and fatally damaging non-lymphoid tissues. These results demonstrate that CTLA-4 has a dual function in maintaining T cell homeostasis: CTLA-4 in regulatory T cells inhibits inappropriate naïve T cell activation and CTLA-4 in conventional T cells can prevent the harmful accumulation of inappropriately activated pathogenic T cells in vital organs. In addition, we have identified Disabled-2 (Dab2), a TGFβ signaling intermediate, as a FOXP3 target gene that is expressed exclusively in Treg cells and is critical for in vitro and in vivo regulation by Treg cells. During T cell development, DAB2 is also expressed in a Foxp3-independent manner in thymic precursor cells, and acts as a sensor of TGFβ signals that is required for programming normal TGFβ responsiveness in T cell progenies. Naïve CD4+ T cells that differentiate from Dab2-deficient precursors favor Th17 cell generation at the expense of FOXP3+ Treg cells as a result of altered sensitivity to TGFβ. Importantly, retinoic acid can restore TGFβ signaling capacity of naïve CD4+ T cells generated from Dab2-deficient precursors, emphasizing the cooperative nature of retinoic acid and TGFβ signaling pathways in promoting Treg cell development and maintenance.

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a plethora of fundamental biological processes ranging from muscle contraction to the formation of memories. The studies described in this work focus on the transcriptional regulation of the CHRNB4 gene, which encodes the ß4 subunit of neuronal nAChRs. We previously identified a regulatory sequence (5´– CCACCCCT –3´), or “CA box”, critical for CHRNB4 promoter activity in vitro. Here I report transcription factor interaction at the CA box along with an in vivo analysis of CA box transcriptional activity. My data indicate that Sp1, Sp3, Sox10 and c-Jun interact with the CHRNB4 CA box in the context of native chromatin. Using an in vivo transgenic approach in mice, I demonstrated that a 2.3-kb fragment of the CHRNB4 promoter region, containing the CA box, is capable of directing cell-type specific expression of a reporter gene to many of the brain regions that endogenously express the CHRNB4 gene. Site-directed mutagenesis was used to test the hypothesis that the CA box is critical for CHRNB4 promoter activity in vivo. Transgenic animals were generated in which LacZ expression is driven by a mutant form of the CA box. Reporter gene expression was not detected in any tissue or cell type at ED18.5. Similarly, I observed dramatically reduced reporter gene expression at PD30 when compared to wild type transgenic animals, indicating that the CA box is an important regulatory feature of the CHRNB4 promoter. ChIP analysis of brain tissue from mutant transgenic animals demonstrated that CA box mutation results in decreased interaction of the transcription factor Sp1 with the CHRNB4 promoter. I have also investigated transcription factor interaction at the CHRNB4 promoter CT box, (5´– ACCCTCCCCTCCCCTGTAA –3´) and demonstrated that hnRNP K interacts with the CHRNB4 promoter in an olfactory bulb derived cell line. Surprisingly, siRNA experiments demonstrated that hnRNP K knockdown has no impact on CHRNA5, CHRNA3 or CHRNB4 gene expression. Interestingly, knockdown of the transcription factor Purα results in significant decreases in CHRNA5, CHRNA3 and CHRNB4 mRNA levels. These data indicate that Purα can act to enhance expression of the clustered CHRNA5, CHRNA3 and CHRNB4 genes. Together, these results contribute to a more thorough understanding of the transcriptional regulatory mechanisms underlying expression of the CHRNB4 as well as the CHRNA5 and CHRNA3 genes, critical components of cholinergic signal transduction pathways in the nervous system.

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a plethora of fundamental biological processes ranging from muscle contraction to the formation of memories. The studies described in this work focus on the transcriptional regulation of the CHRNB4 gene, which encodes the ß4 subunit of neuronal nAChRs. We previously identified a regulatory sequence (5´– CCACCCCT –3´), or “CA box”, critical for CHRNB4 promoter activity in vitro. Here I report transcription factor interaction at the CA box along with an in vivo analysis of CA box transcriptional activity. My data indicate that Sp1, Sp3, Sox10 and c-Jun interact with the CHRNB4 CA box in the context of native chromatin. Using an in vivo transgenic approach in mice, I demonstrated that a 2.3-kb fragment of the CHRNB4 promoter region, containing the CA box, is capable of directing cell-type specific expression of a reporter gene to many of the brain regions that endogenously express the CHRNB4 gene. Site-directed mutagenesis was used to test the hypothesis that the CA box is critical for CHRNB4 promoter activity in vivo. Transgenic animals were generated in which LacZ expression is driven by a mutant form of the CA box. Reporter gene expression was not detected in any tissue or cell type at ED18.5. Similarly, I observed dramatically reduced reporter gene expression at PD30 when compared to wild type transgenic animals, indicating that the CA box is an important regulatory feature of the CHRNB4 promoter. ChIP analysis of brain tissue from mutant transgenic animals demonstrated that CA box mutation results in decreased interaction of the transcription factor Sp1 with the CHRNB4 promoter. I have also investigated transcription factor interaction at the CHRNB4 promoter CT box, (5´– ACCCTCCCCTCCCCTGTAA –3´) and demonstrated that hnRNP K interacts with the CHRNB4 promoter in an olfactory bulb derived cell line. Surprisingly, siRNA experiments demonstrated that hnRNP K knockdown has no impact on CHRNA5, CHRNA3 or CHRNB4 gene expression. Interestingly, knockdown of the transcription factor Purα results in significant decreases in CHRNA5, CHRNA3 and CHRNB4 mRNA levels. These data indicate that Purα can act to enhance expression of the clustered CHRNA5, CHRNA3 and CHRNB4 genes. Together, these results contribute to a more thorough understanding of the transcriptional regulatory mechanisms underlying expression of the CHRNB4 as well as the CHRNA5 and CHRNA3 genes, critical components of cholinergic signal transduction pathways in the nervous system.

[EN] ABSTRACT Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling.<br>[ES] RESUMEN La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos explorado el papel de los receptores PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) de Arabidopsis (Arabidopsis thaliana) en la ruta de señalización de ABA en raíz. Así, hemos descubierto que el receptor de ABA PYL8 juega un papel no redundante en la regulación de la percepción de ABA en raíz. Inesperadamente, dada la naturaleza multigénica y la redundancia funcional parcial observada en la familia PYR/PYL/RCAR, el mutante pyl8 fue el único mutante sencillo de pérdida de función de los receptores PYR/PYL/RCAR que mostraba una sensibilidad reducida a la inhibición del crecimiento mediada por ABA en raíz. Este efecto se debe a la falta de inhibición mediada por PYL8 de varias fosfatasas del grupo A tipo 2C (PP2Cs), ya que PYL8 es capaz de interactuar in vivo con al menos cinco PP2Cs, denominadas HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 según lo han revelado la purificación por afinidad en tándem (TAP por sus siglas en inglés) y estudios proteómicos de espectrometría de masas. La transducción de la señal del ABA localizada en la membrana plasmática celular juega un papel crucial en los pasos iniciales de la señalización de la fitohormona, pero los mecanismos moleculares que unen los componentes básicos de la señalización y la membrana plasmática no están claros. Estudiando las interacciones de los receptores del ABA PYR/PYL/RCAR con la membrana plasmática hemos encontrado que éstos pueden interaccionar transitoriamente con ella de forma dependiente de calcio gracias a una familia de proteínas con dominios C2 relacionadas con la ruta de señalización de ABA (denominadas C2-domain ABA-related (CAR) proteins). Específicamente, se encontró que PYL4 interacciona de manera independiente de ABA con CAR1 tanto en la membrana plasmática como en el núcleo de las células vegetales. La proteína CAR1 pertenece a una familia multigénica constituida por 10 miembros en Arabidopsis thaliana, desde CAR1 hasta CAR10, y que solo se encuentra en plantas. Los ensayos de complementación bi-molecular de fluorescencia y de co-immunoprecipitación confirmaron la interacción en células vegetales tanto de PYL4-CAR1 como de otras parejas de PYR/PYL-CAR. La cristalización de la proteína CAR4 reveló que, además de un dominio C2 clásico de unión a lípidos dependiente de calcio, las proteínas de la familia CAR presentan un dominio específico que probablemente es responsable de la interacción con los receptores PYR/PYL/RCAR y de su posterior reclutamiento a las vesículas de fosfolípidos. Esta interacción es relevante para la función de los receptores PYR/PYL/RCAR en la señalización del ABA, ya que diferentes mutantes triples car de pérdida de función, que tienen afectados los genes CAR1, CAR4, CAR5, y CAR9, demostraron una reducción de la sensibilidad al ABA en ensayos de establecimiento de plántula y crecimiento de la raíz. En resumen, hemos identificado nueva familia de proteínas que son capaces mediar las interacciones transitorias dependientes de Ca2+ con vesículas de fosfolípidos, lo que a su vez afecta localización de PYR/PYL/RCAR y regula positivamente la señalización de ABA.<br>[CAT] RESUM La senyalització per l'hormona vegetal àcid abcíssic (ABA) exerceix un paper crític en la regulació del creixement de l'arrel i també en l'arquitectura del sistema radical. La promoció del creixement de l'arrel en condicions d'estrés hídric, regulada per ABA és clau per la supervivència de les plantes sota condicions limitants d'aigua. Amb aquest treball, hem investigat el paper dels receptors PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) d'Arabidopsis (Arabidopsis thaliana) en el camí de senyalització d'ABA en arrel. Així, hem descobert que el receptor d'ABA PYL8 exerceix un paper no redundant en la regulació de la percepció d'ABA en arrel. Inesperadament, donada la naturalesa multigènica i la redundància funcional parcial que s'observa en la família PYR/PYL/RCAR, el mutant pyl8 va ser l'únic mutant senzill de pèrdua de funció dels receptors PYR/PYL/RCAR que mostrava una sensibilitat reduïda a la inhibició del creixement mitjançada per l'ABA en l'arrel. Doncs aquest efecte es deu a la falta d'inhibició regulada per PYL8 de diverses fosfatases del grup A tipus 2C (PP2Cs), ja que PYL8 té la capacitat d'interactuar in vivo almenys amb cinc PP2Cs, anomenades HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABAHYPERSENSITIVE GERMINATION3 segons ho han revelat per una banda la purificació per afinitat en tàndem (TAP són les seues sigles en anglés) i per altra banda, estudis proteòmics d'espectrometria de masses. Pel que fa a la transducció del senyal del l'ABA, la qual es localitza en la membrana plasmàtica cel¿lular, juga un paper molt important en els primers instants de la senyalització de la fitohormona, no obstant això els mecanismes moleculars que uneixen els components bàsics d'aquesta senyalització amb la membrana plasmàtica, no es troben del tot clars. Per tant, s'han estudiat les interaccions que tenen els receptors del ABA PYR/PYL/RCAR amb la membrana plasmàtica, i hem trobat que aquests tenen la capacitat d'interaccionar transitòriament amb la membrana de forma dependent al calci, gràcies a una família de proteïnes amb domini C2, les quals es troben relacionades amb la ruta de senyalització d'ABA(anomenades C2domain ABArelated (CAR) proteins).Específicament, es va trobar que PYL4 interacciona d'una manera independent al ABA amb CAR1, tant en la membrana plasmàtica, com en el nucli de les cèl¿lules vegetals. La proteïna CAR1 pertany a la família multigènica constituïda per 10 components en Arabidopsis thaliana, des de CAR1 fins CAR10, que tan sols es troba en plantes. Els assajos de complementació bimolecular de fluorescència i de co-immunoprecipitació, van confirmar la interacció en cèl¿lules vegetals, tant de PYL4CAR1 com d'altres parelles de PYR/PYL-CAR. La cristal¿lització de la proteïna CAR4 va revelar que, a més d'un domini C2 clàssic de unió a lípids dependent del calci, les proteïnes de la família CAR presenten un domini PYR/PYL/RCAR, i del seu posterior reclutament a les vesícules fosfolipídiques. Doncs, aquesta interacció és rellevant en la funció dels receptors PYR/PYL/RCAR, ja que participa en la senyalització del l'ABA. Aquesta interacció es clau per a la funció dels receptors, ja que diferents mutants triples car de pèrdua de funció, els quals posseïxen afectats els gens CAR1, CAR4, CAR5 i CAR9, van mostrar una reducció de la sensibilitat a l'ABA en assajos d'establiment de plàntula i creixement de l'arrel. En conclusió, hem identificat una nova família de proteïnes amb la capacitat d'organitzar les interaccions transitòries dependents del calci amb vesícules de fosfolípids, fet que al seu torn afecta la localització de PYR/PYL/RCAR i regula positivament la senyalització d'ABA.<br>Rodríguez Solovey, LN. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58862<br>TESIS

Thesis (M.A.)--Boston University<br>The human immune system is capable of detecting and removing foreign invaders such as viruses, microorganisms, and other harmful materials. A key component of this immune response is leukocyte recruitment—a process, in which leukocytes travel from the bloodstream to the site of injury or infection. SIRPα, a protein mainly known to be expressed in myeloid leukocytes, has been shown to contribute to this process by regulating transendothelial migration (TEM)—leukocyte passage through the vascular endothelium. Interestingly, a recent study has detected low levels of SIRPα on surface of cultured endothelial cells. The aim of this study was to confirm endothelial expression of SIRPα and to investigate its role in leukocyte TEM. SIRPα expression on the endothelial cell was confirmed by immunofluorescence microscopy, indirect immunofluorescence and flow cytometry, and by western blot analysis. shRNA silencing and function blocking antibodies were used to block the adhesive function of SIRPα in an in vitro TEM assay under physiological shear flow conditions. The interventions did not alter leukocyte TEM and we conclude that SIRPα does not play a significant role in leukocyte TEM in vitro.

Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.

Orientador: Kleber Gomes Franchini<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas<br>Made available in DSpace on 2018-08-11T19:13:47Z (GMT). No. of bitstreams: 1 Cardoso_AlissonCampos_M.pdf: 1504948 bytes, checksum: 1996e50c40d74c7a53a5058831fb03ab (MD5) Previous issue date: 2008<br>Resumo: Estudos anteriores demonstraram que em miócitos cardíacos submetidos a estímulos mecânicos ocorre pronta fosforilação e ativação da FAK. Resultados de estudos recentes, realizados em corações de ratos indicaram que em resposta a estímulos mecânicos, a FAK, além de ser ativada, localiza-se no núcleo dos miócitos cardíaco. Estudos conduzidos em corações de ratos Wistar adultos, utilizando a técnica de Imunoprecipitação de cromatina (ChIP) com anticorpo anti- FAK, identificaram uma seqüência intrônica de 182pb do gene fosfolambam (plnil), contendo sítio para a ligação do fator de transcrição MEF2. Portanto, no presente estudo, investigamos se a região intrônica do gene que codifica o fosfolamban tem função regulatória transcricional. Utilizando técnicas de EMSA (Ensaio de retardamento da mobilidade eletroforética), ensaios de Precipitação e de gene reporter, avaliamos a interação entre a FAK e plnil além da função regulatória transcricional dessa seqüência. Como resultado, demonstramos que ocorre pronta ativação da FAK e seu acúmulo no núcleo de miócitos cardíacos de ventrículo esquerdo de ratos submetidos a coarctação da aorta. Através de ensaios de EMSA, demonstramos que proteínas nucleares de ventrículo esquerdo de ratos submetidos a sobrecarga pressórica, apresentaram um aumento na interação com a sonda plni1 em relação aqueles de ratos controles. Também, ensaios de EMSA indicaram uma interação entre MEF2 e a sonda plnil, mas não entre a FAK ou seus domínios (FERM e Cterminal) com a sonda plnil. Ensaios de precipitação com fragmentos recombinantes da FAK (GST-FERM e GSTCterminal) com extratos nucleares de coração de ratos coarctados indicaram uma associação entre FAK e MEF2. Ensaios adicionais demonstraram que a interação entre FAK e MEF2 é dependente do domínio Cterminal e do estado fosforilado da FAK. Estudos de transfecção com gene reporter, utilizando plasmídeo contendo a seqüência plnil, em cultura de miócitos cardíacos submetidos ao estiramento, demonstraram que a região intrônica plnil possui função regulatória transcricional e esse papel é dependente da ligação do fator de transcrição MEF2 ao seu sítio específico no DNA. Portanto, esses dados indicam que FAK e MEF2 podem estar envolvidos na regulação da expressão do gene pln através de regulação mediado pela região intrônica plnil.<br>Abstract: Previous studies have shown that mechanical stress induces phosphorylation and activation of FAK in cardiac myocytes. Recent studies carried out in rat overloaded hearts indicated that FAK re-locates in the nucleus of the cardiac myocytes. By assays in the nuclei of overloaded cardiac myocytes with chromatin immunoprecipitation (ChIP) approach with anti-FAK antibody, we identified an intronic sequence of phospholamban gene (plnil), containing a MEF2 consensus site. In the present study, we investigated whether Plnil has any regulatory function in the pln. To accomplish this, we combinated techniques such as EMSA (Electrophoreses Mobility Shift Assay), reporter gene and pulldown assays. FAK was shown to be rapidly activated and to accumulate in the nuclei of cardiac myocytes taken from overloaded left ventricle. Using EMSA assays, we demonstrated that nuclear extracts of left ventricle rats overloaded, interacted with the plnil probe. EMSA assays, also indicated an interaction between MEF2 and the plnil probe, but no interaction was found between FAK or its domains (FERM and Cterminal) with the plni1. Pulldown assays with FAK recombinant fragments (GST-FERM and GST-Cterminal) and nuclear extracts from left ventricle overloaded indicated that FAK and MEF2 physically interact through FAK Cterminal domain. Reporter gene assays, using a construction of plnil coupled luciferase transfected to cardiac myocytes culture underwent stretching, had demonstrated that the intronic region has transcriptional regulatory function and this role is dependent of the transcription factor MEF2 binding site in the DNA. Therefore, these data indicate that FAK and MEF2 interact in the nuclei of cardiac myocytes and that FAK/MEF2 complex may regulate phospholamban gene expression through the plnil.<br>Mestrado<br>Medicina Experimental<br>Mestre em Fisiopatologia Médica

La protéine GPR50, qui fait partie de la famille des récepteurs de la mélatonine, est classée, avec une centaine d’autres protéines à sept domaines transmembranaires (7TM), dans la catégorie des récepteurs couplés aux protéines G hétérotrimériques (RCPG) orphelins, c’est-à-dire pour lesquels aucun ligand n’a pu être identifié. De plus en plus d’études montrent que les 7TM peuvent avoir des fonctions indépendantes d’un ligand. C’est le cas de GPR50 qui inhibe les fonctions du récepteur de la mélatonine MT1 en interagissant directement avec lui. Nous avons cherché à identifier d’autres partenaires associés à GPR50 en appliquant la technique de purification par affinité en tandem et avons mis en évidence son interaction avec un récepteur du facteur de croissance Transforming Growth Factor ß (TGFβ), le récepteur de type I (TβRI).Nous décrivons ici la formation d’un complexe entre GPR50 et le récepteur TβRI au niveau de la membrane plasmique, avec pour conséquence l’induction d’une activité constitutive du récepteur et des voies de signalisation en aval en l’absence de TGFβ, mais également en l’absence du récepteur TßRII qui est habituellement indispensable pour l’activation de TβRI par phosphorylation. Cette activité constitutive se traduit par la phosphorylation des protéines Smad2 et Smad3, leur intégration dans un complexe avec Smad4, la translocation du complexe dans le noyau et finalement l’activation de la transcription de leurs gènes-cibles. Nous avons décrypté les mécanismes moléculaires de cette activation constitutive en montrant que GPR50 entre en compétition, pour l’interaction avec TβRI, avec le régulateur négatif FKBP12, une protéine inhibitrice de l’activité basale du récepteur en l’absence de ligand. Nous avons identifié dans la queue intracytoplasmique de GPR50 un motif répétitif similaire à la séquence de FKBP12 impliquée dans son interaction avec TβRI , motif qui constitue la base moléculaire de cette compétition.Nous avons étudié les conséquences fonctionnelles de cette activation en surexprimant GPR50 de manière stable dans la lignée cellulaire MDA-MB-231, dérivée d’un cancer de sein. Nous avons observé dans ces cellules des effets pro-migratoires et anti-prolifératifs similaires à ceux causés par l’administration de TGFβ.En conclusion, ce travail décrit un nouveau mode d’activation du récepteur TβRI en l’absence de ligand, mais identifie également une nouvelle fonction indépendante d’un ligand pour le RCPG orphelin GPR50. En perspective de ce travail, nous allons essayer d’identifier des conditions biologiques où cette interaction pourrait prendre place afin de confirmer ces résultats dans un contexte plus physiologique<br>During the last years, it became more and more accepted, that orphan G Protein coupled receptors (GPCRs) with a transmembrane spanning heptahelical core (7TM) can have ligand-independent functions. One of those 100 orphan GPCRs is GPR50, a 7TM protein with a long cytosolic domain. Recently, studies revealed ligand-independent functions for GPR50, where it has the capacity to modulate the activity of other proteins upon complex formation. By applying a tandem affinity purification approach we sought to identify further putative interacting partners of GPR50. One of the identified binding partners is the transforming growth factor β (TGFβ) receptor type I (TβRI).The TGFβ-dependent signal transduction pathway of serine/threonine kinases is a pathway with direct signal flow from ligand over the receptor to its substrates, the Smads which translocate into nucleus where they bind DNA and regulate gene expression. An important question concerns the generation of specificity and fine-tuning of TGFβ-dependent signaling. Throughout the years, an important number of proteins which regulate the activity of the TGFβ signal transduction pathway in a positive or negative manner have been identified. Most of them act in a cell-context-dependent manner, allowing the regulation of TGFβ signaling adapted to the particular circumstances.We report here the complex formation of GPR50 and TβRI on the plasma membrane. The consequence of this interaction is the GPR50-mediated induction of a constitutive activation of the TβRI and its downstream signaling in a TGFβ ligand-independent manner. This has been monitored by Smad2/3 phosphorylation, Smad2/3-Smad4 complex formation and their subsequent translocation into the nucleus, where they activate Smad-dependent gene expression. In order to decipher the molecular mechanism that allows this activation, we showed that GPR50 competes with the negative regulator, that prevents leaky TGFβ signaling, the gatekeeping molecule FKBP12, for binding to the TβRI. We identified a motif in FKBP12 involved in the interaction with TβRI with similarities to a motif in GPR50, providing a molecular basis for the replacement of FKBP12 by GPR50 in the TβRI complex. We showed that GPR50 is capable of activating the TβRI even in the absence of the TβRII, which normally is required for activating the TβRI by phosphorylation. This reveals a previously unknown mode of activation of the TβRI in absence of the TGFβ ligand and TβRII. In order to identify the functional consequences of this crosstalk, we studied migration and growth of MDA-MB-231 breast cancer cells stably overexpressing GPR50. In these cells, TGFβ-like pro-migratory and anti-proliferative effects have been observed.Future research will help to identify tissues and biological circumstances, where this crosstalk could take place for putting this novel mode of regulation of TGFβ signaling pathway into a context-dependent-manner. Additionally our work established another ligand-independent task for the orphan 7TM protein GPR50, consolidating its function as binding partner and activity modulator

Thesis (M.S.)--West Virginia University, 2000.<br>Title from document title page. Document formatted into pages; contains ix, 70 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 63-68).

The cells and molecules that comprise the immune system are essential for mounting an effective response against microbes. A successful immune response limits pathology within the host while simultaneously eliminating the pathogen. The key to this delicate balance is the correct recognition of the pathogen and the appropriate response of immune cells. Cellular activation originates through receptors that relay information about the state of the microenvironment to different compartments within the cell. The rapid relay of information is called signal transduction and employs a network of signaling mediators such as kinases, phosphatases, adaptor molecules, and transcription factors. IL-2 inducible T cell kinase (Itk) is a non-receptor tyrosine kinase that is an integral component of signal transduction downstream of many immunoreceptors. This dissertation describes two distinct pathways that utilize Itk in both phases of the immune response. T cells use the TCR to sense a multitude of peptide-based ligands and to transmit signals inside the cell to activate cellular function. In this regard, the diversity of ligands the T cells encounter can be portrayed as analog inputs. Once a critical threshold is met, signaling events transpire in close proximity to the plasma membrane to activate major downstream pathways in the cell. The majority of these pathways are digital in nature resulting in the on or off activation of T cells. We find, however, that altering the TCR signal strength that a T cell receives can result in an analog-based response. Here, the graded expression of a transcription factor, IRF4, is modulated through the activity of Itk. We link this graded response to an NFAT-mediated pathway in which the digital vs. analog nature has been previously uncharacterized. Finally, we demonstrate that the repercussions of an analog signaling pathway is the altered expression of a second transcription factor, Eomes, which is important in the differentiation and function of T cells. These results suggest that Itk is crucial in the modulation of TCR signal strength. Mast cells primarily rely on the IgE-bound FcεR1 for pathogen recognition. Crosslinking this receptor activates mast cells and results in degranulation and cytokine production via an expansive signaling cascade. Upon stimulation, Itk is recruited to the plasma membrane and phosphorylated. Little else is known about how Itk operates inside of mast cells. We find that mast cells lacking Itk are hyperresponsive to FcεR1-mediated activation. This is most apparent in the amount of IL-4 and IL-13 produced in comparison to wild-type mast cells. Increased cytokine production was accompanied by elevated and sustained signaling downstream of the FcεR1. Finally, biochemical evidence demonstrates that Itk is part of an inhibitory complex containing the phosphatase SHIP-1. These results indicate a novel function for Itk as a negative regulator in FcεR1- mediated mast cell activation.

The cells and molecules that comprise the immune system are essential for mounting an effective response against microbes. A successful immune response limits pathology within the host while simultaneously eliminating the pathogen. The key to this delicate balance is the correct recognition of the pathogen and the appropriate response of immune cells. Cellular activation originates through receptors that relay information about the state of the microenvironment to different compartments within the cell. The rapid relay of information is called signal transduction and employs a network of signaling mediators such as kinases, phosphatases, adaptor molecules, and transcription factors. IL-2 inducible T cell kinase (Itk) is a non-receptor tyrosine kinase that is an integral component of signal transduction downstream of many immunoreceptors. This dissertation describes two distinct pathways that utilize Itk in both phases of the immune response. T cells use the TCR to sense a multitude of peptide-based ligands and to transmit signals inside the cell to activate cellular function. In this regard, the diversity of ligands the T cells encounter can be portrayed as analog inputs. Once a critical threshold is met, signaling events transpire in close proximity to the plasma membrane to activate major downstream pathways in the cell. The majority of these pathways are digital in nature resulting in the on or off activation of T cells. We find, however, that altering the TCR signal strength that a T cell receives can result in an analog-based response. Here, the graded expression of a transcription factor, IRF4, is modulated through the activity of Itk. We link this graded response to an NFAT-mediated pathway in which the digital vs. analog nature has been previously uncharacterized. Finally, we demonstrate that the repercussions of an analog signaling pathway is the altered expression of a second transcription factor, Eomes, which is important in the differentiation and function of T cells. These results suggest that Itk is crucial in the modulation of TCR signal strength. Mast cells primarily rely on the IgE-bound FcεR1 for pathogen recognition. Crosslinking this receptor activates mast cells and results in degranulation and cytokine production via an expansive signaling cascade. Upon stimulation, Itk is recruited to the plasma membrane and phosphorylated. Little else is known about how Itk operates inside of mast cells. We find that mast cells lacking Itk are hyperresponsive to FcεR1-mediated activation. This is most apparent in the amount of IL-4 and IL-13 produced in comparison to wild-type mast cells. Increased cytokine production was accompanied by elevated and sustained signaling downstream of the FcεR1. Finally, biochemical evidence demonstrates that Itk is part of an inhibitory complex containing the phosphatase SHIP-1. These results indicate a novel function for Itk as a negative regulator in FcεR1- mediated mast cell activation.

Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(農学)<br>甲第17236号<br>農博第1962号<br>新制||農||1006(附属図書館)<br>学位論文||H24||N4719(農学部図書室)<br>29982<br>京都大学大学院農学研究科応用生命科学専攻<br>(主査)教授 植田 和光, 教授 阪井 康能, 教授 植田 充美<br>学位規則第4条第1項該当

Human complex diseases, like Diabetes and Cancer, affect many people worldwide today. Despite existing knowledge, many of these diseases are still not preventable. Complex diseases are known to be caused by a combination of genetic factors, as well as environmental and life style factors. The scope of this investigation covered the genomics of Type 1 Diabetes (T1D). There are 49 human genomic regions that are known to carry markers (disease-associated single nucleotide mutations) for T1D, and these were extensively studied in this research. The aim was to find out in how far this disease may be caused by problems in gene regulation rather than in gene coding. For this, the genetic factors associated with T1D, including the single point mutations and susceptibility regions, were characterised on the basis of their genomic attributes. Furthermore, mutations that occur in binding sites for transcription factors were analysed for change in the conspicuousness of their binding region, caused by allele substitution. This is called SNP (Single nucleotide polymorphism) sensitivity. From this study, it was found that the markers for T1D are mostly non-coding SNPs that occur in introns and non-coding gene transcripts, these are structures known to be involved in gene regulatory activity. It was also discovered that the T1D susceptibility regions contain an abundance of intronic, non-coding transcript and regulatory nucleotides, and that they can be split into three distinct groups on the basis of their structural and functional genomic contents. Finally, using an algorithm designed for this study, thirty-seven SNPs that change the representation of their surrounding region were identified. These regulatory mutations are non-associated T1D-SNPs that are mostly characterised by Cytosine to Thymine (C-T) transition mutations. They were found to be closer in average distance to the disease-associated SNPs than other SNPs in binding sites, and also to occur frequently in the binding motifs for the USF (Upstream stimulatory factor) protein family which is linked to problems in Type 2 diabetes.

Les relais de phosphorylation de type histidine/aspartate constituent des voies de signalisation impliquées dans la perception et la transduction des signaux jusqu’à la mise en place de réponses spécifiques. Ils mettent en jeu un récepteur ou Histidine aspartate Kinase (HK), des protéines navettes en charge de la transmission du phosphate (HPt) et des Régulateurs de Réponse (RR). L’implication d’un tel système dans la transduction du signal de la contrainte osmotique est avérée chez la levure et fortement suspectée chez Arabidopsis. Ce travail de thèse visait d’une part à caractériser l’implication de cette voie de transduction de la contrainte osmotique chez le peuplier, avec l’identification de partenaires HPt et RR en aval du récepteur HK1 et d’autre part à caractériser le mode de fonctionnement d’un RR de type-B. HK1, un osmosenseur membranaire détecterait le signal et le transmettrait à trois HPt préférentielles. De plus, un partenariat d’interaction se dégagerait entre ces trois HPt et certains RR-B. La régulation transcriptionnelle observée lors d’une contrainte osmotique pour deux des représentants des RR-B témoigne d’une possible implication de ces RR dans cette voie. Ces protéines sont des facteurs de transcription dont la fonction a été confirmée in planta pour l’un d’entre eux. La dimérisation du domaine receveur du RR et son interaction avec le domaine de fixation à l’ADN ou domaine GARP apparaissent comme des points de contrôle clés dans la régulation de l’activité effectrice des RR-B. De plus, la capacité d’un RR-B à se fixer sur ses motifs de reconnaissance (boîtes AGAT) a pu être vérifiée in vitro et la présence de ces séquences a d’ailleurs été retrouvée dans des gènes régulés par la contrainte osmotique. Ce travail prospectif ouvre des perspectives concernant l’implication des RR-B dans la voie de transduction du signal de la contrainte osmotique, et propose notamment des mécanismes fins pour l’élaboration d’une réponse hautement spécifique<br>Multistep His-to-Asp phosphorelay systems are signaling pathways devoted to signal perception and transduction for establishment of specific responses. These systems are composed of three successive partners: Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). One of the best characterized corresponding systems is the osmo-responsive pathway in yeast. Such systems are also suspected in Arabidopsis. This work aimed to characterize the involvement of an osmosensing pathway in Populus by identifying HPt and RR elements downstream of HK1 and to reveal the underlying mechanisms for the activity of a RR-B. HK1, membrane osmosensor, is expected to be responsible for signal detection and propagation by triggering the activation of three preferential HPt. Furthermore, an interacting partnership between those HPts and particular B-type RRs was observed. Two of them appear to be regulated by an osmotic stress, suggesting their possible involvement in this pathway. The B-type RR members, the final output elements of the pathway, act as transcription factors, as shown for at least for one of them in planta. Taken together, the dimerization of the RR receiver domain and its interaction with its DNA binding domain (GARP), are likely key checkpoints in the regulation of RR-B activity. Besides, the ability of one RR-B to bind its cognate specific DNA sequences (AGAT boxes) was confirmed in vitro and those were found in promoters of osmotic response genes. This work opens up prospects for the involvement of RR-B in the osmotic stress signaling pathway and suggests mechanisms tuning induction of specific responses

Dissertação de mestrado em Bioinformática<br>Transcription factors (TFs) are proteins that mediate the cellular response to the changes of the surrounding environment. Studying their functional domains and protein structure is fundamental in order to gain insight of the way they are triggered and how they shape genetic transcription. The current work aimed for classifying both TFs and functional domains, understanding which features can be related to the different functions of the TFs. By using UniProtJAPI, a JAVA library that allows remote access to UniProt, the information of 200 Escherichia coli’s (E. coli) TFs has been retrieved. This data was manually curated, in order to remove domain duplicates and other excess information, and to add missing domains. The obtained functional domains were classified according to their molecular function, while the TFs were classified according to their regulatory function. TFs that exclusively induce gene expression were classified as activators, while TFs that only perform gene repression were classified as repressors. On the other hand, TFs that perform both the activation and repression of transcription were classified as duals. The information was then analysed altogether in order to understand what relationships between the TFs’ function and functional domains could exist. Several analysis were performed, which include statistical tests and clustering methods. Along with the analysis of the full list of TFs, TFs that are part of two-component signal transduction systems and global TFs were given special focus, due to their important role in cellular function. The results showed that there is a relationship between the functional domains and the regulatory function of the different TFs. This may be related to the evolutionary relationships between repressors and activators. It is also understandable that dual regulators are closely related to activators and repressors than what activators and repressors are to each other. Moreover, TFs of two-component signal transduction systems are similar to each other, given that they perform similar functions. Their domain architectures are also predictable and do not vary from what was expected of these TFs. However, in global TFs the results are opposite of the ones obtained for two-component system TFs: their structures are very different from each other and each TF is specific. The amount of different domains is high when comparing to the full sample of TFs, since the number of domains exceeds the number of TFs. Domains of all classification types are present in their structure and the domain architectures are varied, which reflects their different activities within the cell.<br>Os factores de transcrição (TFs) são proteínas que mediam resposta celular perante alterações do meio em que se inserem. Estudar os seus domínios funcionais e estrutura proteica é fundamental para compreender a forma como as suas funções são desencadeadas e como moldam a regulação da transcrição. Este trabalho teve como objectivos a classificação dos TFs de acordo com a sua função, assim como a classificação dos domínios funcionais. Através do uso da UniProtJAPI, uma biblioteca de JAVA que permite o acesso remoto à UniProt, foi recolhida informação de 200 TFs da Escherichia coli (E. coli). Estes dados foram curados manualmente, com o objectivo de remover domínios duplicados e outra informação em excesso, assim como de adicionar domínios em falta. Os domínios funcionais obtidos foram classificados de acordo com a sua função molecular, enquanto que os TFs foram classificados de acordo com a sua função regulatória. TFs que exclusivamente induzem a expressão genética foram classificados como activadores, enquanto que TFs que apenas reprimem a expressão genética foram classificados como repressores. Por usa vez, TFs que tanto induzem como reprimem a expressão genética foram classificados como duais. A informação dos domínios e dos TFs foi considerada como um todo de forma a compreender quais as possíveis relações entre a função regulatória dos TFs e os domínios funcionais. Várias análises foram efectuadas, das quais testes estatísticos e métodos de clustering. Para além da análise de todos os TFs, foi também feita uma análise de TFs que fazem parte de two-component transduction systems e TFs globais, devido à sua importância na actividade celular. Os resultados demonstram que existe uma relação entre os domínios funcionais e a função regulatória dos TFs. Esta pode ter a ver com as relações evolucionárias dos activadores e repressores. É, também, perceptível que os reguladores duais relacionam-se com mais proximidade dos activadores e dos repressores do que os activadores e os repressores se relacionam entre si. Para além disso, TFs de two-component transduction systems têm estruturas semelhantes , uma vez que desempenham funções idênticas. As duas arquitecturas de domínios também são previsíveis e não variam do que era esperado. Contudo, para os TFs globais, os resultados são antagónicos: as suas estruturas são diferentes umas das outras e cada TF é específico. A quantidade de domínios diferentes é elevada em comparação com a amostra completa de TFs, uma vez que o número de domínios excede o número de TFs. Domínios de todas as classificações estão presentes na estrutura dos TFs globais e as arquitecturas de domínios são variadas, o que reflecte as suas actividades específicas na célula.

Indiana University-Purdue University Indianapolis (IUPUI)<br>Monomers like triethylene glycol dimethacrylate (TEGDMA) leach from dental composites and adhesives due to incomplete polymerization or polymer degradation. The release of these monomers causes a variety of reactions that can lead to cell death. This death can be either necrotic, which is characterized mainly by inflammation and injury to the surrounding tissues, or apoptotic, which elicits little inflammatory responses, if any at all. TEGDMA-induced apoptosis in human pulp has been reported recently. However, the molecular mechanisms and the apoptotic (pro and anti) proteins involved in this process remain unclear. The objective of this study was to determine the apoptotic proteins expressed or suppressed during TEGDMA-induced apoptosis. Human pulp fibroblasts (HPFs) were incubated for 24 hours with different TEGDMA concentrations (0.125-1.0 mM). Cytotoxicity was determined using the cytotoxicity Detection KitPLUS (Roche Applied Science, Mannheim, Germany). TEGDMA was shown to cause cell cytotoxicity at concentrations of 0.50 mM and up. The highest concentration with no significant cytotoxicity was used. Cells were incubated with or without 0.25 mM TEGDMA for 6 h and 24 h. Cell lysates were then prepared and the protein concentrations determined using the Bradford protein assay. A Human Apoptosis Array kit (Bio-Rad Hercules, CA ) was utilized to detect the relative levels of 43 apoptotic proteins. The results of this study showed statistically significant increases of multiple examined pro-apoptotic proteins. The anti-apoptotic proteins were also altered. Pro-apoptotic proteins involved in the intrinsic and extrinsic apoptotic pathways were increased significantly. The results indicated that TEGDMA has effects on both the extrinsic and intrinsic apoptotic pathways.

Indiana University-Purdue University Indianapolis (IUPUI)<br>In response to different environmental stresses, phosphorylation of eIF2 (eIF2P) represses global translation coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of the integrated stress response, a program of gene expression involved in metabolism, nutrient uptake, anti-oxidation, and the activation of additional transcription factors, such as CHOP/GADD153, that can induce apoptosis. Although eIF2P elicits translational control in response to many different stress arrangements, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2P. In this study we addressed the underlying mechanism for variable expression of ATF4 in response to eIF2P during different stress conditions and the biological significance of omission of enhanced ATF4 function. We show that in addition to translational control, ATF4 expression is subject to transcriptional regulation. Stress conditions such as endoplasmic reticulum stress induce both transcription and translation of ATF4, which together enhance expression of ATF4 and its target genes in response to eIF2P. By contrast, UV irradiation represses ATF4 transcription, which diminishes ATF4 mRNA available for translation during eIF2∼P. eIF2P enhances cell survival in response to UV irradiation. However, forced expression of ATF4 and its target gene CHOP leads to increased sensitivity to UV irradiation. In this study, we also show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter resulting in its transcriptional repression. The LIP isoform of C/EBPβ, but not the LAP version is regulated following UV exposure and directly represses ATF4 transcription. Loss of the LIP isoform results in increased ATF4 mRNA levels in response to UV irradiation, and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2P and translational control, combined with transcription factors regulated by alternative signaling pathways, can direct programs of gene expression that are specifically tailored to each environmental stress.

碩士<br>中山醫學大學<br>生化暨生物科技研究所<br>94<br>To date, it is well accepted that Regulator of G-protein signalling（RGS） proteins terminate signalling by accelerating the intrinsic G alpha-GTPase activity and recycling the G-protein complex back to its inactive GDP-bound heterotrimeric configuration. The RGS protein can be divided into 15 subfamilies（RGS1~RGS15） in accordance with the structure and the biological function. The RGS domain, a conserved 120-aa region exists in all RGS proteins, functions as a negative regulator through interacting with activated Gα subunit and turn off the signaling. In addition, RGS proteins contain other domains that can associate with other protein to enhance the G-protein signaling. In this study, we attempted to investigat the expression pattern and function of RGS7 in zebrafish during development. Using RT-PCR analysis, RGS7 was expressed from fertilization to 96 hours post-fertilization of Zebrafish. Expression of RGS7 was found in brain、aorta ventralis、intermediate cell mass and somite by whole-mount in situ hybridization. Microinjection of RGS7-specific antisense morpholino oligonucleotide into fertilized zebrafish embryos leaded to interfere normal heart development and the heart function. Phenotype such as weakness of heart beat, swollen pericardial cavity and abnormal heart tube formation was found in morphants. Taken together, there results suggest that RGS7 protein plays an important role in the early development of the zebrafish heart.

H HARP (heparin-affin regulatory peptide), γνωστή και ως πλειοτροπίνη (PTN), είναι ένας 18 kDa αυξητικός παράγοντας, ο οποίος έχει υψηλή συγγένεια για την ηπαρίνη. Η HARP έχει πολλαπλές βιολογικές δράσεις, όπως συμμετέχει στη ρύθμιση του κυτταρικού πολλαπλασιασμού, στη μετανάστευση και τη διαφοροποίηση. Επιπλέον η έκφραση της σχετίζεται με την φυσιολογική και καρκινική αγγειογένεση in vitro και in vivo. Στην παρούσα εργασία μελετήθηκε η έκφραση της HARP και των υποδοχέων της, ALK και RPTPβ/ζ, στις διάφορες ημέρες ανάπτυξης της CAM εμβρύου όρνιθας. Επίσης, μελετήθηκε η μείωση της έκφρασης της ενδογενούς HARP, με πλασμίδιο που φέρει την αντινοηματική αλληλουχία (AS-HARP), στην αγγειογένεση in vivo, στη φωσφορυλίωση των Εrk1,2 και στη λεμφαγγειογένεση της CAM εμβρύου όρνιθας. Ανάλυση κατά Western και RT-PCR στις διάφορες ημέρες ανάπτυξης του εμβρύου έδειξε ότι η έκφραση της HARP συμβαδίζει με τη δημιουργία νέων αγγείων στη CAM, ενώ η έκφραση των υποδοχέων της HARP στην CAM φαίνεται να είναι αυξημένη στα πρώτα στάδια ανάπτυξης του ιστού. Επίσης, η μείωση της έκφρασης της HARP μετά τη χορήγηση του πλασμιδίου AS-HARP, μείωσε τα επίπεδα της πρωτεΐνης, το μήκος των αγγείων και τη φωσφορυλίωση των Erk1/2 στο in vivo μοντέλο της CAM εμβρύου όρνιθας. Αντίθετα, η μείωση της έκφρασης της HARP μετά τη χορήγηση του πλασμιδίου AS-HARP, δεν επηρέασε τη λεμφαγγειογένεση της CAM εμβρύου όρνιθας. Σαν τελικό συμπέρασμα προκύπτει ότι η έκφραση της ενδογενούς HARP στην CAM εμβρύου όρνιθας είναι σημαντική για τη φυσιολογική αγγειογένεση in vivo.<br>Heparin-affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is an 18 kDa growth factor that has a high affinity for heparin. HARP is involved in the control of cellular proliferation, migration and differentiation. Moreover, there is a strong correlation between HARP expression and tumor growth and angiogenesis. In the present work, we studied the expression of HARP and its receptors, ALK and RPTPβ/ζ, during development of the chicken embryo chorioallantoic membrane (CAM), in relation to angiogenesis. By western blot analysis and RT-PCR, it was shown that HARP, ALK and RPTPβ/ζ expression increased at days of on-going angiogenesis and decreased at later time points. Transfection of CAMs with an anti-sense HARP gene construct led to a significant decrease in HARP amounts compared to vector control transfected CAMs, a significant decrease in the length of CAM blood vessels, and a decrease in the phosphorylation of Erk1/2. Contrary, transfection of CAMs with the anti-sense HARP gene construct had no influence in lymphangiogenesis of the chicken embryo chorioallantoic membrane (CAM). These data suggest that endogenous HARP is involved in angiogenesis in vivo.

Mycobacterium tuberculosis, the causative organism of the disease tuberculosis (TB) in humans, leads to nearly two million deaths each year. This versatile pathogen can exist in highly distinct physiological states such as asymptomatic latent TB infection where bacilli lie dormant or as active TB disease in which the bacilli replicate in macrophages. The pathogenic lifestyle requires the tubercle bacillus to sense and respond to multiple environmental cues to ensure its survival. Such stimuli include hypoxia, nutrient limitation, presence of reactive oxygen and reactive nitrogen intermediates, pH alterations, and cell wall/ membrane stress. Two component systems (TCSs) form the primary apparatus for sensing and responding to environmental cues in bacteria. A prototypical TCS is composed of a sensory protein called sensor kinase (SK) and a response generating protein called response regulator (RR). M. tuberculosis encodes 11 genetically paired TCSs, 2 orphan sensor kinases and six orphan response regulator proteins. Studies of the TB bacilli using transcriptional profiling and gene knockouts have revealed that TCSs play an important role in facilitating successful adaptation to diverse environmental conditions encountered within the host. The mtrAB and prrAB genes encoding corresponding TCSs have been shown to be essential for survival, mprAB for persistence and devRS for hypoxic adaptation. Further, inactivation of the TCSs regX3-senX3, tcrXY, trcRS, phoPR or kdpDE was shown to affect the growth and/or virulence of M. tuberculosis in animal infection models. The SK and RR proteins of TCSs are modular and contain variable input and output domains coupled to conserved ‘transmitter’ and ‘receiver’ domains. Despite the modular nature and extensive homology of SK and RR proteins across TCSs, which may allow non-cognate interactions, it is believed that crosstalk across different TCSs is not favored and that individual pathways are generally well insulated. The existing profiling studies have been performed on the TCSs of bacterial species containing a relatively large number of TCSs. In those studies, specificity and insulation have been the norm and thus become the prevalent paradigm of TCS signaling. In vitro genome wide phosphotransfer profiling has revealed only a few cross- communication nodes in the TCSs of Escherichia coli (~3%), while none in Caulobacter crescentus (in 352 interactions tested, in short time duration) and Myxococcus xanthus (in 250 interactions tested). Yet, many instances of cross talk have been reported in literature. For example, E. coli TCSs PmrAB and EnvZ-OmpR show cross-communication with QseBC and ArcBA, and many more. In M. tuberculosis, indirect evidence of the existence of such cross regulation has originated from studies where mutations in phoPR have been shown to affect the expression of the TCS devRS and its regulon. It is thus interesting to examine the extent of crosstalk in the TCSs of M. tuberculosis, which has an exceptionally small number of TCS proteins compared to E. coli. As mentioned earlier, M. tuberculosis H37Rv has 11 cognate pairs of TCSs, 2 orphan sensor kinases and 6 orphan response regulators. To study the entire landscape, we aimed to study all 221 connections between SK and RR proteins including 12 cognate interactions. While 10 of the cognate TCS interactions were established in the literature, two putative systems KdpDE and NarSL and 5 orphan response regulators were still uncharacterized, therefore we initiated our work with the characterization of these TCSs. At the biochemical level, the KdpDE two component system of M. tuberculosis is not well studied, though one report showed interaction of the C-terminal domain of KdpD SK and KdpE RR using yeast two hybrid assay and another reported the interaction of the SK with LRP protein. Besides these associations, there is no evidence for the functionality of KdpDE system. Similarly, NarSL system also has not been characterized and it not known whether these putative two component proteins are functional. The initial part of the study includes the characterization of these two TCSs, NarS-NarL and KdpD-KdpE, at biochemical and physiological levels. In our studies we demonstrated that KdpDE system is a bonafide two component system of M. tuberculosis, and KdpD SK undergoes autophosphorylation at His642 residue in presence of Mg+2 ions and then it transfers phosphoryl group to a conserved Asp52 residue on the KdpE RR protein. The acid-base stability analysis revealed the nature of chemical bonds present in the KdpD and KdpE proteins, and further confirmed that KdpD and KdpE are typical SK and RR respectively. SPR analysis demonstrated that KdpD and KdpE proteins interact under basal non-phosphorylated conditions and the interaction affinity reduced when SK was phosphorylated. The reduction in the interaction affinity indicated towards a possible dissociation of SK and RR protein during phosphotransfer, which allows RRs to exert their regulatory effect. On the similar line, the phosphorylation defective SK (KdpDH642Q) had least affinity with KdpE suggesting that perhaps this mutant SK, fails to interact with the RR. We have also shown that both the kdpD and kdpE genes are in the same operon and are up regulated in potassium ions limitation and osmotic stress conditions. Overall, using the biochemical approaches, we have established that Rv1027c–Rv1028c operon of M. tuberculosis encodes a functional and a typical KdpDE two component signal transduction system. Using the similar biochemical and biophysical approaches, we have demonstrated that NarS-NarL proteins constitute a functional TCS and His241 and Asp61 are the phosphorylatable residues. In contrast to KdpDE which shows typical behaviour of TCS, NarSL TCS showed atypical behaviour. Malhotra and group’s work on NarSL suggested that there is cross-regulation between NarS/NarL and DevS/DosT/DevR systems. We addressed this possibility on three separate levels, by examining (i) the cross-phosphorylation of DevR and NarL RRs by non-cognate sensor kinases NarS and DevS/DosT respectively, (ii) the interaction between DevR and NarL RR proteins, and (iii) examining the effect of DevR-NarL interactions on their DNA binding properties. Our studies ruled out the presence of any physiologically relevant phosphorylation mediated cross-talk between NarS/NarL and DevS/DosT/DevR. We identified that the cross talk between these TCSs could be explained on the basis of interaction between NarL and DevR RRs and their subsequent binding to the target gene promoter regions for concerted regulation of gene expression. We also identified that DevR activation is critical for cooperative action with NarL. This process comes out as a novel mechanism of gene regulation via heteromerization of RRs. We hypothesized that formation of NarL-DevR heteromers may arise because of high sequence similarities. Conclusively, our study provides insights into the functionality of M. tuberculosis NarL/NarS TCS and regulatory function of NarL protein which acts in concert with another RR, DevR. Overall, NarS-NarL system showed an atypical, novel mode of gene regulation involving RR heteromerization. Subsequent to the basic biochemical characterization of NarSL and KdpDE system, the genome wide phosphotransfer profiling was done to identify the cross-connections between TCSs. Remarkably, we found that specificity was the exception rather than the rule. While only three of the TCS pairs were completely specific, all the other nine TCS pairs exhibited crosstalk, including a few that were highly promiscuous. We classified the interactions as specific, one-to-many, and many-to-one signaling circuits. We also profiled all the RRs including the orphans for their ability to accept phosphoryl group from a low molecular weight donor, acetyl phosphate, and interestingly found that only two RRs DevR and NarL were capable of accepting phosphoryl group from such a donor. Interestingly, none of the orphan RRs accepted phosphoryl group from any donor, neither SKs nor low molecular weight phospho donors, warranting further analysis of their roles and presence in the M. tuberculosis genome. Our exhaustive map of the crosstalk between the TCSs of M. tuberculosis sets the stage for a renewed view of TCS signaling and proposes a dispersive-integrative landscape for TCS signaling rather than one of insulation. As an extension of our basic characterization work of NarSL TCS, we also attempted to understand the localization pattern of NarS sensor kinase in M. smegmatis cells using fluorescence approaches. It is known that many bacterial receptors including sensor kinases form clusters or show specific localization patterns inside the cell. We found that NarS shows distinct cellular localization pattern. However, the functional significance of this localization pattern is not obvious yet and warrants further investigations. We also developed a few non-radioactive methods to study interaction between two component systems to overcome the limitations associated with radioactive experiments in studying TCSs. We developed fluorescence resonance energy transfer (FRET) to study in vitro interaction between two component proteins which was sensitive to the phosphorylation status of the proteins. Using fluorescently tagged SKs and RRs, we determined a change in FRET for KdpDE and NarSL TCS pairs in vitro. Our study thus also provides an alternative approach to study TCS signaling, using an easier, non-radioactive and high throughput approach. In summary, our study presents the evidence of an alternative paradigm of bacterial signaling, where significant crosstalk between the underlying TCSs prevails. The new paradigm is expected to have important implications in our understanding of the virulence and pathogenesis of bacterial infections. Overall, our studies (i) allowed the establishment of functionality of all paired TCSs encoded in the genome of M. tuberculosis including NarSL and KdpDE TCSs, (ii) identified the novel mechanism of gene regulation by NarL RR and DevR, (iii) demonstrated the existence of TCS signaling which is contrary to the existing notion of specificity (iv) showed the distinct localization pattern of NarS and (v) developed non-radioactive approaches to study two component interactions.