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The Global ETD Search service is a free service for researchers
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Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
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XGef interacts with and is involved in Ringo's influence on meiotic maturation in Xenopus laevis oocytesRunge, Erika January 2009 (has links)
Thesis advisor: Laura Hake / The completion of meiosis in Xenopus oocytes requires the coordinated translation of stored mRNAs. CPEB, the cytoplasmic polyadenylation element binding protein, controls the translation of developmentally important early-class maternal mRNAs. Resumption of meiosis through stimulation with progesterone leads to the phosphorylation and activation of CPEB. This results in the lengthening of the poly(A) tails and translation of mRNAs containing the cytoplasmic polyadenylation element (CPE). XGef, a putative guanine nucleotide exchange factor, binds to and is required for CPEB activation. Translation of c-mos, a MAPK kinase kinase, is controlled by CPEB, and activation of the Mos/MAPK pathway is required for meiotic maturation. In addition, the synthesis of Ringo protein, an atypical cdk binding protein and activator, is required for progesterone-induced maturation, though Ringo is able to stimulate resumption of meiosis independent of progesterone. Although much work has been done to understand the key events leading to activation of maturation promoting factor (MPF) and meiotic maturation, the events immediately following progesterone stimulation remain unclear, particularly regarding the role of XGef. The work that follows describes experiments performed to further understand the role of XGef in meiotic maturation through both Ringo and MAPK activity. It was found that XGef and Ringo interact directly and form a complex throughout early meiosis. XGef is involved in Ringo’s influence during meiosis, specifically through MEK-activation of MAPK. Notably, XGef functions in a common pathway and complex with Ringo most likely to influence CPEB phosphorylation and activation. / Thesis (BS) — Boston College, 2009. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.
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Functional Analysis of Proteins Involved in Translational RegulationRaher, Michael J January 2003 (has links)
Thesis advisor: Laura E. Hake / Cytoplasmic polyadenylation regulates translational activation of mRNA stored in immature Xenopus oocytes. This event is necessary for the beginning of oocyte maturation, and later for critical processes in early embryonic development. A major protein required for polyadenylation is the cytoplasmic polyadenylation element-binding protein (CPEB), which recruits a factor that promotes the interaction between Poly(A) polymerase and the end of the mRNA. Polyadenylation in turn leads to translation through interactions between CPEB and other proteins. Using a yeast two-hybrid screen, several of these proteins were identified and cloned, including two of note. X295, a zinc-finger containing novel protein, and DEK, which has significant homology with the Homo sapiens DEK involved in certain juvenile leukemias. Through the cloning of the genes encoding these proteins, transcription of mRNA, and protein overexpression in oocytes, a series of protein-protein interaction binding assays were performed. Immunoblotting of SDS-PAGE analyzed samples shows that GST-CPEB and HA-X295 interact in ovo, and suggests a possible in ovo interaction of endogenous CPEB and endogenous X295. In similar experiments, DEK and CPEB do not interact, suggesting they may not interact in ovo. / Thesis (BS) — Boston College, 2003. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.
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XGef functions independently of exchange factor activity to influence RINGO/CDK1 signaling and CPEB activation during Xenopus oocyte maturationKuo, Peiwen January 2009 (has links)
Thesis advisor: Laura E. Hake / Metazoan development depends on cytoplasmic polyadenylation, a key mechanism that controls the translation of maternally deposited mRNAs. In Xenopus laevis oocytes, CPEB regulates the translation of several developmentally important mRNAs, which drive meiotic progression and the production of fertilizable eggs. Most of our current knowledge of this process, also referred to as oocyte maturation, has been acquired from experiments conducted in Xenopus laevis oocytes. Despite over 30 years of research devoted to the exploration of progesterone signaling during maturation, the very early events that occur from progesterone receptor engagement to CPEB activation are not well understood. XGef, a putative Rho family guanine nucleotide exchange factor (GEF), interacts with CPEB and facilitates CPEB activation and timely meiotic progression. To further our understanding of XGef function during meiotic progression, the requirement for exchange factor activity and the activities of several Rho GTPases during maturation were examined. Despite previous reports of XGef activation of Cdc42 in mammalian cell culture, XGef does not stimulate the activation of Cdc42 in maturing Xenopus oocytes. Further, Cdc42 activity does not affect CPEB phosphorylation and overexpression of a dominant negative Cdc42 mutant does not affect maturation. Inhibition of Toxin B sensitive Rho GTPases, including Cdc42, Rac1 and Rho A-C, also fails to affect CPEB activation or meiotic progression. Lastly, the overexpression of XGef exchange deficient point mutants did not affect maturation compared to oocytes overexpressing wildtype XGef. Together, these results suggest that as a facilitator of CPEB activation and meiotic progression, XGef functions independently of exchange factor activity and Rho GTPase activation. Additionally, we found that XGef activity influences the function of RINGO/CDK1, a novel component of the progesterone signaling pathway. XGef inhibition depresses RINGO-induced GVBD, whereas XGef overexpression enhances this process. XGef interacts with RINGO in oocyte extracts and the interaction is direct in vitro. Our protein interaction data, in total, suggest that a XGef/RINGO/MAPK/CPEB complex forms in ovo to facilitate CPEB activation. Lastly, inhibition of RINGO activity directly compromises CPEB phosphorylation during early maturation, which suggests that RINGO/CDK1 directly mediates CPEB-activation. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
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Expression of anxiety-related genes, including the cytoplasmic polyadenylation element binding protein (CPEB), in the rat limbic systemVan Cleemput, Jamie Michelle 03 May 2006
Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety.
Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.
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Expression of anxiety-related genes, including the cytoplasmic polyadenylation element binding protein (CPEB), in the rat limbic systemVan Cleemput, Jamie Michelle 03 May 2006 (has links)
Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety.
Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.
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Identification of a new deadenylation negative feedback loop that regulates meiotic progressionBelloc Rocasalbas, Eulàlia 15 December 2008 (has links)
Els oòcits de vertebrats es troben aturats a la profase I de la primera meiosi (PI). Durant el procés anomenat oogènesi, els oòctits sintetitzen i emmagatzemen grans quantitats d'ARN missatgers(ARNm)que els seran necessaris per la compleció de la meiosi.I,per posteriorment, aturar-se de nou a la metafase de la segona divisió meiòtica (MII) per l'activitat del factor citostàtic(CSF).D'aquestes divisions en destaca el fet que transcorren en absència de transcripció, i per tant depenen totalment en l'activació traduccional dels ARNm anteriorment esmentats que han estat acumulats durant l'oogènesi. L'activació traduccional d'aquests missatgers és principalment induïda per l'elongació de les cues d'adenines(cues de poli(A)), aquest procés és mediat per les seqüències de poliadenilació citoplasmàtiques (CPE)presents a la regió 3' no tradudïda (3'UTR)dels ARNm. El moment i la longitud de la poliadenilació dels ARNm que contenen CPEs estan finament regulats, de manera que en combinació amb la degradació de proteïnes, s'estableixen els patrons específics d'expresió de les proteïnes que condueixen la meiosi (Shmitt et al., 2002; de Moor and Richter, 1997; Ballantyne et al., 1997; Mendez et al., 2002; Charlesworth et al., 2002). Fins a la data, no s'havia descrit que la deadenilació (escurçament de la cua de poli(A)) fos necessària per la progressió meiòtica. En aquesta tesi s'ha descrit, a partir d'un cribatge d'abast genòmic, una ruta de retroalimentació negativa requerida per a la sortida de la primera metafase meiòtica. La nova ruta identificada, a més té la particularitat d'actuar a nivell traduccional regulant l'expressió de proteïnes que participen directament en la progressió meiòtica. L'element central d'aquesta nova ruta és la proteïna C3H-4, que a la vegada és regulada per poliadenilació citoplasmàtica. C3H-4 crea la retroalimentació negativa interaccionant amb elements ARE de les regions 3'UTR, promovent la deadenilació del ARNm al qual s'uneix. D'entre les seves dianes hem identificat Emi1 i Emi2, ambdós reguladors de l'activitat de l'APC/C, crítica per la divisió cel·lular.
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Spindle-Localized CPE-Mediated Translation Controls Mediotic Chromosome SegregationEliscovich, Carolina 11 June 2008 (has links)
La progresión meiótica y el desarrollo embrionario temprano están programados, en parte, por la activación tradcuccional de mRNAs maternos como lo son los que codifican para las proteinas de ciclina B1 o mos. Estos mRNAs no son traducidos al mismo tiempo ni en el mismo lugar. Por lo contrario, su traducción está especificamente regulada por elementos de poliadenilación citoplasmática (CPEs) presentes en sus 3'UTRs. Los elementos CPEs reclutan a la proteina de unión a CPE (CPE-binding protein CPEB (Colegrove-Otero et al., 2005; de Moor et al., 2005; Mendez and Richter, 2001; Richter, 2007)). Esta proteina de unión al RNA no sólo determina cuándo y en qué medida un mRNA será activado traduccionalmente por poliadenilación citoplasmática (Mendez et al., 2000a; Mendez et al., 2000b; Mendez et al., 2002) sino que también participa, junto con el represor de la traducción Maskin, en el transporte y la localización de sus mRNAs diana hacia los sitios de localización subcelular donde su traducción ocurrirá (Huang et al., 2003; Huang and Richter, 2004). Durante el desarrollo embrionario de Xenopus, CPEB se encuentra localizada en el polo animal de los oocitos y más tarde, sobre el huso mitótico y centrosomas en el embrión (Groisman et al., 2000). Se ha demostrado que embriones de Xenopus inyectados con agentes que interrumpen la traducción dependiente de poliadenilación citoplasmática, detienen la división celular y presentan estructuras mitóticas anormales (Groisman et al., 2000). En este trabajo que derivó en mi tesis doctoral, hemos demostrado que la activación traduccional localizada en el huso mitótico de mRNAs regulados por CPEB que codifican para proteinas con una conocida función en aspectos estructurales del ciclo celular como la formación del huso mitótico y la segregación cromosómica, es esencial para completar la primera división meiótica y para la correcta segregación cromosómica en oocitos de Xenopus.
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