Eukaryotic protein translation elongation is tightly controlled by several regulation factors. Eukaryotic translation elongation factor 1B (eEF1B) is the GTP exchange factor for eukaryotic translation elongation factor 1A (eEF1A), which is a G-protein transporting aminoacyl-tRNA to the A site of the ribosome in a GTP dependent manner. The structure of the heavy complex composed of eEF1B and eEF1A (eEF1H) has been widely studied and several models have been proposed, but it is yet not clear how the subunits of the two proteins interact with each other. eEF1B is made up of three subunits, eEF1Bα, eEF1Bδ and eEF1Bγ, and each subunit has been found to be over expressed in different types of cancer. A copy number variant near the eEF1Bδ gene is associated with amyotrophic lateral sclerosis. The two isoforms of eEF1A, eEF1A1 and eEF1A2, are 92% identical, but only eEF1A1 was found to interact with eEF1B subunits in yeast two hybrid (Y2H) experiments. The aims of this PhD project are to investigate the potential involvement of eEF1B in disease, as well as the relationship between eEF1B and eEF1A2. All three eEF1B subunits were present in almost all the cell types and mouse tissues tested. eEF1Bδ showed different variants, the heaviest of which is tissue specific and expressed only in brain and spinal cord. eEF1Bα and eEF1Bδ showed certain abnormalities in transformed cell lines, although in the breast cancer tissues tested no apparent change in eEF1B expression was found. Knockdown of eEF1B did not significantly affect NSC34 cell viability over short periods. In spinal cord sections from motor neurone disease (MND) patients, half of the cases showed a change of eEF1B protein expression compared to normal spinal cord, with either a higher level in glial cells, or a lower level in motor neurones. eEF1B and eEF1A2 were found to be co-expressed in mouse motor neurones, and proximity ligation assay also detected physical interactions between both eEF1A isoforms and eEF1B subunits in mammalian cells, contrary to the previous Y2H study. Experiments in a mouse model with no eEF1A2 expression also support this finding. In heart and skeletal muscle from wasted mice where eEF1A is absent the expression of eEF1Bα and eEF1Bδ was down regulated at both protein and mRNA level, suggesting that eEF1A2 and eEF1B not only physically interact, but also show an interdependence in expression. Overall the results from cultured cells, mouse and human tissues in this study demonstrate the potential involvement of eEF1B in MND, and its interaction with eEF1A, which contributes to the understanding of the non-canonical functions of eEF1B and the structure of eEF1H.

No description available.

Peptídeos sinais são moléculas envolvidas no crescimento, desenvolvimento e defesa de plantas. RALF (Rapid Alkalinization Factor) é um peptídeo sinal ubíquo no reino vegetal e que está envolvido com a expansão celular. Peptídeos RALF em Arabidopsis estão organizados em uma família multigênica de 37 membros, alguns com expressão tecido-específica, outros expressos em toda a planta. Este trabalho se insere dentro de um projeto maior que tem por objetivo esclarecer a função dos peptídeos RALF em plantas e determinar seu mecanismo de ação. Este trabalho teve dois objetivos específicos distintos. O primeiro consistiu em caracterizar as isoformas AtRALF19, AtRALF23, AtRALF31, AtRALF33 e AtRALF34 utilizando-se plantas mutantes, plantas superexpressoras e a análise dos promotores. O segundo objetivo específico foi identificar proteínas que interagem com o peptídeo AtRALF1 com o uso da técnica de purificação por afinidade em tandem (TAP) in planta. As análises fenotípicas das plantas transgênicas mostraram que plantas que superexpressam o gene que codifica o AtRALF33 apresentam fenótipo semi-anão, células foliares com área menor e com menor número de lóbulos. As plantas mutantes atralf33 e atralf23 apresentaram hastes e folhas maiores e células foliares com área maior. Plantas mutantes atralf34 não mostraram diferenças significativas quando comparadas com plantas selvagens e a análise do promotor do AtRALF34 mostrou uma expressão específica em estômatos, hipocótilo e ápice radicular. Com relação a purificação por afinidade, plantas mutantes mcca foram transformadas com a construção 35S:AtRALF1:HPB e usadas para obtenção dos extratos proteicos. / Peptides signals are molecules involved with growth, development and defense in plants. RALF (Rapid Alkalinization Factor) is an ubiquous peptide in plant kingdom and it is involved with cell expansion. RALF peptides are organized in a multigenic family with 37 members, some are tissue-specific expressed, others are expressed in whole plant. This work is part of a larger project that has the approach to clarify the RALF peptides functions in plants and to determine its mechanism of action. This work has two distinct approaches. The first specific approach was to characterize the isoforms AtRALF19, AtRALF23, AtRALF31, AtRALF33 and AtRALF34 using mutant plants, overexpression plants and promoters analysis. The second specific approach was to identify proteins that interact with AtRALF1 peptide using in planta tandem affinity purification (TAP). The phenotype analysis of transgenic plants showed that the overexpression of the gene which codifies AtRALF33 plants presented a semi-dwarf phenotype, smaller leaf cells area and number of lobes. The mutant plants atralf33 and atralf23 presented larger stalks and leaf cells. The mutant plants atralf34 did not show significant differences when compared to wild-type plants. The promoter analysis of AtRALF34 showed a specific expression in stomata, hypocotyls and root shoot. Regarding the TAP, mcca mutant plants were transformed with the construction 35S:AtRALF1:HPB.

Osseointegrated transfemoral implants have been introduced as an alternative to conventional, socket-based prostheses for above knee amputees. This method is showing great promise; however, the frequency of device failure is of concern. As a means to understand some of these failures, this study investigates the mechanical behaviour of three osseointegrated transfemoral implant systems; an in-house prototype design and two assemblies comprised of implant-grade parts provided by Integrum AB, Sweden. A series of mechanical tests were administered to evaluate the effects of tightening specifications and connection geometry on the material response, and mechanical behaviour of the assemblies. The tests included torque-elongation measurements, torque to failure tests, and strain analyses during loading. This research was carried out in an attempt to better understand the behaviour of components under loading, in order to optimize the assembly specifications and improve the useful life of the system. The results obtained confirm that the Integrum assemblies exhibit improved performance when tightened to a level beyond the current specified tightening torque of 12 Nm. Increased tightening torque provides an improved connection between components, leading to increased torque retention, decreased peak tensile strain values and a more gradual, primarily compressive distribution of strains throughout the assembly. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2009-09-29 12:14:18.438

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Molecular Biology and Microbiology. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2008. / Härtill 4 uppsatser.

Peptídeos sinais são moléculas envolvidas no crescimento, desenvolvimento e defesa de plantas. RALF (Rapid Alkalinization Factor) é um peptídeo sinal ubíquo no reino vegetal e que está envolvido com a expansão celular. Peptídeos RALF em Arabidopsis estão organizados em uma família multigênica de 37 membros, alguns com expressão tecido-específica, outros expressos em toda a planta. Este trabalho se insere dentro de um projeto maior que tem por objetivo esclarecer a função dos peptídeos RALF em plantas e determinar seu mecanismo de ação. Este trabalho teve dois objetivos específicos distintos. O primeiro consistiu em caracterizar as isoformas AtRALF19, AtRALF23, AtRALF31, AtRALF33 e AtRALF34 utilizando-se plantas mutantes, plantas superexpressoras e a análise dos promotores. O segundo objetivo específico foi identificar proteínas que interagem com o peptídeo AtRALF1 com o uso da técnica de purificação por afinidade em tandem (TAP) in planta. As análises fenotípicas das plantas transgênicas mostraram que plantas que superexpressam o gene que codifica o AtRALF33 apresentam fenótipo semi-anão, células foliares com área menor e com menor número de lóbulos. As plantas mutantes atralf33 e atralf23 apresentaram hastes e folhas maiores e células foliares com área maior. Plantas mutantes atralf34 não mostraram diferenças significativas quando comparadas com plantas selvagens e a análise do promotor do AtRALF34 mostrou uma expressão específica em estômatos, hipocótilo e ápice radicular. Com relação a purificação por afinidade, plantas mutantes mcca foram transformadas com a construção 35S:AtRALF1:HPB e usadas para obtenção dos extratos proteicos. / Peptides signals are molecules involved with growth, development and defense in plants. RALF (Rapid Alkalinization Factor) is an ubiquous peptide in plant kingdom and it is involved with cell expansion. RALF peptides are organized in a multigenic family with 37 members, some are tissue-specific expressed, others are expressed in whole plant. This work is part of a larger project that has the approach to clarify the RALF peptides functions in plants and to determine its mechanism of action. This work has two distinct approaches. The first specific approach was to characterize the isoforms AtRALF19, AtRALF23, AtRALF31, AtRALF33 and AtRALF34 using mutant plants, overexpression plants and promoters analysis. The second specific approach was to identify proteins that interact with AtRALF1 peptide using in planta tandem affinity purification (TAP). The phenotype analysis of transgenic plants showed that the overexpression of the gene which codifies AtRALF33 plants presented a semi-dwarf phenotype, smaller leaf cells area and number of lobes. The mutant plants atralf33 and atralf23 presented larger stalks and leaf cells. The mutant plants atralf34 did not show significant differences when compared to wild-type plants. The promoter analysis of AtRALF34 showed a specific expression in stomata, hypocotyls and root shoot. Regarding the TAP, mcca mutant plants were transformed with the construction 35S:AtRALF1:HPB.

During mouse development, the primordia of the posterior body including the trunk and tail tissues of the embryo forms largely from a bipotential cell population that resides in the posterior growth zone in vertebrate embryos. This bipotential cell population contains neuromesodermal progenitors (NMP) which are found in the tail bud which replaces the primitive streak after gastrulation and contributes to axial elongation by the formation of both the spinal cord and paraxial mesoderm derivatives. The three vertebrate Cdx genes, Cdx1, Cdx2 and Cdx4, encode transcription factors that play important roles in axial elongation since the triple Cdx mutant embryos fail to generate any tissue posterior to the occipital primordia. A comparison of Cdx mutant phenotypes suggests that Cdx2 is the most important contributor to axial elongation since Cdx2 heterozygous mutants exhibit foreshortened tails and Cdx2 conditional mutants exhibit axial truncation and complete loss of tail bud structures. Cdx2 target genes, such as Wnt3a, Cyp26a1 and T, are also essential for axial elongation. Cdx1 null mutants are viable and exhibit homeosis of cervical and anterior thoracic vertebrae, while Cdx4 null mutants are phenotypically normal. In addition, it has been shown that simultaneous loss of multiple copies of Cdx alleles disrupts axial elongation more severely than each single mutation which suggests there is overlapping function among the Cdx family. The genetic network underlying regulation of axial elongation by the Cdx family is not fully understood due in part to this functional overlap. In this thesis, I employed a conditional Cre-loxP system to derive conditional mutants lacking all Cdx functions. Additionally, Pax2-GFP transgenic mice where GFP is expressed under the control of Pax2 locus were used to enrich tail bud NMP cells for RNA-seq and ChIP-seq analysis for Cdx2. Using this approach, I revealed new target genes and pathways that are regulated by Cdx members and likely involved in axial elongation.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / During RNA Polymerase II (RNAPII) transcription, a dynamic network of protein-protein interactions (PPIs) coordinates the regulation of initiation, elongation, and termination. Taking a proteomics approach to study RNAPII transcription can offer a comprehensive view of the regulatory mechanisms mediated by PPIs within the transcription complex. However, traditional affinity purification mass spectrometry (APMS) methods have struggled to quantitatively capture many of the more dynamic, less abundant interactions within the elaborate RNAPII transcription interactome. To combat this challenge, we have developed and optimized a quantitative AP-MS based method termed Disruption-Compensation (DisCo) Network Analysis that we coupled with Tandem Mass Tag (TMT) labeling. In this application, TMT-DisCo was applied to investigate the PPIs that regulate RNAPII transcription. In the first study, TMT-DisCo network analysis was used to analyze how perturbation of subunits of four major transcription elongation regulators —Spt6, Spt5 (DSIF), Cdc73 (PAF-Complex), and Spt16 (FACT)— affect the RNAPII PPI network. TMT-DisCo was able to measure specific alterations of RNAPII PPIs that provide insight into the normal functions of Spt6/Spt5/Cdc73/Spt16 proteins within the RNAPII elongation complex. The observed changes in the RNAPII interactome also reveal the distinct mechanisms behind the phenotypes of each perturbation. Application of TMTDisCo provides in vivo, protein-level insights into synthetic genetic interaction data and in vitro structural data, aiding in the understanding of how dynamic PPIs regulate complex processes. The second study focused on the essential RNAPII CTD phosphatases, Ssu72 and Fcp1. TMT-DisCo captures how the ssu72-2 allele affects the ability of RNAPII to proceed through elongation, resulting in more arrested RNAPII that requires proteasomal degradation. Reduction of Ssu72 phosphatase activity shifts cells away from RNAPII reinitiation/ recycling and toward de novo expression and newly assembled RNAPII, aided by chaperones. RNAPII in fcp1-1 cells was observed to increase in interaction with the 26S proteasome, as well as TFIID and mRNA capping enzyme. These data support a model of the nuclear proteasome functioning as a chaperone during transcription initiation, as the fcp1-1 allele leads to inefficient formation of a pre-initiation complex with a hyperphosphorylated RNAPII CTD. / 2024-08-16