L'organisation et la dynamique du cytosquelette d'actine sont finement régulées par une multitude de "actin-binding proteins" (ABPs). Parmi ces dernières, les ADFs (actin-depolymerizing factors) jouent un rôle majeur dans le turnover des filaments d'actine en induisant leur découpage et en facilitant leur dépolymérisation. Arabidopsis thaliana possède 11 protéines ADFs fonctionnelles qui peuvent être classées en 4 sous-classes sur la base de leur profil d'expression et liens phylogénétiques. Nous démontrons que l’ADF5 et l’ADF9 de la sous-classe III sont des ADFs atypiques puisqu’elles n’induisent pas la dépolymérisation des filaments d’actine. Au contraire, elles montrent une forte capacité à stabiliser et ponter les filaments d’actine en longs câbles in vitro ainsi que in vivo. Nous décrivons la caractérisation d’un nouveau mutant knockout d’Arabidopsis. Les données suggèrent un rôle d’ADF9 dans l’élongation cellulaire. Ainsi, l’hypocotyle est significativement plus long dans les mutants adf9 que dans les plantules sauvages, et ce phénotype est amplifié par des conditions de croissance à l’obscurité dans lesquelles le gène ADF9 est normalement préférentiellement exprimé. L’analyse des cellules épidermiques d’hypocotyle indique que ce phénotype est essentiellement dut à une augmentation de l’élongation cellulaire. De manière surprenante, les plantules mutantes adf9 présentent également des racines plus courtes que les contrôles, suggérant un lien complexe entre l’organisation du cytosquelette d’actine et l’élongation cellulaire. Finalement, la capacité réduite du cal issue des plantules adf9 à proliférer suggère également un rôle d’ADF9 dans la division cellulaire. / Actin cytoskeleton organization and dynamics are tightly regulated by many actin-binding proteins (ABPs). Among ABPs, the actin-depolymerizing factors (ADFs) play a major role in actin filament turnover by promoting actin filament severing and facilitating pointed end depolymerization. Arabidopsis thaliana has 11 functional proteins that can be classified into four subclasses according to their expression profile and phylogenetic relationships. We provide evidence that subclass III ADF5 and ADF9 are unconventional ADFs since they do not display typical actin filament depolymerizing activities. Instead, they exhibit opposite activities with a surprisingly high ability to stabilize and crosslink actin filaments into long and thick actin bundles both in vitro and in live cells. Competition experiments with ADF1 support that ADF9 antagonizes the depolymerizing activity of conventional ADFs. We report the characterization of a not yet described knockout Arabidopsis mutant. Data strongly suggests a role for ADF9 in cell elongation. Indeed, hypocotyls are significantly longer in adf9 mutant than in wild- type seedlings, and this phenotype is enhanced in dark growth conditions in which the ADF9 gene is normally preferentially expressed. The analysis of hypocotyl epidermal cells indicates that this phenotype is essentially due to an increase of cell expansion. Surprisingly, adf9 seedlings exhibit shorter roots than control plants, suggesting a complex link between actin cytoskeleton organization and cell elongation. Finally, the reduced ability of adf9- derived calli to proliferate supports a role for ADF9 in cell division as well.

[ES] Los péptidos DEVIL/ROTUNDIFOLIA (DVL/RTFL) constituyen una familia de péptidos de pequeño tamaño codificados por la familia génica DVL/RTFL de 24 miembros en A. thaliana. Estos genes fueron caracterizados por los fenotipos que confiere su sobreexpresión, que provoca cambios pronunciados en la morfología de la planta con hojas de roseta más redondeadas, plantas de menor estatura, peciolos cortos e inflorescencias compactas. Estos fenotipos afectan de un modo fascinante a la morfología de los frutos, que varía según qué miembro de la familia se sobreexprese, demostrando tener un papel en el desarrollo de múltiples órganos de la planta. Se ha visto que los péptidos DVL/RTFL se localizan en la membrana plasmática y que comparten homología en sus secuencias, con un dominio conservado en el extremo C-terminal, estando además ampliamente conservados en el mundo vegetal. Tanto su localización como dominio funcional conservado resultan ser esenciales para su actividad. Sin embargo y, a pesar de los fenotipos sorprendentes causados por la sobreexpresión de diferentes genes DVL/RTFL, las líneas de pérdida de función no aportan información sobre la función biológica de la familia DVL/RTFL. En el laboratorio donde se ha realizado este trabajo, se ha avanzado en los últimos años en la caracterización de estos péptidos, su modo de interacción con la membrana celular y la determinación de sus patrones de expresión, así como en la identificación de mutantes de pérdida de función. Para continuar en estas direcciones, en este proyecto se generaron combinaciones de mutantes múltiples estables en diferentes genes DVL/RTFL combinando mutantes de inserción de T-DNA con mutantes generados por CRISPR/Cas9 (dvl3dvl5dvl6dvl1dvl4rtfl9dvl8dvl11rtfl11dvl19). A pesar de que la pérdida de función de múltiples genes DVL/RTFL no mostró fenotipos morfológicos evidentes, análisis transcriptómicos y proteómicos apoyaron la hipótesis de la elevada redundancia génica entre los miembros de esta familia y de que podrían tener un papel en la regulación de procesos como el crecimiento y desarrollo del tubo polínico o el crecimiento distal de la célula. Experimentos donde analizamos la germinación de polen, el crecimiento de tubo polínico o el desarrollo de pelos radiculares no mostraron que los péptidos DVL/RTFL afectaran de un modo significativo estos procesos , pero sí sugirieron que tienen un rol generalizado aportando estabilidad o robustez al proceso de morfogénesis vía elongación celular. Adicionalmente se llevaron a cabo estudios de topología de membrana que permitieron confirmar su localización en la membrana plasmática, de tal manera que estos péptidos no se integraban en la membrana, sino que estarían asociados a su interfase. La posibilidad de que la asociación se llevase a cabo a través de otras proteínas hizo que se comprobasen in planta interacciones de los péptidos DVL1 y DVL11 con proteínas candidatas identificadas en un escrutinio de doble híbrido de levadura y relacionadas con procesos de tráfico intra e intercelular, división y elongación celular. Su interacción confirmada con proteínas como SRC2, BSK6 o CDC48 llevó a estudiar la posible relación funcional con éstas y en especial con CDC48 por su implicación en procesos de división, expansión y diferenciación celular, sin obtener resultados concluyentes. Los escasos resultados obtenidos en Arabidopsis nos condujo a estudiar el papel del único homólogo DVL/RTFL en M. polymorpha. Tras generar y caracterizar líneas de pérdida de función y sobreexpresoras MpDVL, hemos podido confirmar la conservación funcional de los péptidos DVL/RTFL en especies de plantas tan alejadas evolutivamente, así como determinar que no se trata de péptidos esenciales para el desarrollo de la planta pero que sí parecen tener un papel en los procesos de morfogénesis vía elongación celular aportando robustez al sistema. Este trabajo pone de manifiesto que la necesidad de profundizar en el estudio de los péptidos DVL/RTFL. / [CA] Els pèptids DEVIL/ROTUNDIFOLIA (DVL/DVL) constitueixen una família de pèptids de xicoteta grandària codificats per la família gènica DVL/RTFL de 24 membres en A. thaliana. Aquests gens van ser caracteritzats pels fenotips que confereix la seua sobreexpressió, que provoca canvis pronunciats en la morfologia de la planta amb fulles de roseta més arredonides, plantes de menor alçada, pecíols curts i inflorescències compactes. A més, aquests fenotips afecten d'una manera fascinant a la morfologia dels fruits, que varia segons quin membre de la família es sobreexprese, demostrant tindre un paper en el desenvolupament de múltiples òrgans de la planta. També s'ha vist que els pèptids DVL/RTFL es localitzen en la membrana plasmàtica i que comparteixen homologia en les seues seqüències, amb un domini conservat en l'extrem C-terminal, estant a més àmpliament conservats en el món vegetal. Tant la seua localització com domini funcional conservat resulten ser essencials per a la seua activitat gènica adequada. No obstant això i, malgrat els fenotips sorprenents observats en la sobreexpressió de diferents gens DVL/RTFL, les línies de pèrdua de funció no aporten informació sobre la funció biològica de la família DVL/RTFL. En el laboratori on s'ha fet aquest treball, s'ha avançat en els últims anys en la caracterització d'aquests pèptids, la seua manera d'interacció amb la membrana cel·lular i la determinació dels seus patrons d'expressió, així com en la identificació de mutants de pèrdua de funció. Per a continuar en aquestes direccions, en aquest projecte es van generar combinacions de mutants múltiples estables en diferents gens DVL/RTFL combinant mutants d'inserció de T-DNA amb mutants generats per CRISPR/Cas9 (dvl3dvl5dvl6dvl1dvl4rtfl9dvl8dvl11rtfl11dvl19). A pesar que la pèrdua de funció de múltiples gens DVL/RTFL no va mostrar fenotips morfològics evidents, anàlisis transcriptòmics i proteòmics van donar suport a la hipòtesi de l'elevada redundància gènica entre els membres d'aquesta família i que a més tenen un paper en la regulació de processos com són la morfogènesi, el creixement i desenvolupament del tub pol·línic o el creixement distal de la cèl·lula. Experiments de germinació de pol·len, creixement de tub pol·línic o desenvolupament de pèls radiculars duts a terme van demostrar que els pèptids DVL/RTFL no tenien un paper significatiu en aquests processos, però sí que van suggerir que tenen un rol generalitzat aportant estabilitat o robustesa al procés de morfogènesi via elongació cel·lular. Addicionalment es van dur a terme estudis de topologia de membrana que van permetre confirmar la seua localització en la membrana plasmàtica, de tal manera que aquests pèptids no s'integraven en la membrana si no que estarien associats a la seua interfase. La possibilitat que aquesta associació es duguera a terme a través d'altres proteïnes va fer que es comprovaren in planta interaccions dels pèptids DVL1 i DVL11 amb proteïnes candidates extretes d'un escrutini de doble híbrid de llevat i relacionades amb processos de trànsit intra i intercel·lular, divisió i elongació cel·lular. La seua interacció confirmada amb proteïnes com SRC2, BSK6 o CDC48 va portar a estudiar la possible relació funcional amb aquests i especialment amb CDC48 per la seua implicació en processos de divisió, expansió i diferenciació cel·lular, sense obtindre resultats concloents. Els escassos resultats obtinguts en Arabidopsis ens va conduir a estudiar el paper de l'únic homòleg DVL/RTFL en M. polymorpha. Després de generar i caracteritzar línies de pèrdua de funció i de sobreexpressió MpDVL, hem pogut confirmar la conservació funcional dels pèptids DVL/RTFL en espècies de plantes tan allunyades evolutivament, així com determinar que no es tracta de pèptids essencials per al desenvolupament de la planta però que sí que semblen tindre un paper en els processos de morfogènesis via elongació cel·lular aportant robustesa al sistema. / [EN] DEVIL/ROTUNDIFOLIA (DVL/DVL) peptides constitute a family of small peptides encoded by the 24-member DEVIL/ROTUNDIFOLIA (DVL/RTFL) gene family in Arabidopsis thaliana. These genes were characterized by the phenotypes conferred by their overexpression, which causes pronounced changes in plant morphology with round rosette leaves, shorter plants, short petioles, and compact inflorescences. In addition, these phenotypes dramatically affect fruit morphology, which varies depending on which family member is overexpressed, proving to play a role in the development of multiple plant organs. It has also been shown that DVL/RTFL peptides are located in the plasma membrane, that they share sequence homology, mostly in a conserved C-terminal domain, and that they are also widely conserved among land plants. Both the localization at the membrane and conserved functional domain are essential for proper gene activity. However, despite the surprising overexpression phenotypes observed, the loss-of-function mutants do not provide information on the DVL/RTFL biological function. In the lab where this work has been carried out, progress has been made in the characterization of these peptides, determining how they interact with the plasma membrane, how they are expressed and accumulated, as well as identifying loss-of-function mutants. To continue in these directions, in this project we have generated combinations of multiple stable DVL/RTFL mutants by combining T-DNA insertion mutants with mutants generated by CRISPR/Cas9 (dvl3 dvl5 dvl6 dvl1 dvl4 rtfl9 dvl8 dvl11 rtfl11 dvl19). Even though the loss of function of multiple DVL/RTFL genes did not show evident morphological phenotypes, transcriptomic and proteomic analyzes supported the hypothesis of a high gene redundancy among the members of this family and suggested that they might have a role in the regulation of processes such as pollen tube growth and development or cell tip growth. However, pollen germination, pollen tube growth or root hair development experiments did not demostrate that DVL/RTFL peptides had a significant role in these processes, but they suggested that they may have a general role in providing stability or robustness to the morphogenesis process via cell elongation. Additionally, membrane topology studies were carried out to confirm their location in the plasma membrane, in such a way that these peptides were not integrated but would be associated with the membrane interface. The possibility that this association was carried out through other proteins led to the in planta verification of DVL1 and DVL11 peptide interactions with candidate proteins identified in a previous yeast two-hybrid screening and related to intracellular and intercellular trafficking processes cell division and elongation. The confirmed interaction with proteins such as SRC2, BSK6 or CDC48 led us to study the possible functional relationship with these, and especially with CDC48 due to its involvement in cell division, expansion, and differentiation processes, but unfortunately, we did not obtain conclusive results. The unconclussive results obtained in Arabidopsis led us to study the role of the unique DVL/RTFL homologue in Marchantia polymorpha. After generating and characterizing MpDVL loss-of-function and overexpression lines, we have been able to confirm the functional conservation of the DVL/RTFL peptides in so evolutionarily distant plant species, as well as to determine that they are not essential for plant development, but they seem to have a role in the morphogenesis processes via cell elongation, providing robustness to the system. This work highlights the need of furthering the study of DVL/RTFL peptides to discover the mechanism by which they participate in plant development processes and to determine their biological function in depth. / Esta Tesis Doctoral ha sido financiada por la Generalitat Valenciana con una Subvención para la Contratación de Personal Investigador Predoctoral (ACIF/2018/260), el Ministerio de Ciencia e Innovación (proyectos BIO2015-64531-R y RTI2018-099239-B-I00), la Generalitat Valenciana (proyecto PROMETEU/2019/004) y el ExpoSeed H2020-MSCA-RISE-2015-691109 / Alarcia García, A. (2024). Los péptidos DEVIL: estudio de su papel en el control de la proliferación celular y la morfogénesis de las plantas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202903

Abnormal expression of the eukaryotic translation elongation factor 1A (eEF1A) has been implicated in disease states such as motor neuron degeneration and cancer. Two variants of eEF1A are found in mammals, named eEF1A1 and eEF1A2. These two variants are encoded by different genes, produce proteins which are 92% identical but have very different patterns of expression. eEF1A1 is almost ubiquitously expressed while eEF1A2 is expressed only in specialised cell types such as motor neurons and muscle. A spontaneous mutation in eEF1A2 results in the wasted mouse phenotype which shows similar characteristics in the mouse to those seen in human motor neuron degeneration. This mutation has been shown to be a 15.8kb deletion resulting in the complete loss of the promoter region and first non coding exon of eEF1A2 which completely abolishes protein expression. The main aim of this project was to further investigate the role of eEF1A2 in motor neuron degeneration. Firstly, although the wasted phenotype is considered to be caused by a recessive mutation, I established a cohort of aged heterozygote mice to evaluate whether any changes are seen later in life that might model late onset motor neuron degeneration. A combination of behavioural tests and pathology was used to compare wild type and heterozygous mice up to 21 months of age. Whilst results indicate that there is no significant difference between ageing heterozygotes and wildtype controls, there is an indication that female heterozygote mice perform slightly worse that wildtype controls on the rotarod (a behavioural test for motor function). Secondly, I aimed to investigate the primary cause of the wasted pathology by generating transgenic wasted mice expressing neuronal eEF1A2 only. This would complement previous experiments in the lab which studied transgenic wasted mice expressing eEF1A2 in muscle only. Unfortunately the expression of eEF1A2 in the transgenic animals was not neuronal specific. However a transgenic line with expression of eEF1A2 in neurons and skeletal muscle but not cardiac muscle has been generated which clearly warrants further investigation. Thirdly, I wished to assess whether eEF1A2 has any role in human motor neuron degeneration. To achieve this, eEF1A2 expression was investigated in spinal cords from human motor neuron disease (MND) patients. Preliminary data suggests that motor neurons from some MND patients express significantly less eEF1A2 than motor neurons of control samples. Further work is required to confirm these findings. Finally, I investigated the individual roles of eEF1A1 and eEF1A2 in the heat shock response. I used RNAi to ablate each variant separately in cells and subsequently measured the ability of each variant individually to mount a heat shock response. Results indicate a clear role for eEF1A1 but not eEF1A2 in the induction of heat shock. This may explain in part why motor neurons exhibit a poor heat shock response as they express eEF1A2 and not eEF1A1. These experiments shed light on our understanding of the role of eEF1A2 in motor neuron degeneration and uncover many new avenues of future investigation.

In eukaryotes, mRNA decay pathways are important for cellular response to various physiological conditions and also function in co-translational quality control systems that target translationally aberrant mRNAs for degradation. My work on identification and characterization of novel components and pathways of mRNA degradation and quality control in Saccharomyces cerevisiae is summarized below.I have identified Edc3p as a novel protein important for mRNA decay. Deletion of Edc3p leads to a defect in mRNA decay in strains deficient in decapping enzymes and, in combination with a block to the 3' to 5' decay pathway, causes exaggerated growth defects and synthetic lethality. An Edc3p-GFP fusion protein localizes in processing bodies, which are specialized cytoplasmic foci containing decapping proteins. Together, these observations indicate that Edc3p directly interacts with the decapping complex to stimulate the mRNA decapping rate.Quality control during mRNA translation is critical for regulation of gene expression. My work shows that yeast mRNAs with defects in translation elongation, due to strong translational pauses, are recognized and targeted for degradation via an endonucleolytic cleavage in a novel process referred to as No-Go Decay (NGD). The cellular mRNA decay machinery degrades the 5' and 3' cleavage products produced by NGD. NGD is translation-dependent, occurs in a range of mRNAs and can be induced by a variety of elongation pauses. These results indicate NGD may occur at some rate in response to any stalled ribosome.I also show that two highly conserved proteins, Dom34p and Hbs1p, homologous to the eukaryotic release factors eRF1 and eRF3 respectively, are required for NGD. Further characterization of the No-Go decay pathway indicates that Dom34p function during NGD is conserved across species. Identification of RPS30, a small ribosomal protein as a trans-acting factor during NGD suggests that the ribosome may have a novel role during NGD. Other experiments indicate that the No-Go decay pathway may cross talk with the unfolded protein response pathway. The identification of No-Go decay as a novel quality control pathway during translation elongation supports the existence of a global cellular mechanism for maintenance of translational quality control.

Protein synthesis is a highly complex process executed by the ribosome in coordination with mRNA, tRNAs and translational protein factors. Several antibiotics are known to inhibit bacterial protein synthesis by either targeting the ribosome or the proteins factors involved in translation. Fusidic acid (FA) is a bacteriostatic antibiotic that blocks polypeptide chain elongation by locking elongation factor-G (EF-G) on the ribosome. Mutations in fusA, the gene encoding bacterial EF-G, confer high-level of resistance towards FA.  Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by acquiring secondary mutations. In order to understand the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that, the causes for fitness loss in the FA-resistant mutant F88L are resulting from significantly slower tRNA translocation and ribosome recycling. Analysis of the crystal structures, together with the results from our biochemical studies enabled us to propose that FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome. EF-G is a G-protein belonging to the GTPase super-family. In all the translational GTPases, a conserved histidine (H92 in E. coli EF-G) residue, located at the apex of switch II in the G-domain is believed to play a crucial role in ribosome-stimulated GTP hydrolysis and inorganic phosphate (Pi) release. Mutagenesis of H92 to alanine (A) and glutamic acid (E) showed different degree of defect in different steps of translation. Compared to wild type (WT) EF-G, mutant H92A showed a 10 fold defect in ribosome mediated GTP hydrolysis whereas the other mutant H92E showed a 100 fold defect. However, both the mutants are equally defective in single round Pi release (100 times slower than WT). When checked for their activity in mRNA translocation, H92A and H92E were 10 times and 100 times slower than WT respectively. Results from our tripeptide formation experiments revealed a 1000 fold defect for both mutants. Altogether, our results indicate that GTP hydrolysis occurs before tRNA translocation, whereas Pi release occurs probably after or independent of the translocation step. Further, our results confirm that, His92 has a vital role residue in ribosome-stimulated GTP hydrolysis and Pi release.

Baigiamajame magistro darbe tirta popieriaus paviršiaus ir mechaninių savybių kaita spausdinant fleksografiniais dažais ir keičiant dažų sluoksnio storį. Tyrimams buvo pasirinktos šešios kreidinio ir nekreidinio popieriaus rūšys. Buvo tirtos šių mechaninių ir paviršiaus savybių kaita: spalvinės charakteristikos, atsparumas tempimui ir pailgėjimas, atsparumas lankstymui, PPS šiurkštumas, trinties savybės, popieriaus sugertis. Buvo nustatyta, kad fleksografiniai dažai didina popieriaus šiurkštumą, trūkio jėga ir atsparumas lankstymui visada yra didesni išilgine popieriaus kryptimi, atsparumas tempimui nepriklauso nuo dažų sluoksnio storio, o atsparumas lankstymui mažėja. Darbą sudaro 6 dalys: įvadas, literatūros apžvalga, tyrimo metodika, tyrimų rezultatai ir jų aptarimas, išvados ir siūlymai, literatūros sąrašas. Darbo apimtis – 83 psl. teksto be priedų, 82 iliustracijos, 1 lentelė, 28 bibliografinių šaltinių. / In the Master thesis were investigated the change of the surface and mechanical properties during flexo printing with different ink thickness. 6 papers coated and uncoated grades were tested. Following properties were investigated: colour properties, tension strength and elongation, folding strength, PPS roughness and friction properties. It was found that flexographic ink increases paper PPS roughness, tensile strength and folding resistance is higher in the machine direction in all cases, tensile strength does not depend on the ink thickness and folding resistance becomes smaller. Master thesis includes 6 chapters: introduction, publications review, experimental methods, results and discussion, conclusions and recommendations, references. Master thesis consists of 83 p. (without appendixes), 82 fig., 1 table, and 28 references.

Elongation of the vertebrate rostrocaudal axis depends on localised populations of axial progenitors. Previous work has demonstrated the presence of Neuromesodermal (NM) progenitors that behave as multipotent stem cells, which contribute to the neurectoderm and mesoderm throughout axis elongation. They have been localised to the Node-­‐Streak Border (NSB) in the primitive streak region, and the Chordoneural Hinge (CNH) in its descendant, the tail bud. At primitive streak stages, the Caudal Lateral Ectoderm (CLE) on either side of the primitive streak itself is also fated for neurectoderm and mesoderm. However, fate mapping studies in mouse and chick have suggested that these progenitors are more transitory than those in the NSB and CNH, leading to the idea that two different types of progenitor cell exist in the primitive streak region; long-­‐term (stem cell-­‐like) and transient progenitors. In this thesis, I have examined the potency of the CLE cells by heterotopically grafting them into the NSB. Anterior CLE cells are exquisitely sensitive to their position and differentiate predominantly as neurectoderm, mesoderm, or both, depending on their exact location in the NSB. Most significantly, their descendants are retained in the CNH, indicating that CLE cells show equal potential to NSB progenitors on transplantation to the border environment. The relationship between fate and potency within the streak stage embryo suggest a mechanism by which stem cells are maintained not only by their intrinsic stem cell program, but are also influenced by their location. Furthermore, I have investigated the expression of candidate markers of NM-­‐progenitors, and have found that the combined expression of Sox2 and T genes in the progenitor area coincides with observed NM-­‐potency, and could serve as a marker for this stem cell population. Over time, axial elongation comes to a halt and NM-­‐progenitors are thereafter not longer active. It is still unclear how exactly this process occurs and specifically whether axial elongation ceases because NM progenitors are eliminated. I have investigated the events occurring immediately before the end of axial elongation. Morphological and gene expression analysis shows that apoptosis reaches a peak only after the complete axis has been laid down, and is not dramatically elevated in the progenitors themselves before that. In order to test signalling pathways that lead to progenitor maintenance, I have developed an in vitro tail growth assay that recapitulates in vivo development, as measured by several morphometric criteria. I show that, even though FGF signalling is critical for most cells in the tail bud including NM-­‐progenitors, it is not sufficient for NM-­‐ progenitor maintenance. In contrast, exposing tail buds to elevated Wnt/β-­‐catenin signalling does prolong the lifetime of NM-­‐progenitors in the ageing tail bud, as judged by the presence of Sox2-­‐T double-­‐positive cells. In this regard we have found that the time of cessation coincides with the disappearance of Sox2-­‐T double-­‐positive cells, the disappearance of Wnt3a and concomitant neuralisation of the progenitor region. This data suggest an important governing role for Wnt signalling in both maintenance and fate decision of NM progenitors. Thus the disappearance of Wnt signalling in the tail bud over time could well be the main reason for triggering the halt of caudal elongation.

eIF5A seems to be involved in both, translation initiation and elongation. It was also reported to affect assembly of P-bodies. Given similarities of P-bodies with stress granules (SGs) we decided to test the role of eIF5A in dynamics of heat-induced SGs and its implications for the cell recovery. For the evaluation of eIF5A function in SGs formation was used the temperature- sensitive (ts) mutant eIF5A-3 (C39Y/G118D) cultivated under permissive temperature 25řC and Rpg1-GFP fusion protein as a marker of SGs. The cells were exposed to robust heat shock at 46řC for 10 minutes. The ability of the mutant cells to recover was tested by propidium iodine staining and colony forming units plating. We found that the eIF5A-3 mutant forms heat-induced SGs more loosely aggregated, indicating that the fully functional eIF5A is necessary for SGs assembly. However, it does not seem to affect the rate of SGs dissolution. Survival tests indicate that eIF5A-3 mutant cells are susceptible to dying in a similar way as WT cells; nevertheless, their ability to resume proliferation is significantly better. We also observed a loss of the ts phenotype of the eIF5A-3 mutant. This loss cannot be explained by reversion of mutated eIF5A sequence into normal. Probable cause lies in the adaptive evolution. Our results indicate role of...

No description available.

Rop/Rac proteins are plant-specific monomeric guanosine triphosphate-binding proteins (G-proteins) with important functions in plant development. Until recently, only three cotton (Gossypium hirsutum) Rop/Rac G-protein genes were sequenced, representing subfamilies III and IV of the plant monomeric Gprotein family. In this project, members of subfamilies II and I were cloned, sequenced, and named GhRac2 and GhRac3, respectively. Using real-time reverse transcription PCR, expression of GhRac2 was highest during fiber elongation, decreasing significantly when cellulose biosynthesis began. Transcript abundance of GhRac3 doubled between fiber elongation and secondary wall synthesis, remaining constant until 20 days post-anthesis. Expression of GhRac2 and GhRac3 was compared between the unfertilized ovules of Gossypium hirsutum, Texas Marker 1 and two near-isogenic fiber-impaired mutants. Expression of GhRac2 and GhRac3 was significantly higher in wild type ovules than in Ligon lintless, a mutant impaired in fiber elongation, but was not different in Naked Seed, a mutant impaired in fiber initiation.