No description available.

No description available.

Flavopridol (FVP) is a well known pharmacological inhibitor of Cyclin Dependent Kinases (CDKs), with significant selectivity for Cyclin Dependent Kinase 9 (CDK9). Treatment of cells with FVP results in inhibition of transcription elongation. CDK9 is a serine/threonine kinase that associates with T-type cyclins. These complexes are designated transcription elongation factors (P-TEFb). P-TEFb controls transcription elongation by phosphorylating the carboxyl terminal domain (CTD) of RNA polymerase II (RNAPII) and negative elongation factors. Whether P-TEFb is required for the elongation of most genes transcribed by RNAPII or fraction of them is still debatable. The aim of my Thesis is to understand the early and late effects of FVP on primary response gene expression. Two different microarray analyses with RNA probes obtained from T98G and BJ-TERT cells were performed by Drs. Graña and Garriga to determine the effect of inhibiting CDK9 on global mRNA expression using a dominant negative mutant of CDK9 (dnCDK9) and FVP. These gene profiling experiments showed that FVP and dnCDK9 downregulate the expression of several genes. However, these studies also showed upregulation of a group of primary response genes (PRGs). The goal of this thesis was to bring some light into this unexpected phenomenon. I have found that several PRGs including FOS, JUNB, EGR1 and GADD45B, are rapidly and potently downregulated before they are upregulated upon FVP treatment in exponentially growing cells. In serum starved cells restimulated with serum, FVP also inhibits the expression of these genes, but subsequently, JUNB, GADD45B and EGR1 are upregulated in the presence of FVP. Chromatin Immunoprecipitation of RNAPII revealed that EGR1 and GADD45B are apparently transcribed at the FVP-treatment time points where their corresponding mRNAs accumulate. These results suggest a possible stress response triggered by CDK9 inhibition. I also show that serum starvation does not affect the localization of RNAPII immediately downstream of the promoter of a PRG where RNAPII remains paused in the absence of mitogenic stimulation, suggesting that initiation is not rate limiting for transcription of at least certain PRGs in the absence of mitogens and remains dependent on transcription elongation. In sum, I have shown that certain PRG/IRGs are transcribed in the presence of FVP and their transcription might be independent of CDK9 suggesting a possible alternative mechanism of their transcription. I also determined that transcription initiation is not affected by serum starvation, as paused RNAPII appears to remain bound downstream of a PRG promoter in quiescent cells independently of the length of mitogenic starvation. / Molecular Biology and Genetics

Formins are potent regulators of actin dynamics that can remodel the actin cytoskeleton to control cell shape, cell cytokinesis, and cell morphogenesis. The defining feature of formins is the formin homology 2 (FH2) domain (Paul and Pollard, 2008), which promotes actin filament assembly while processively moving along the polymerizing filament barbed end. INFT-1 is one of six formin family members present in Caenorhabditis elegans (Hunt-Newbury et al., 2007) and is most closely related to vertebrate INF2, an inverted formin with regulatory domains in the C- rather than N-terminus. Nematode INFT-1 contains both formin homology 1 (FH1) and formin homology 2 (FH2) domains. However, it does not share the regulatory N-terminal Diaphanous Inhibitory Domain (DID) domain and C-terminal Diaphanous Autoregulatory Domain (DAD) domain found in mammalian INF2. In contrast to mammalian INF2, the sequence of INFT-1 starts immediately at FH1 domain and C-terminal region of INFT-1 shares little homology with INF2, suggesting that elegans INFT-1 is regulated by other mechanisms. We used fluorescence spectroscopy to determine the effect of INFT-1 FH1FH2 on actin assembly and total internal reflection fluorescence microscopy to investigate how INFT-1 formin homology 1 and formin homology 2 domains (FH1FH2) mediate filament nucleation and elongation. INFT-1 FH1FH2 nucleates actin filament and promote actin assembly. However, INFT-1 FH1FH2 reduces filament barbed-end elongation rates in the absence or presence of profilin. Evidences demonstrated that INFT-1 is non-processive, indicating a unique mechanism of nucleation. INFT-1 nucleation efficiency is similar to the efficiency of Arabidopsis FORMIN1 (AFH1), another non-processive formin. High phosphate affected the assembly activity of INFT-1 FH1FH2 in the absence or presence of profilin. INFT is thus the second example of a non-processive formin member and will allow a more detailed understanding of the mechanistic difference between processive and non-processive formins. / Master of Science

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation. Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome. Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer. Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen. These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.

Le ketchup, la moutarde, la mousse à raser, sont des fluides à seuil, ils s’écoulent uniquement lorsqu’on leur applique une contrainte supérieure à une valeur critique, appelée contrainte seuil. Sur des surfaces lisses, ces fluides peuvent s’écouler sous de petites contraintes : on a alors un phénomène de glissement. En étudiant par rhéométrie les écoulements de ces matériaux des séquences originales et une technique d’imagerie directe (vélocimétrie en IRM), on montre que le glissement ne se produit qu’au-delà d’une contrainte critique. Selon les cas, cette contrainte critique est due soit à un effet de bord, soit à un effet de surface. L’excès de contrainte par rapport à cette contrainte critique varie linéairement avec la vitesse de glissement. De ce fait le glissement peut être représenté comme le cisaillement d’une couche de liquide le long de la paroi, mais la réalité est plus complexe compte tenu de la structure du matériau au contact avec la paroi. Curieusement l’épaisseur de cette couche de liquide « équivalente » ne semble pas varier avec la concentration, la taille des gouttes, la force normale, etc. Ceci suggère que cette épaisseur est gouvernée par des forces plus élevées que la lubrification et la pression osmotique. Nous étudions également le glissement pour des écoulements plus complexes. Pour cela on impose une élongation au fluide à seuil par une expérience de traction avec des surfaces lisses. La force normale mesurée pour différents matériaux avec des structures différentes montre que la condition de transition solide-liquide en élongation est différente que ce que prédit la théorie standard, et l’épaisseur de la couche de glissement est de plusieurs ordres de grandeur supérieure à celle trouvée en cisaillement simple / Ketchup, mustard, shaving creams flow only when submitted to stresses greater than a critical stress – yield stress, these are yield stress fluids. On smooth surfaces, these fluids can flow under very small stresses; this phenomenon is the wall slip. Using gels, emulsions, clay suspensions, etc., and from rheometrical tests with original protocols and internal measurements (MRI velocimetry), we show that a minimal stress must be reached to initiate wall slip and, depending on cases, this value is either due to an edge effect or to an adhesion of the suspended elements to the wall. Above this critical value, the excess of stress is found to vary linearly with the slip velocity, except at the transition of the yield stress or using a microtextured surface: in that cases the relation becomes quadratic. The wall slip can be interpreted as the shear flow of a thin liquid layer between the yield stress fluid and the wall. However, given the complexity of the material structure in contact with the wall, the exact picture of the slip layer requires further investigations. The apparent thickness of the liquid layer seems to be independent of the concentration, the mean droplet size, the external normal forces, etc., suggesting that it depends on interactions between the suspended droplets and the surface which are much stronger than the lubricating and osmotic pressures. We also study wall slip under more complex flow conditions, by inducing an elongational flow during a traction test with smooth surfaces. The normal force measured for various materials with different microstructures shows that the yielding condition in an elongational flow is different from the standard theory, and the apparent thickness of the wall slip layer is several orders of magnitude larger than that found in shear flows

Coding sequences (5,334 nt total) from elongation factor-1α, elongation factor-2, and the largest subunit of RNA polymerase II were determined for 6 species of Tardigrada, 2 of Arthropoda, and 2 of Onychophora. Parsimony and likelihood analyses of nucleotides and amino acids yielded strong support for Tardigrada and all internal nodes (i.e., 100% bootstrap support for Tardigrada, Eutardigrada, Parachela, Hypsibiidae, and Macrobiotidae). Results are in agreement with morphology and an earlier molecular study based on analysis of 18S ribosomal sequences. Divergence times have been estimated from amino acid sequence data using an empirical Bayesian statistical approach, which does not assume a strict molecular clock. Divergence time estimates are pre-Vendian for Tardigrada/Arthropoda, Vendian or earlier for Eutardigrada/Heterotardigrada, Silurian to Ordovician for Parachela/Apochela, Permian to Carboniferous for Hypsibiidae and Macrobiotidae, and Mesozoic for Isohypsibius/Thulinia (both within Hypsibiidae) and Macrobiotus/Richtersius (both within Macrobiotidae).

No description available.

The length of the primary root and the density of lateral roots determine the architecture of the root. In this thesis the effect of NAA, ABA and the NO donor SNP alone as well as the combination of ABA or NAA with SNP on lateral root development was investigated. The interaction between CPTIO, a NO scavenger, and NAA or SNP is also reported. Following preliminary experiments in which it was observed that the aerial part of the seedling influenced LR growth and that there was a possible inhibitory effect of light on cultured root tips, experiments were conducted with excised roots tips in the dark. NAA was shown to have the potential to initiate LRs across a wide concentration gradient with the total number of LRs and initiated lateral root primordia (LRP) remaining constant across the range of concentrations tested. Over the last decade, nitric oxide (NO), a bioactive molecule, has been reported to be involved in the regulation of many biological pathways. The presence of NO in the system provided via sodium nitroprusside (SNP), promoted LRP initiation based on the NAA concentration gradient; but without changing the total LR initiation, that is LRs plus primordia density remained constant along the concentration gradient of NAA. The absence of LR and LRP in the treatments of CPTIO (a NO scavenger) with SNP or NAA suggests that NO regulates LRP initiation triggered by NAA, which is in agreement with the recent paper published after the commencement of this study (Correa-Aragunde et al., 2006). In agreement with previous studies, ABA inhibited lateral root development by reducing LR density and the number of LRs. The experiments with fluridone, an ABA biosynthesis inhibitor, may indicate that endogenous ABA was at sufficient concentrations in the excised root tips to inhibit primordia initiation. In this study, evidence is presented for the first time to show that SNP can relieve the inhibitory effect of ABA on LR density and number of LRs suggesting the NO, released from SNP, acts downstream of ABA. Overall these data confirm a critical role for NO in LR initiation.

The distinct stages of cotton fiber development and maturation serve as a single-celled model for studying the molecular mechanisms of plant cell elongation, cell wall development and cellulose biosynthesis. However, this model system of plant cell development is compromised for proteomic studies due to a lack of an efficient protein extraction method during the later stages of fiber development, because of a recalcitrant cell wall and the presence of abundant phenolic compounds. Here, we compared the quality and quantities of proteins extracted from 25 dpa (days post anthesis) fiber with multiple protein extraction methods and present a comprehensive quantitative proteomic study of fiber development from 10 dpa to 25 dpa. Comparative analysis using a label-free quantification method revealed 287 differentially-expressed proteins in the 10 dpa to 25 dpa fiber developmental period. Proteins involved in cell wall metabolism and regulation, cytoskeleton development and carbohydrate metabolism among other functional categories in four fiber developmental stages were identified. Our studies provide protocols for protein extraction from maturing fiber tissues for mass spectrometry analysis and expand knowledge of the proteomic profile of cotton fiber development.