No description available.

The CTP:glycerol3-phosphate cytidylyltransferase (TarD) from Staphylococcus aureus catalyzes the formation of the nucleotide activated form of glycerol 3-phosphate (CDP-glycerol) used in the construction ofteichoic acid, a structure shown to be essential in Bacillis subtilis 168. The CTP:glycerol 3-phosphate cytidylyltransferase from B. subtilis 168 (TagD) involved in teichioc acid biosynthesis has high sequence identity (69 %) and similarity (86 %) to TarD and its characterization has been well documented. In these studies, TagD was shown to carry out the CTP:glycerol 3-phosphate cytidylyltransferase reaction via a random mechanism where there is negative cooperativity in the binding of substrates but not in catalysis. The work described here illustrates that the kinetic reaction mechanism for TarD is vastly different from TagD in spite of their high sequence similarity. Recombinant TarD was over-expressed in Escherichia coli and purified to homogeneity. Steady state hi-substrate experiments were performed utilizing a high-performance liquid chromatography assay in order to deduce the kinetic mechanism for TarD. In this analysis, data were globally best fit to the model that describes the formation of a ternary complex of substrates (CTP and glycerol 3- phosphate) and enzyme before catalysis. This examination yielded Km values for CTP and glycerol 3-phosphate of 21 ± 4.1 ).lM and 36 ± 5.8 ).lM respectively, while the kcat was measured to be 2.6 ± 0.2 s -I. From the pattern observed in product inhibition studies, a classic ordered Bi Bi reaction mechanism was inferred where glycerol 3-phosphate is the initial substrate to bind followed by CTP and the release ofCDP-glycerol precedes the release of pyrophosphate. A Keq of 16 ± 15 was calculated using data obtained from exploring the kinetic parameters of the reverse reaction where data was also fit to the equation that describes the formation of a ternary complex before catalysis. The equilibrium constant was also determined experimentally to be 6. To illustrate the biological role ofTarD with respect to TagD, the integration plasmid, pSWEET, was used to introduce a copy of tarD, under xylose control, into the chromosome of a strain of B. subtilis 168 possessing a temperature sensitive mutation (tag-12) mapped to tagD. Successful complementation of the temperature sensitive mutant by tarD at the restrictive temperature indicated that despite their apparent uniqueness in kinetic mechanism, TarD and TagD have similar roles in vivo. / Thesis / Master of Science (MSc)

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

No description available.

No description available.

No description available.

Viruses in the genus Orthobunyavirus (family Bunyaviridae) are classified serologically into 18 serogroups. The viruses have a tripartite genome of negative sense RNA composed of large (L), medium (M) and small (S) segments. The L segment encodes the polymerase protein, the M segment encodes two glycoproteins, Gc and Gn, and a non-structural protein (NSm), and the S segment encodes nucleocapsid (N) and NSs proteins, in overlapping reading frames (ORF). The NSs proteins of Bunyamwera and California serogroup viruses have been shown to play a role in inhibiting host cell protein synthesis and preventing induction of interferon in infected cells. To-date, viruses in only 4 serogroups: Bunyamwera, California, Group C and Simbu, have been studied intensively. Therefore, this study was conducted with the aim to sequence the S RNA segments of representative viruses in the other 14 orthobunyavirus serogroups, to analyse virus-encoded proteins synthesised in infected cells, and to investigate their ability to cause shutoff of host protein synthesis. S RNA segment sequences were obtained from cloned RT-PCR products. They were compared with the available sequences and each other. Complete S RNA sequences of Anopheles A (ANAV) and Tacaiuma virus (TCMV) [Anopheles A serogroup], Anopheles B (ANBV) and Boraceia virus (BORV) [Anopheles B serogroup], Eretmapodites (E147V) and Nyando virus (NDV)[Nyando serogroup], Bwamba virus (BWAV) [Bwamba serogroup], M’Poko virus (MPOV) [Turlock serogroup], Tete (TETEV) and Batama virus (BMAV) [Tete serogroup], and Gamboa (GAMV) and San Juan 2441 virus (SJ244V) [Gamboa serogroup], and partial sequences of Patois virus (PATV) [Patois serogroup], Guama (GMAV) and Bertioga virus (BERV) [Guama serogroup], Capim virus (CAPV) [Capim serogroup] and Palestina virus (PLSV) [Minatitlan serogroup] were obtained. Complete S segment sequences revealed that viruses in the same serogroup have same length of N and NSs proteins, except for the viruses in Gamboa serogroup which were found to have two lengths of NSs protein. Viruses in 4 serogroups (Anopheles A, Anopheles B, Tete and Capim) were found not to encode an NSs ORF, presenting the first report of naturally isolated orthobunyaviruses without an NSs protein. Most of these viruses were found to have longer N proteins compared to those with NSs protein, with the largest N protein observed to date in TETEV and BMAV (258 amino acids). Other viruses 3 (EREV, NDV, GAMV, SJ2441V, BWAV and MPOV) were found to encode both N and NSs proteins in their S segment with the largest and smallest NSs protein detected to date in SJ2441V (137 amino acids) and MPOV (70 amino acids) respectively. The conserved CA rich motif in 5’ non coding region (NCR) of Bunyamwera and California serogroups viruses was absent in BWAV and MPOV, while ANBV and BORV were found to have two copies of this motif. Repeated sequences, as observed previously in the 5’ NCR of genomic-sense RNA of Lumbo virus (LUMV), were also detected in BWAV and TCMV S RNA segments. Sequence comparisons and phylogenetic analyses of the sequences determined in this study were in agreement with previous serological classification of the viruses, except for BERV and TCMV. BERV, in the Guama serogroup, was found to have a closer relationship with CAPV compared to GMAV. However high sequence identities (>70%) were observed between these 3 viruses, suggesting that they are derived from the same ancestor. N protein and nucleotide sequence identities of TCMV with ANAV were only 53% and 59% respectively. However, Neighbour-Joining (NJ) plot based on complete N amino acid sequence and Maximum Parsimony (MP) plot based on partial N sequence supported previous serological classification which placed this virus in the same clade as ANAV. This study first reports on the proteins synthesised by Bakau, Bwamba, Koongol, Gamboa, Minatitlan, Olifantsvlei and Tete serogroup viruses. Analysis of radio-labelled cell extracts revealed similar protein migration patterns for all the studied viruses compared with other viruses in the genus Orthobunyavirus. Shutoff of host cell protein synthesis, similar to that seen in Bunyamwera virus (BUNV)-infected cells was only observed in ACAV, BAKV, BWAV, CAPV, PAHV, PATV and WONV-infected cells. However, this shutoff was found not related to the presence of NSs protein. In general, viruses in the same serogroup were found to have almost same size of plaque and plaque-size did not correlate with the presence of NSs protein and the virulence of the virus in the mice. In vitro transcription and translation (TnT) using rabbit reticulocyte and wheat germ lysate expression systems further confirmed the sequencing results that no NSs protein was expressed from S cDNA clones of ANAV, TCMV, ANBV, BORV, BMAV and TETEV. S RNA segments shutoff almost similar to BUNV-infected cells was observed in A549 cells infected with TCMV, suggesting that TCMV might use a different mechanism to induce shutoff. No significant shutoff was observed in Hep2, Hep2/V and C6/36 cells infected with any of the viruses. RT-PCR specific for IFN- ß mRNA in 293 infected cells and IFN reporter gene assays revealed that TCMV was capable of counteracting IFN production similar to wt BUNV, whereas the other NSs minus viruses (ANAV, ANBV, BORV, TETEV and BMAV) were found to be capable of inducing IFN in infected cells. However, only low level of IFN- ß mRNA and weak activation of the IFN- ß promoter was detected in ANAV and BMAV- infected cells.

Les microtubules sont des polymères cylindriques de tubuline-αβ, membres du cytosquelette eucaryote. Ils possèdent une dynamique intrinsèque nécessaire à de nombreuses fonctions cellulaires telle que la mitose. L’hydrolyse du nucléotide GTP dans les polymères de tubuline-αβ ainsi que les interactions entre la tubuline et les protéines partenaires ou les molécules à visées pharmacologiques, jouent un rôle critique sur la dynamique des microtubules. Durant cette thèse, des approches de modélisation moléculaire ont été utilisées pour mieux appréhender les interactions tubuline-ligand à l’échelle atomique et contribuer au développement de nouvelles molécules actives. Des simulations de dynamiques moléculaires ont été réalisées pour étudier l’effet de différents nucléotides dans la tubuline-β sur la structure et la dynamique du protofilament de tubuline. Nous proposons un rôle du résidu αE254 dans la coordination du magnésium catalytique. Nous observons également des changements conformationnels aux interfaces latérales et un réarrangement de structure aux interfaces longitudinales qui peuvent affecter la stabilisation du microtubule. Des travaux menés au laboratoire ont montré que la colchicine et le carbendazime se fixent dans des poches voisines dans la sous-unité tubuline-β et inhibent la prolifération cellulaire. Nous avons proposé un site de fixation du carbendazime dans les complexes tubuline-colchicine à l’aide de l’amarrage moléculaire et de simulations de dynamiques moléculaires. Ces expériences ont mené au design de molécules hybrides composées des noyaux colchicine et carbendazime reliés par un linker. Une de ces molécules hybrides a été synthétisée et testée avec succès sur des lignées de cellules HeLa. Enfin, nous avons construit des peptides cycliques dérivées d’I19L, un peptide anti-microtubule identifié au laboratoire. Des simulations de dynamique moléculaire et des calculs d’énergie libre de liaisons ont permis d’évaluer ces peptides. Enfin, des mutations ont été proposées afin d’optimiser l’interaction entre le meilleur peptide et la tubuline. / Microtubules are cylindrical polymers of αβ-tubulin heterodimers, members of the eukaryotic cytoskeleton. They possess an intrinsic dynamics which is necessary to any cellular functions such as the mitosis. It has long been recognized that GTP hydrolysis in αβ-tubulin polymers plays a critical role in this dynamics as well as the interactions between tubulin and the protein partners or the drugs. In this thesis, molecular modeling approaches are applied to three theoretical studies to gain insight at the atomic scale about tubulin-ligand interactions and to contribute to the development of new active compounds. Molecular dynamics simulations were used to study the effect of the different nucleotide states at β-tubulin on the protofilament structure and dynamics. We propose a role for residue αE254 in catalytic magnesium coordination. We also observe conformational changes and structure rearrangement at lateral and longitudinal interfaces that can affect the microtubule stabilization. Previous work carried out in the laboratory showed that colchicine and carbendazime bind neighboring pockets in the β-tubulin subunit and inhibit cell proliferation. We proposed a binding site of carbendazime on the tubulin-colchicine complex, using docking and molecular dynamics simulation, which lead to the design of hybrid molecules composed of both colchicines and carbendazime moieties attached with a linker. One of these hybrid molecules has been synthesized and successfully tested on HeLa cells. Finally, we designed four cyclic peptides based on I19L, an anti-microtubule peptide identified at the laboratory. Molecular dynamic simulations and binding free energy calculations were used to evaluate these peptides. Mutations were then proposed on the best peptide to increase its interactions with tubulin.

This dissertation summarizes the development of experimental and analytical tools for single cell RNA sequencing (scRNA-Seq), including 1) scPLATE-Seq, a FACS- and plate-based scRNASeq platform, which is accurate, robust, fully automated and cost-efficient; 2) metaVIPER, an algorithm for transcriptional regulator activity inference based on scRNA-Seq profiles; and 3) iterClust, a statistical framework for iterative clustering analysis, especially suitable for dissecting hierarchy of heterogeneity among single cells. Further this dissertation summarizes biological questions answered by combining these tools, including 1) understanding inter- and intra-tumor heterogeneity of human glioblastoma; 2) elucidating regulators of β-cell de-differentiation in type-2 diabetes; and 3) developing novel therapeutics targeting cell-state regulators of breast cancer stem cells.

Graduation date: 1995