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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    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.
141

Structure of eukaryotic DNA polymerase epsilon and lesion bypass capability

Sabouri, Nasim January 2008 (has links)
To transfer the information in the genome from mother cell to daughter cell, the DNA replication must be carried out only once and with very high fidelity prior to every cell division. In yeast there are several different DNA polymerases involved in DNA replication and/or DNA repair. The two replicative DNA polymerases, DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon), which both include a proofreading 3´→5´exonuclease activity, can replicate and proofread the genome with a very high degree of accuracy. The aim of this thesis was to gain a better understanding of how the enigmatic DNA polymerase epsilon participates in DNA transactions. To investigate whether Pol epsilon or Pol delta is responsible for the synthesis of DNA on the lagging strand, the processing and assembly of Okazaki fragments was studied. Pol delta was found to have a unique property called “idling” which, together with the flap-endonuclease (FEN1), maintained a ligatable nick for DNA ligase I. In contrast, Pol epsilon was found to lack the ability to “idle” and interact functionally with FEN-1, indicating that Pol epsilon is not involved in processing Okazaki fragments. Together with previous genetic studies, it was concluded that Pol delta is the preferred lagging strand polymerase, leaving Pol epsilon to carry out some other function. The structure of Pol epsilon was determined by cryo-electron microscopy, to a resolution of ~20 Å. Pol epsilon is composed of a globular “head” domain consisting of the large catalytic subunit Pol2p, and a “tail” domain, consisting of the small subunits Dpb2p, Dpb3p, and Dpb4p. The two separable domains were found to be connected by a flexible hinge. Interestingly, the high intrinsic processivity of Pol epsilon depends on the interaction between the tail domain and double-stranded DNA. As a replicative DNA polymerase, Pol epsilon encounters different lesions in DNA. It was shown that Pol epsilon can perform translesion synthesis (TLS) through a model abasic site in the absence of external processivity clamps under single-hit conditions. The lesion bypass was dependent of the sequence on the template and also on a proper interaction of the “tail”domain with the primer-template. Yeast cells treated with a DNA damaging agent and devoid of all TLS polymerases showed improved survival rates in the presence of elevated levels of dNTPs. These genetic results suggested that replicative polymerases may be engaged in the bypass of some DNA lesions. In vitro, Pol epsilon was found to bypass 8-OxoG at elevated dNTP levels. Together, the in vitro and in vivo results suggest that the replicative polymerases may be engaged in bypass of less bulky DNA lesions at elevated dNTP levels. In conclusion, the low-resolution structure presented represents the first structural characterization of a eukaryotic multi-subunit DNA polymerase. The replicative DNA polymerase Pol epsilon can perform translesion synthesis due to an interaction between the tail domain and double-stranded DNA. Pol epsilon may also bypass less bulky DNA lesions when there are elevated dNTP concentrations in vivo.
142

Protein-Nucleic Acid Interactions in Nuclease and Polymerases

rob, abdur 05 May 2011 (has links)
DNA polymerase binds to the double stranded DNA and extends the primer strand by adding deoxyribonucletide to the 3’-end. Several reactions in the polymerase active site have been reported by Kornberg in addition to the polymerization. We observed DNA polymerase I can act as a pyrophosphatase and hydrolyze deoxyribonucletide. In performing the pyrophosphatase activity, DNA polymerase I requires to interact with RNA. RNA in general, was found to activate the DNA polymerase I as pyrophosphatase. This hydrolysis causes depletion of dNTP and inhibits DNA polymeration synthesis in vitro. In this RNA-dependent catalysis, DNA polymerase I catalyzes only dNTP but not rNTP. We have also observed that many other DNA polymerases have this type of the RNA-dependent pyrophosphatase activity. Our experimental data suggest that the exonuclease active sites most likely play the critical role in this RNA-dependent dNTP hydrolysis, which might have a broader impact on biological systems. On the basis of the crystal structure of a ternary complex of RNase H (Bacillus halodurans), DNA, and RNA, we have introduced the selenium modification at the 6-position of guanine (G) by replacing the oxygen (SeG). The SeG has been incorporated into DNA (6 nt. - 6 nucleotides) by solid phase synthesis. The crystal structure and biochemical studies with the modified SeG-DNA indicate that the SeDNA can base-pair with the RNA substrate and serve as a template for the RNA hydrolysis. In the crystal structure, it has been observed that the selenium introduction causes shifting (or unwinding) of the G-C base pair by 0.3 Å. Furthermore, the Se-modification can significately enhance the phosphate backbone cleavage (over 1000 fold) of the RNA substrate, although the modifications are remotely located on the DNA bases. This enhancement in the catalytic step is probably attributed to the unwinding of the local duplex, which shifts scissile phosphate bond towards the enzyme active site. Our structural, kinetic and thermodynamic investigations suggest a novel mechanism of RNase H catalysis, which was revealed by the atom-specific selenium modification.
143

An Embedded Shading Language

Qin, Zheng January 2004 (has links)
Modern graphics accelerators have embedded programmable components in the form of vertex and fragment shading units. Current APIs permit specification of the programs for these components using an assembly-language level interface. Compilers for high-level shading languages are available but these read in an external string specification, which can be inconvenient. It is possible, using standard C++, to define an embedded high-level shading language. Such a language can be nearly indistinguishable from a special-purpose shading language, yet permits more direct interaction with the specification of textures and parameters, simplifies implementation, and enables on-the-fly generation, manipulation, and specification of shader programs. An embedded shading language also permits the lifting of C++ host language type, modularity, and scoping constructs into the shading language without any additional implementation effort.
144

Development and characterization of Mantle Cell Lymphoma specific IgGs

Gärdefors, Katarina January 2008 (has links)
Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.
145

Analysis of a direct energy conversion system using medium energy helium ions

Carter, Jesse James 16 August 2006 (has links)
A scaled direct energy conversion device was built to convert kinetic energy of singly ionized helium ions into an electric potential by the process of direct conversion. The experiments in this paper aimed to achieve higher potentials and higher efficiencies than ever before. The predicted maximum potential that could be produced by the 150 kV accelerator at the Texas A&M Ion Beam Lab was 150 kV, which was achieved with 92% collection efficiency. Also, an investigation into factors affecting collection efficiency was made. It was concluded that charge was being lost due to charge exchange occurring near the surface of the target which caused positive target atoms to be ejected from the face and accelerated away. Introducing a wire mesh near the face of the target with an electric potential, positive or negative, which aimed to control secondary ion emissions, did not have an effect on the collection efficiency of the system. Also, it was found that the gas pressure inside the chamber did not have an effect on the collection efficiency. The goal of achieving higher electric potentials and higher efficiencies than previous direct conversion work was met.
146

Development and characterization of Mantle Cell Lymphoma specific IgGs

Gärdefors, Katarina January 2008 (has links)
<p>Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.</p>
147

A l'échelle des mots l'écriture théâtrale brève en France (1980-2007) /

Koutchevsky, Alexandre Plassard, Didier January 2009 (has links)
Thèse de doctorat : Etudes théâtrales : Rennes 2 : 2009.
148

Development and Optimization of Kinetic Target-Guided Synthesis Approaches Targeting Protein-Protein Interactions of the Bcl-2 Family

Kulkarni, Sameer Shamrao 01 January 2012 (has links)
Kinetic target-guided synthesis (TGS) and in situ click chemistry are among unconventional discovery strategies having the potential to streamline the development of protein-protein interaction modulators (PPIMs). In kinetic TGS and in situ click chemistry, the target is directly involved in the assembly of its own potent, bidentate ligand from a pool of reactive fragments. Herein, we report the use and validation of kinetic TGS based on the sulfo-click reaction between thio acids and sulfonyl azides as a screening and synthesis platform for the identification of high-quality PPIMs. Starting from a randomly designed library consisting of nine thio acids and nine sulfonyl azides leading to eighty one potential acylsulfonamides, the target protein, Bcl-XL selectively assembled four PPIMs, acylsulfonamides SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5, which have been shown to modulate Bcl-XL/BH3 interactions. To further investigate the Bcl-XL templation effect, control experiments were carried out using two mutants of Bcl-XL. In one mutant, phenylalanine Phe131 and aspartic acid Asp133, which are critical for the BH3 domain binding, have been substituted by alanines, while arginine Arg139, a residue identified to play a crucial role in the binding of ABT-737, a BH3 mimetic, has been replaced by an alanine in the other mutant. Incubation of these mutants with the reactive fragments and subsequent LC/MS-SIM analysis confirmed that these building block combinations yield the corresponding acylsulfonamides at the BH3 binding site, the actual "hot spot" of Bcl-XL. These results validate kinetic TGS using the sulfo-click reaction as a valuable tool for the straightforward identification of high-quality PPIMs. Protein-protein interactions of the Bcl-2 family have been extensively investigated and the anti-apoptotic proteins (Bcl-2, Bcl-XL, and Mcl-1) have been validated as crucial targets for the discovery of potential anti-cancer agents. At the outset, Bcl-2 and Bcl-XL were considered to play an important role in the regulation of apoptosis. Accordingly, several small molecule inhibitors targeting Bcl-2 and/or Bcl-XL proteins were primarily designed. A series of acylsulfonamides targeting these proteins were reported by Abbott laboratories, ABT-737 and ABT-263 being the most potent candidates. Remarkably, these molecules were found to exhibit weaker binding affinities against Mcl-1, another anti-apoptotic protein. Further experimental evidence suggests that, inhibitors targeting Mcl-1 selectively or in combination with other anti-apoptotic proteins would lead to desired therapeutic effect. As a result, numerous small molecules displaying activity against Mcl-1 have been identified so far. Specifically, acylsulfonamides derived from structure activity relationship by interligand nuclear overhauser effect (SAR by ILOEs), a fragment-based approach, have been recently reported with binding affinities in the nanomolar range. In the meantime, we have reported that the kinetic TGS approach can also be applied to identify acylsulfonamides as PPIMs targeting Bcl-XL. Taken together, structurally novel acylsulfonamides can be potentially discovered as Mcl-1 inhibitors using the kinetic TGS approach. Thus, a library of thirty one sulfonyl azides and ten thio acids providing three hundred and ten potential products was screened against Mcl-1 and the kinetic TGS hits were identified. Subsequently, control experiments involving Bim BH3 peptide were conducted to confirm that the fragments are assembled at the binding site of the protein. The kinetic TGS hits were then synthesized and subjected to the fluorescence polarization assay. Gratifyingly, activities in single digit micromolar range were detected, demonstrating that the sulfo-click kinetic TGS approach can also be used for screening and identification of acylsulfonamides as PPIMs targeting Mcl-1. The amide bond serves as one of nature's most fundamental functional group and is observed in a large number of organic and biological molecules. Traditionally, the amide functionality is introduced in a molecule through coupling of an amine and an activated carboxylic acid. Recently, various alternative methods have been reported wherein, the aldehydes or alcohols are oxidized using transition metal catalysts and are treated with amines to transform into the corresponding amides. These transformations however, require specially designed catalysts, long reaction times and high temperatures. We herein describe a practical and efficient amidation reaction involving aromatic aldehydes and various azides under mild basic conditions. A broad spectrum of functional groups was tolerated, demonstrating the scope of the reaction. Consequently, the amides were synthesized in moderate to excellent yields, presenting an attractive alternative to the currently available synthetic methods.
149

Using in situ click chemistry to modulate protein-protein interactions: Bcl-XL as a case study

Malmgren, Lisa M 01 June 2007 (has links)
Protein-protein interactions are central to most biological processes. Although in the field of drug discovery there is a great interest in targeting protein-protein interactions, the discovery and development of small-molecules, which effect these interactions has been challenging. The purpose of this project is to determine if in situ click chemistry is a practical approach towards testing whether Bcl-XL is capable of assembling it's own inhibitory compounds. Abbott laboratories developed compound ABT-737, which binds with high affinity (Ki < 1 nM) to the binding sites of Bcl-XL.³⁶ Based on ABT-737, two acetylene anchor molecules AM3 and AM4 have been synthesized. These anchor molecules are distinguished by the reactivity of the their carbon-carbon triple bond. Compound AM3 contains an electron withdrawing carbonyl in the alpha-position to the acetylene resulting in an activating effect towards the [1,3]-dipolar cycloaddition compared to compound AM4. To determine the reactivity of the activated system, ¹ H-NMR kinetic studies were performed to compare the relative rates of these two systems by reacting model alkynes 1,2,3, and 4 with azide AZ7. It was shown that the activated systems, 1 and 3, produce triazoles in an accelerated rate compared to the unactivated systems 2 and 3. To test for the self-assembly of inhibitory triazoles, the acetylenes AM3 and AM4 were incubated with Bcl-XL and 14 azide building blocks (AZ1-AZ12) for 12 hours at 37 degrees C. Subjecting these mixtures to LC/MS-SIM led to the discovery of two hit compounds, 35 and 36, of which 35 has been chemically synthesized confirming the hit. Future work includes the synthesis of all hit compounds. Since hit triazoles can be syn or anti, both need to be synthesized for each hit to investigate which regioisomer Bcl-XL generates. Tests to confirm if hit compounds are actually modulating Bcl-XL activity will be done using conventional bio-assays. This will validate that Bcl-XL is capable of assembling its own inhibitor via the in situ click chemistry approach to drug discovery.
150

Studies on Application of Silyl Groups in Ring-Closing Metathesis Reactions and Fragment-Based Probe Discovery

Wang, Yikai 19 December 2012 (has links)
In efforts to search for tool compounds that are capable of probing normal and disease-associated biological processes, both quality and identity of the screening collection are very important. Towards this goal, diversity-oriented synthesis (DOS) has been explored for a decade, which aims to populate the chemical space with diverse sets of small molecules distinct from the traditional ones obtained via combinatorial chemistry. In the practice of DOS, macrocyclic ring-closing metathesis (RCM) reactions have been widely used. However, the prediction and control of stereoselectivity of the reaction is often challenging; chemical transformation of the olefin moiety within the product is in general limited. Chapter I of this thesis describes a methodology that addresses both problems simultaneously and thus extends the utility of the RCM reactions. By installing a silyl group at the internal position of one of the olefin termini, the RCM reaction could proceed with high stereoselectivity to afford the (E)-alkenylsiloxane regardless of the intrinsic selectivity of the substrate. The resulting alkenylsiloxane can be transformed to a variety of functionalities in a regiospecific fashion. The conversion of the (E)-alkenylsiloxanes to alkenyl bromides could proceed with inversion of stereochemistry for some substrates allowing the selective access of both the E- and Z-trisubstituted macrocyclic alkenes. It was also found that the silyl group could trap the desired mono-cyclized product by suppressing nonproductive pathways. Chapter II of this thesis describes the application of the concept of DOS in the area of fragment-based drug discovery. Most fragment libraries used to date have been limited to aromatic heterocycles with an underrepresentation of chiral, enantiopure, \(sp^3\)-rich compounds. In order to create a more diverse fragment collection, the build/couple/pair algorithm was adopted. Starting from proline derivatives, a series of bicyclic compounds were obtained with complete sets of stereoisomers and high \(sp^3\) ratio. Efforts are also described toward the generation of diverse fragments using methodology described in Chapter I. The glycogen synthase kinase \((GSK3\beta)\) was selected as the proof-of-concept target for screening the DOS fragments. / Chemistry and Chemical Biology

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