Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Chemistry." Includes bibliographical references (p. 147-163).

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 105-106). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38818

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 133-138).

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-114). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 243-262).

Abstract Fluorescent molecules used as detection probes and sensors provide vital information about the chemical events in living cells. Despite the large variety of available fluorescent dyes, new improved fluorogenic systems are of continued interest. The Diaryl-substituted Maleimides (DMs) exhibit excellent photophysical properties but have remained unexplored in bioscience applications. Herein we present the identification and full spectroscopic characterization of 3,4-bis(2,4-difluorophenyl)-maleimide and its first reported use as a donor component in Forster resonance energy transfer (FRET) systems. The FRET technique is often used to visualize proteins and to investigate protein-protein interactions in vitro as well as in vivo. The analysis of the photophysical properties of 3,4-bis(2,4-difluorophenyl)-maleimide revealed a large Stokes shift of 140 nm in MeOH, a very good fluorescence quantum yield in DCM (Ffl 0.61), and a high extinction coefficient ε(340) 48,400 M-1cm-1, thus ranking this molecule as superior over other reported moieties from this class. In addition, 3,4-bis(2,4-difluorophenyl)-maleimide was utilized as a donor component in two FRET systems wherein different molecules were chosen as suitable acceptor components - a fluorescent quencher (DABCYL) and another compatible fluorophore, tetraphenylporphyrin (TPP). It has been demonstrated that by designing a FRET peptide which contains the DM donor moiety and the acceptor (quencher) motif, a depopulation of the donor excited state occurred via intermolecular FRET mechanism, provided that the pairs were in close proximity. The Forster-Radius (R0) calculated for this FRET system was 36 % and a Forster-Radius (R0) of 26 % was determined for the second FRET system which contained TPP as an acceptor. The excellent photophysical properties of this fluorophore reveal a great potential for further bioscience applications. The 3,4-bis(2,4-difluorophenyl)-maleimide fluorescent moiety was also implemented in an alternative application targeting the enzyme carbonic anhydrase (CAs) are metalloenzymes that regulate essential physiologic and physio-pathological processes in different tissues and cells, and modulation of their activities is an efficient path to treating a wide range of human diseases. Developing more selective CA fluorescent probes as imaging tools is of significant importance for the diagnosis and treatment of cancer related disorders. The kinetic TGS approach is an efficient and reliable lead discovery strategy in which the biological target of interest is directly involved in the selection and assembly of the fragments together to generate its own inhibitors. Herein, we investigated whether the in situ click chemistry approach can be implemented in the design of novel CA inhibitors from a library of non-sulfonamide containing scaffolds, which has not been reported in the literature. In addition, we exploit the incorporation of the (recently reported by us) fluorescent moiety 3,4-bis(2,4-difluorophenyl)-maleimide) as a potential biomarker with affinity to CA, as well as two coumaine derivatives representing a newly discovered class of inhibitors. The screening of a set of library with eight structurally diverse azides AZ1-AZ8 and fifteen functionalized alkynes AK1-AK12 led to the identification of 8 hit combinations among which the most prominent ones were those containing the coumarine and fluorescent maleimide scaffolds. The syn- and anti-tirazole hit combinations, AK1AZ2, AK1AZ3, AK4AZ2, and AK4AZ3 were synthesized, and in a regioisomer-assignment co-injection test it was determined that the enzyme favored the formation of the anti-triazoles for all identified combinations. The mechanism of inhibition of these triazoles was validated by incubating the alkyne/azide scaffolds in the presence of Apo-CA (non-Zn containing) enzyme. It was demonstrated that the Zn-bound water/hydroxide was needed in order to hydrolyze the coumarins which generated the actual inhibitor, the corresponding hydroxycinnamic acid. The time dependent nature of the inhibition activity typical for all coumarine-based inhibitors was also observed for the triazole compounds whose inhibition constants (Ki) were determined in two independent experiments with pre-incubation times of 3 and 25 minutes, respectively. It was observed that the lower Ki values were determined, the longer the pre-incubations lasted. Thus, a novel type of coumarin-containing triazoles were presented as in situ generated hits which have the potential to be used as fluorescent bio-markers or other drug discovery applications. The proteins from the Bcl-2 family proteins play a central role in the regualtion of normal cellular homeostasis and have been validated as a target for the development of anticancer agents. Herein, in a proof-of-concept study based on a previous kinetic TGS study targeting Bcl-XL, it was demonstrated that a multi-fragment kinetic TGS approach coupled with TQMS technology was successfully implemented in the identification of known protein-protein modulators. Optimized screening conditions utilizing a triple quadruple mass spectrometer in the Multiple Reaction Monitoring (MRM) mode was demonstrated to be very efficient in kinetic TGS hit identification increasing both the throughput and sensitivity of this approach. The multi-fragment incubation approach was studied in detail and it was concluded that 200 fragment combinations in one well is an optimal and practical number permitting good acylsulfonamide detectability. Subsequently, a structurally diverse liberty of forty five thio acids and thirty eight sulfonyl azides was screened in parallel against Mcl-1 and Bcl-XL, and several potential hit combinations were identified. A control testing was carried out by substituting Bcl-XL with a mutant R139ABcl-XL, used to confirm that the potential kinetic TGS hit combinations were actually forming at the protein's hot spot and not elsewhere on the protein surface. Although, the synthesis of all these kinetic TGS hit compounds is currently ongoing, preliminary testing of several acylsulfonamides indicate that they disrupt the Bcl-XL/Bim or Mcl-1/Bim interaction.

ARID proteins are highly conserved among eukaryotes and are involved in chromatin remodeling, differentiation and development. The founding member of the ARID3 subfamily, Bright/ARID3A, is an activator of the immunoglobulin heavy chain locus. Bright has been shown to immortalize mouse embryonic fibroblasts and induce malignant transformation when co-expressed with oncogenic Ras. A genomic locus that encodes a gene paralogous to Bright has been identified as Brightlike/ARID3C. In addition to the highly conserved ARID and REKLES domains, Brightlike contains a conserved sumoylation motif. Brightlike orthologous genes have been identified in all vertebrate genomes examined. Its absence from EST databases suggested that it is a rare transcript, and accordingly, its expression in adult mice appears to be restricted to spleen, testes and thymus. Brightlike is also regulated by alternative pre-mRNA splicing, differential subcellular distribution, and post-translational modification by SUMO. The two isoforms of Brightlike appear to have differential expression in lymphocyte populations. Brightlike and Bright bind to the same DNA motif. Unexpectedly, Bright and Brightlike do not form heterocomplexes on DNA nor compete for binding, suggesting they have independent functions in vivo. Brightlike increases the proliferation potential of mouse embryonic fibroblasts and rescues cells from premature senescence, suggestive of a proto-oncogene. / text

Unnatural amino acids (UAAs) have been used in bacteria and yeast to pinpoint protein binding sites, identify binding partners, PEGylate proteins site-specifically (vs. randomly), and attach small molecule fluorophores to proteins. The process of UAA incorporation involves the manipulation of the genetic code, which is established by the proper function of aminoacyl tRNA synthetases (RSs) and their cognate transfer RNAs (tRNAs). It has been discovered that certain regions of RS proteins can either block or enable cross-species reactivity of RSs. In essence, a bacterial RS can function with a human tRNA by transferring the human CP1 region to the bacterial RS, and vice versa. This knowledge has been used to engineer a tRNA capable of recognizing a stop codon (tRNA*), rather than an amino acid codon, and a cognate RS capable of recognizing only tRNA* and no endogenous tRNAs. We have previously described the use of this methodology to engineer a UAA incorporation system capable of amber stop codon suppression in HEK293T cells. Since UAAs are so useful, and their use has now been enabled in mammalian systems, we applied UAA incorporation to pluripotent cells. Stem and pluripotent cells have been the focus of cutting edge research for years, but much of the work done on these cell lines is done in the ignorance of basic biological processes underlying differentiation, dedifferentiation, and tumorigenesis. In order to facilitate the study of these basic biological processes and enable more adept manipulation of differentiation, dedifferentiation, and tumorigenesis, the development and use of two separate UAA incorporation systems is described herein. The overarching goal of this project is to facilitate the study of protein-protein interactions in stem and pluripotent cells. Since we have also previously described the development of a mammalian two-hybrid system, the use of that system in pluripotent cells is also described. / text

The organization of two or more biological macromolecules into a functioning assembly is critical for many biological functions to occur. This phenomenon is the result of subtle interplay between complimentary structural and electrostatic factors. While a growing protein data bank of solved protein structures provides experimental evidence for studying the structural factors that stabilize protein-protein interface, there has been little advance in experimental determination of the electrostatic contributions. This lack of experimental investigation into protein electrostatics results in an inability to describe or predict how protein-protein complexes are arranged and stabilized. This problem is addressed in this dissertation by use of vibrational Stark effect (VSE) spectroscopy in which the spectral transitions of a vibrational probe are directly related to the strength and direction of the electric fields in the vicinity of the probe. The work presented here details an approach using VSE spectroscopy coupled with molecular dynamics simulation (MD) to interpret the role that electrostatics play in organizing the signaling protein Ras' interactions with its downstream effectors Raf and Ral guanosine dissociation simulator (RalGDS). Each chapter describes a specific set of experiments and MD simulations designed to understand the nature of protein-protein interactions. In Chapter 3, changes in the absorption energy of the nitrile probe at nine positions along the Ras-Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface was found. The areas of conserved Stark shifts are used to help describe electrostatic factors that stabilize the Ras-Ral interface. In Chapter 4, VSE is used to describe an electrostatic origin to the binding tilt between complexes formed between Ras and its two effectors Raf and Ral. There are three regions of conserved Stark effect shifts upon docking with WT Ras between the two effectors, indicating that the docked complexes conserve electrostatic fields, resulting in different binding orientation of otherwise structurally similar proteins. Chapter 5 details the use of MD simulation in correlation with VSE data for 18 mutants of the Ras at the oncogenic position 61 site. The combination of experimental and simulations support the hypothesis that position 61 on Ras is used to coordinate an active site water molecule during native guanosine triphosphate (GTP) hydrolysis. / text

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy