The dynamic rearrangements of RNA and protein complexes and the fidelity of pre-mRNA splicing are governed by DExD/H-box ATPases. One of the spliceosomal ATPases, Brr2, is believed to facilitate conformational rearrangements during spliceosome activation and disassembly. It features an unusual architecture, with two consecutive helicase-cassettes, each comprising a helicase and a Sec63 domain. Only the N-terminal cassette exhibits catalytic activity. By contrast, the C-terminal half of Brr2 engages in protein interactions. Amongst interacting proteins are the Prp2 and Prp16 helicases. The work presented in this thesis aimed at studying and assigning functional relevance to the bipartite architecture of Brr2 and addressed the following questions: (1) What role does the catalytically inert C-terminal half play in Brr2 function, and why does it interact with other RNA helicases? (2) Which RNAs interact with the different parts of Brr2? (1) In a yeast two-hybrid screen novel brr2 mutant alleles were identified by virtue of abnormal protein interactions with Prp2 and Prp16. Phenotypic characterization showed that brr2 C-terminus mutants exhibit a splicing defect, demonstrating that an intact C-terminus is required for Brr2 function. ATPase/helicase deficient prp16 mutants suppress the interaction defect of brr2 alleles, possibly indicating an involvement of the Brr2 C-terminus in the regulation of interacting helicases. (2) Brr2-RNA interactions were identified by the CRAC approach (in vivo Crosslinking and analysis of cDNA). Physical separation of the N-terminal and C-terminal portions and their individual analyses indicate that only the N-terminus of Brr2 interacts with RNA. Brr2 cross-links in the U4 and U6 snRNAs suggest a step-wise dissociation of the U4/U6 duplex during catalytic activation of the spliceosome. Newly identified Brr2 cross-links in the U5 snRNA and in pre-mRNAs close to 3’ splice sites are supported by genetic analyses. A reduction of second step efficiency upon combining brr2 and U5 mutations suggests an involvement of Brr2 in the second step of splicing. An approach now described as CLASH (Cross-linking, Ligation and Sequencing of Hybrids) identified Brr2 associated chimeric sequencing reads. The inspection of chimeric U2-U2 sequences suggests a revised secondary structure for the U2 snRNA, which was confirmed by phylogenentic and mutational analyses. Taken together these findings underscore the functional distinction of the N- and C-terminal portions of Brr2 and add mechanistic relevance to its bipartite architecture. The catalytically active N-terminal helicase-cassette is required to establish RNA interactions and to provide helicase activity. Conversely, the C-terminal helicase-cassette functions solely as protein interaction domain, possibly exerting regulation on the activities of interacting helicases and Brr2 itself.

Recent advances in technology have led to the determination of numerous notable structures of membrane proteins. While they provide valuable information about the structure of membrane proteins these studies often provide static images with potentially limited dynamics, and structural determination often requires truncation of flexible regions, and often utilizes bacterial homologs given the need for stable, heterologous overexpression. In order to better understand allostery at a molecular level, state-dependent crosslinking studies coupled with multidimensional mass spectrometry (MS) were conducted on glycine receptor (GlyR) stabilized in different allosteric states. Predominant allosteric states were stabilized using wild type or mutated receptor in the presence of selected ligands: resting (no ligand), desensitized (saturating glycine) and open state (non-desensitizing ivermectin (IVM)-gated F207A/A288G GlyR). Photo-crosslinking methodology linked with mass spectrometric analysis was developed on systematically generated single Cys mutations in GlyR with both Cys null and IVM sensitive backgrounds to enable the study of state-dependent structures of GlyR in comparative crosslinking studies. Studies were conducted on A41C and H419C mutants. A41 is shown to be in proximity to the pre-M1 and the M2-M3 loop region crucial for gating. Prior to these studies, very little information on H419 was available as it is located in C-terminal tail of the receptor that is often truncated in structural studies conducted on other related pentameric ligand-gated ion channels. These studies identified specific GlyR crosslinks unique to each conformational state and identified potential motions in the receptor upon gating and desensitization. The defined distance constraints will be used to update our model of human α1-GlyR and provide insight into channel function. Significantly, this methodological approach is amenable to study any allosteric protein and complement other high resolution structural studies in identifying protein dynamics. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry / PhD; / Dissertation;

Ubiquitin (Ub) is a small, 76 amino acid, and post-translational modification (PTM) protein in eukaryotes. Modification of a substrate protein via the covalent attachment of the C-terminal glycine of Ub to the ε-amino group of lysine residues in a substrate is termed ubiquitination. Unlike, other PTM proteins, Ub can form polyUb chains at one or more of its seven lysine residues. (K6, K11, K27, K29, K33, K48, and K68). The consequence of these different polymerization sites is altered biological response with different polyUb linkages conferring different fates to target proteins. Unfortunately, the study of these chains have been limited by the inability to generate homogeneous polyUbs chains linked at known lysine residues. Furthermore, a three step enzymatic cascade consisting of activating-enzymes (E1s), conjugating enzymes (E2s), and ligase enzymes (E3s) tightly controls this modification. In response, our laboratory has developed a system that creates polyUb chains through bacterial expression and "synthetic" building blocks. Now, the main questions are what do these chains interact with in the cell and how do these interactions mediate biological responses? In an attempt to answer these questions, this dissertation looks at different molecular techniques created to capture the transient interactions of monoUb and diUb probes with Ub substrates, such as, ubiquitin binding domains (UBDs) and conjugating E2 enzymes. One molecular technique focuses on the use of incorporating a genetically encoded, photo-crosslinker, p-Benzoyl-L-phenylalanine (pBpa) into diUb probes to capture their interaction with UBDs. This sets the foundation for understanding Ub’s cellular signaling recognition of UBDs. Another technique is creating diUb probes that contain lysine derivatives, Nε-L-Thiaprolyl-L-lysine (ThzK) or Nε-L-Cysteinyl-L-lysine (CysK), and can form a disulfide bonds with E2 enzymes to capture their complex, opening an opportunity to understand mechanistically the role E2 enzymes have with polyUb chain formation. Herein, these techniques are established to help unravel the complexity of Ub signaling.

Consumption of plastics has increased exponentially, in line with the world's population. Not surprisingly this is reflected in enormous growth of the plastic industry especially during the last five decades. Commensurate with this, waste produced from plastics consumption has created a major environmental problem. Many types of waste disposal methods have been used all over the world so far, but all of them have disadvantages. Furthermore, some methods are responsible for the generation of green house gases and further contribution to global warming. Recently, reduction of green house gas emission has become a target of most industries. Plastic recycling and reuse breaks the cycle of endless production of virgin polymer and thus contributes to a net reduction of green house gas emission. Recycling of plastics should produce materials with improved properties to replace virgin plastics for a variety of applications. Improvement in the properties of recycled plastics can be achieved by blending with other plastics, by filler addition and by modification using free radical initiators. Introduction of the free radical initiator (organic peroxide) during reprocessing of the recycled plastics has been found to offer significant property improvements to the recycled materials. Extremely small amounts of a free radical initiator (typically ranging between 0.01 wt% to 0.2 wt%) is capable of enhancing the properties of the recycled plastics to a great extent. This project investigates the use of free radical initiators in the recycling of post consumer recycled high density polyethylene using reactive extrusion. Both molecular and rheological characterisation of recycled and reprocessed materials was carried out and this was followed by tensile testing of the modified materials to satisfy end use applications such as packaging and drainage piping. Post consumer recycled high density polyethylene (R-HDPE) resin and virgin high density polyethylene (V-HDPE) were reactively extruded with low concentrations of dicumyl peroxide (DCP) and 1, 3 1, 4 Bis (tert- butylperoxyisopropyl) Benzene (OP2) respectively in a twin screw extruder in order to produce modified materials with varying composition (0.0 wt%, 0.02 wt%, 0.05 wt%, 0.07 wt%, 0.10 wt% and 0.15 wt%) of both organic peroxides. Morphological characterisation using modulated differential scanning calorimetry (MDSC) demonstrated that there is a decrease in the crystallinity level for all the modified samples. Shear rheological tests were carried out to study the structure of the modified materials within the linear viscoelastic region. Viscoelastic parameters, such as storage modulus (G'), loss modulus (G

The effects of crosslinking on EVA foaming are studied in this thesis. A fundamental approach was applied to describe the influences of crosslinking on EVA/gas viscosities, gas solubility and diffusivity in EVA, EVA foaming nucleation and early stage of bubble growth, which leads to a better understanding of the plastic foaming mechanism. Although crosslinked polyolefin foaming technology has been well applied in industry, more fundamental and thorough studies are demanded to understand the mechanism, which can serve to improve the present technology. The shear and extensional viscosities have been measured for the chemically crosslinked EVA with dissolved gas which could not be found from literature. Furthermore, by controlling the crosslinking agent amount, the polymer melt strength/viscosity can be controlled, so as to obtain optimum foam morphology. The crosslinking also has effects on the diffusivity and solubility of a blowing agent inside EVA. The solubility and the diffusivity of the blowing agent in the EVA decrease with the crosslinking degree increases. The diffusivity decrease makes more gas is utilized for the foaming rather than leak out of the polymer matrix quickly. This thesis also presents the fundamental studies on the effects of crosslinking on cell nucleation and early bubble growth. Theoretical work and in-situ visualization experimental results indicate that partial crosslinking leads to higher cell nucleation density and slower bubble growth, both of which benefit a fine-cell foam morphology generation. Last but not least, an optimized foaming process was conducted to produce chemically crosslinked EVA foams with large expansion ratios in a batch system, using a chemical blowing agent. The results determine that an optimal crosslinking degree is critical for the crosslinked EVA foaming with maximum expansion ratio. Furthermore, all research results not only benefit the foaming of crosslinked EVA, but also serve the better production of other crosslinked polyolefin foams.

The effects of crosslinking on EVA foaming are studied in this thesis. A fundamental approach was applied to describe the influences of crosslinking on EVA/gas viscosities, gas solubility and diffusivity in EVA, EVA foaming nucleation and early stage of bubble growth, which leads to a better understanding of the plastic foaming mechanism. Although crosslinked polyolefin foaming technology has been well applied in industry, more fundamental and thorough studies are demanded to understand the mechanism, which can serve to improve the present technology. The shear and extensional viscosities have been measured for the chemically crosslinked EVA with dissolved gas which could not be found from literature. Furthermore, by controlling the crosslinking agent amount, the polymer melt strength/viscosity can be controlled, so as to obtain optimum foam morphology. The crosslinking also has effects on the diffusivity and solubility of a blowing agent inside EVA. The solubility and the diffusivity of the blowing agent in the EVA decrease with the crosslinking degree increases. The diffusivity decrease makes more gas is utilized for the foaming rather than leak out of the polymer matrix quickly. This thesis also presents the fundamental studies on the effects of crosslinking on cell nucleation and early bubble growth. Theoretical work and in-situ visualization experimental results indicate that partial crosslinking leads to higher cell nucleation density and slower bubble growth, both of which benefit a fine-cell foam morphology generation. Last but not least, an optimized foaming process was conducted to produce chemically crosslinked EVA foams with large expansion ratios in a batch system, using a chemical blowing agent. The results determine that an optimal crosslinking degree is critical for the crosslinked EVA foaming with maximum expansion ratio. Furthermore, all research results not only benefit the foaming of crosslinked EVA, but also serve the better production of other crosslinked polyolefin foams.

Polyfluorene (PF) and its derivates as well-known fluorescent materials are promising materials in optoelectronic applications due to their high quantum yields in the solid state. Nevertheless, the easy chain inter-action in PFs to result in the unfavorable associations (aggregate and excimer) are generally considered to be detrimental to the emission efficiency in the concentrated solid state and/or at high temperatures. In the study, restraining the extent of associations is therefore by embedding fluorene-based alternative and block copolymers in crosslinked network as matrix. Firstly, alternative copolymers with fluorene connected by anthracene (or pyridine or fluorene) ring were prepared through Suzuki coupling. In this way, the steric hindrance between the o-hydrogens in the neighboring aromatic ring causes the twisting of the constructed polymer chain and the resulting twisting chain conformation keeps the polymer chains from the unfavorable inter-chain interactions and reduces the extent of the association. Secondly, the alternative fluorene-anthracene copolymer (a-PFA) from the first approach can be further chemically formulated to obtain a triblock copolymer with the central a-PFA rod block connected by two flexible poly(methyl methacrylate) (PMMA) segments. In this way, the two flexible PMMA chains serve as spatial isolator to keep the central PFA rod from approaching each other and a reduced extent of association is expected for this block polymer of b-PMMA-PFA. Thirdly, all the alternative and block copolymers cited above were immersed in the curable liquid methyl metharcylate (MMA)/ditrimethylolproanetetracrylate (DTTPT) monomer mixtures and photo-irradiated to obtain composites with the fluorescent polymers immersed in the cured crosslinked network. The chain morphology and thus the degree of associations will be successfully frozen by the immobilized crosslinked network. For systems before and after photo-irradiation, the degree of aggregation was evaluated by Uv-vis absorption, photoluminescent (PL) and PL excitation spectroscopy. Polymer concentration was found to be important factor in controlling the degree of aggregation and was discussed in this study. In addition, the cured solid composites after high-temperature annealing were studied and compared with pure PFs to evaluate the effectiveness of this crosslinking strategy in restraining the extent of aggregation. In most cases, quantum yields (£XPLs) also were measured to evaluate the effectiveness of this strategy.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 19, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. Xudong Fan and Dr. Azlin Mustapha. Includes bibliographical references.

To understand the chemistry of life processes in detail is largely a challenge of resolving them in their native, cellular environment. Cell culture, first developed a century ago, has proven to be an essential tool for reductionist studies of cellular biochemistry and development. However, for the technology of cell culture to move forward and address increasingly complex problems, in vitro environments must be refined to better reflect the cellular environment in vivo. This dissertation work has focused on the development of methods to define cellular microenvironments using the high resolution, 3D capabilities of multiphoton lithography. Here, site-specific photochemistry using multiphoton excitation is applied to the photocrosslinking of proteins, providing the means to organize bioactive species into well-defined 3D microenvironments. Further, conditions have been identified that enable microfabrication to be performed in the presence of cells -- allowing cell outgrowth and motility to be directed in real time. In addition to the intrinsic chemical functionality of microfabricated protein structures, 3D protein matrices are shown to respond mechanically to changes in the chemical environment, enabling new avenues for micro-scale actuation to be explored. Complex 2D and 3D protein photocrosslinking is further facilitated by integrating transparency and automated reflectance photomasks into the fabrication system. These advances could be transformative in efforts to fabricate precise cellular scaffolding that replicates the morphological (and potentially biochemical) features of in vivo tissue microenvironments. Finally, these methods are applied to the study of microorganism behavior with single-cell resolution. Microarchitectures are designed that allow the position and motion of motile bacterial to generate directional microfluidic flow -- providing a foundation to develop micro-scale devices powered by cells. / text

Recent improvements in mass spectrometry instrumentation have stimulated the fusion of this technology with protein crosslinking to advance the structural proteomics field. However, analysis of complex datasets from crosslinking experiments remains a bottleneck. The majority of crosslinking studies for structural characterization of protein- protein interactions have been conducted with reagents specific for discrete amino acids. While this approach simplifies data analysis, the requirement for specific functionalities to be present at the interaction interface limits resolution. Herein, we report the application of stapled peptides for the development of photoaffinity reagents for mass spectrometric characterization of BCL-2 family protein interactions. To validate this approach, we synthesized photoreactive stabilized alpha-helices of BH3 domains (pSAHBs) incorporating a benzophenone containing amino acid, and demonstrated that the photo crosslinking specificity of these reagents paralleled the interaction specificity of the native proteins. We show that the standard SEQUEST algorithm is effective at identifying specific amino acids crosslinked by pSAHBs, and that this information can be used to create distance restraints for characterizing interaction interfaces by in silico docking. The pSAHB approach is applied to characterize previously elusive activating interactions between BH3 domains and the proapoptotic proteins BAX and BAK. We demonstrate that full-length BAK requires a direct activation stimulus, and that this involves interaction at a canonical surface groove at the C-terminal face of BAK. We confirmed that initiation of direct BAX activation occurs at a geographically distinct site at the N-terminal face of BAX, but further find that induced release of its C-terminus from the canonical groove exposes these residues for secondary BH3 interaction. These data suggest that BAX may be subject to a two-step activation mechanism within distinct cytosolic and mitochondrial compartments. Finally, we report the structural characterization of an interaction between BAD and glucokinase, the first description of a BH3 domain interaction with a non-BCL-2 family member. We identify the active site region of glucokinase as the BAD interaction site, establishing this region as a novel target for development of glucokinase activators. We conclude that the pSAHB approach represents a rapid and powerful approach to protein interaction site identification that complements conventional structural biology techniques.