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Sulfur-substituted purine and pyrimidine nucleobases—also known as thiobases—are among the world’s leading prescriptions for chemotherapy and immunosuppression. Long-term treatment with some of the purine derivatives of these drugs has recently been correlated with the photo-induced formation of carcinomas. Establishing an in-depth understanding of the photochemical properties of these thiobase drugs may provide a route to overcoming these carcinogenic side effects, or, alternatively, may provide a basis for developing highly-effective compounds for targeted photochemotherapy. In this thesis work, a broad investigation is undertaken, surveying the excited-state dynamics and photochemical reactions of nearly every sulfur-substituted analog of the canonical DNA and RNA nucleobases. The thiobase derivatives are investigated using time-resolved absorption and emission spectroscopies in the femtosecond (10-15 s) to microsecond (10-6 s) time window. Coupling these experiments with quantum chemical calculations, we have developed a molecular-level understanding of how sulfur-substitution so drastically perturbs the photochemical properties of the nucleobases. The structure-property relationships established by this work demonstrate the impact of site-specific sulfur substitution on the population and reaction dynamics of the excited triplet state. Some of the most photoreactive derivatives identified are applied to human epidermoid carcinoma cells and shown to effectively decrease their proliferation upon exposure to a low dose of light. The results presented in this body of work demonstrate the utility of fundamental photochemical investigations for driving the development of next-generation photochemotherapeutics, while simultaneously elucidating overarching principles for the impact of sulfur substitution (thionation) on the photochemical properties of organic chromophores.

In the treatment of severe traumatic brain injury (TBI), a two-stage surgical intervention is routinely performed. In the first surgery, decompressive craniectomy (DC) is performed to remove a large portion of calvarial bone to allow unimpeded brain swelling. A second surgery termed cranioplasty, usually performed weeks to months later, is then employed to rebuild the cranium. Hydrogels have the potential to revolutionize TBI treatment by permitting a single-stage surgical intervention, capable of exhibiting paste-like handling properties in the pre-crosslinked form for in situ placement to fill any size or shape of defect, remaining pliable during brain swelling, and tuned to regenerate bone after swelling has subsided. The motive of the current dissertation was to achieve the first step in designing a single-stage surgical intervention for treatment of TBI following DC, being the design of a hydrogel capable of regenerating bone within a critical size calvarial defect. The current dissertation first evaluated the use of colloidal hydrogels of hyaluronic acid, hydroxyapatite, and extracellular matrix (ECM) materials demonstrating promise for decellularized cartilage as a material for bone regenerative medicine. A next-generation biomaterial was then developed utilizing a methacrylated solubilized decellularized cartilage hydrogel encapsulating osteoconductive particles, the results of which demonstrated diminished bone regeneration that was speculated to be due to cartilage processing (i.e., solubilization). In the final study, pentenoate-functionalized hyaluronic acid (PHA) hydrogels encapsulating devitalized ECM materials were evaluated and I found that PHA hydrogels encapsulating devitalized tendon (DVT) particles successfully regenerated bone in a critical size calvarial defect. Future studies will be conducted to test the use of the PHA-DVT hydrogel in a rat TBI model to evaluate the use of hydrogels in the treatment of TBI following DC. Ultimately, the current dissertation work successfully developed a hydrogel material that exhibits desirable handling properties in the pre-crosslinked form for surgical placement and adequate bone regeneration after 8 weeks of in vivo implantation, with promising future applications to bone regeneration and application to treatment of TBI.

Proteins comprise the majority of the cell and are vital to all cellular functioning. Protein-protein interactions (PPIs) are the communication networks behind cellular processes, often functioning in machine-like complexes with exchangeable subunits or parts to convey different messages. PPIs exhibit a wide range of structural features, surface areas, and affinities with some displaying dynamic interfaces allowing multiple binding partners to interact depending on cellular conditions. This makes some PPIs more difficult to study than others. Understanding these PPIs and exploring larger PPI networks has been a challenge without considering the cellular context in which they belong. Methods to study difficult PPIs in their native environments have thus been instrumental advancing the field. The predominant theme of this work is the demonstration of the utility of genetically incorporated photolabile unnatural amino acids for the study of the difficult PPIs between transcriptional activator-coactivator complexes. Covalent chemical capture of protein binding partners in live cells is combined with mass spectrometry to discover novel PPIs and further expanded to include new ways to visualize direct PPIs on DNA. Caveats to the covalent capture method are also explored with an illustration of capture efficiencies of two common photolabile groups across various PPI binding affinities and surface areas. The work presented here displays a thorough examination of the use and application of chemical capture for the study of PPIs in a cellular context. The methods established within this work add to the foundation for the study of difficult PPIs and demonstrates the ability to understand new networks of low affinity, dynamic interactions. The presentation of novel binding partners for the well-studied transcriptional activator, Gal4, expands traditional beliefs on transcriptional activator participation in binding dynamic complexes as well as highlights the potential of these PPIs for later therapeutic points of intervention. In addition, the groundwork for guidelines on using covalent chemical capture in various PPIs was established which, when completed, will enable not only easier use but also hopefully lead to the ability to tailor selection of a photocrosslinker based on the specific PPIs under study.

The development of high-efficiency nonlinear optical (NLO) polymers has opened up many opportunities in the field of electro-optics. However, current NLO polymers do not meet stability requirements for semiconductor integration. In an effort to improve this, we examined the effects of crosslinking following electric field poling. A series of photocrosslinkable polymers bearing side chain chromophores was synthesized, poled and evaluated on the basis of the thermal stability of Second Harmonic Generation. Photoinitiation allowed for control of the onset of curing. Crosslinking was monitored by FTIR and optimal conversion was achieved by applying a slow temperature ramp during exposure. The ultimate stability of the poled polymers was directly related to the number of crosslinking substituents attached to the chromophore pendant group. With two reactive groups per chromophore significant SHG was retained at temperatures beyond the polymer Tg. In integrated circuit packaging there is a need for directly-patternable polymers of low dielectric constant. Bridging the gap between the high-value silicon chip and circuit board is a substrate comprising alternating layers of metal conductor and polymer dielectric. PMDA-ODA, an aromatic polyimide, meets many of the requirements for integration and can be patterned using a photobase generator (PBG). Due to absorbance by the PMDA-ODA precursor, this PBG must have activity at visible wavelengths. Several oxime urethanes were synthesized and evaluated as candidate long wavelength PBG. These compounds exhibit clean photochemistry and high visible light sensitivity. Unfortunately, carbamate thermal stability is insufficient for patterning PMDA-ODA. For improved material properties, PMDA-TFMB, a fluorinated polyimide, was also evaluated. Importantly, the polymer precursor is sufficiently transparent to employ thermally-stable near-UV photobases. With photobase, 2.5 micron features were resolved in PMDA-TFMB. An ancillary benefit of this methodology is reduced cure temperature (~200 °C), a traditional drawback of polyimides. This material demonstrates a dielectric constant near 3 and a thermal expansion coefficient (CTE) of approximately 6 ppm/°C in-plane. Through-plane thermal expansion is somewhat problematic, with a CTE of approximately 160 ppm/°C, and will likely require a nanoparticle composite strategy. However, this combination of material and lithographic properties make PMDA-TFMB a promising candidate for this application.

Dissecting the Mechanisms of Direct Activation for Proapoptotic BAK and BAX

Signaling lipids such as diacylglycerol (DAG) and the phosphatidylinositol polyphosphates (PIPns) play crucial roles in numerous cellular pathways. However, characterization of their activities is hindered by the complexity of associated signaling pathways and of the membrane environment.  To address this issue, we have developed lipid probes that are effective for characterizing biological events using different applications, including activity-based probing (PIPns and DAG) and microarray analysis (PIPns). The activity-based probes have been applied to label receptor targets in multiple cancer cell proteomes through photocrosslinking followed by click reactions. The probes were found to label several proteins, as judged by on-gel fluorescence, and labeling was abrogated through various controls, such as heat denaturation and competition. Proteomic studies have been successfully performed to identify protein targets through biotin enrichment followed by mass spectrometric analysis.  For microarray analysis, functionalized PIPn probes were synthesized and applied to develop a high throughput microarray analysis to measure protein-lipid binding affinity. These approaches will be invaluable for characterizing PIPn/DAG-regulated events and their involvement in disease. The design, synthesis and application of these lipid probes are included in this dissertation.  In addition, the design and synthesis of other lipid probes are discussed, such as bis(monoacylglycero)phosphate (BMP), and lysophophatidylcholine (LPC) analogs.

A complete understanding of protein synthesis requires description of the cyclical motions and interactions that occur in ribosomal RNA and transfer RNA during translation. The purpose of the following research was to gain understanding of the nature of these motions and interactions, and to develop new tools to measure how ligands alter the conformational flexibility of RNA during the steps of initiation, elongation and termination. Different tRNA substrates were bound to the E. coli 70S to simulate the arrangement of the tRNA-ribosomal complex before and after peptide bond formation, and different UV-induced 16S rRNA-tRNA photocrosslinks were produced in these complexes, illustrating that the 16S rRNA P-site undergoes local deformations during elongation. A statistical study was undertaken to understand the nature of conformational states in the 30S ribosomal subunit. Using the lists of observed UVB/C- and UVA-s4U-induced crosslinks and the T. thermophilus 30S X-ray crystal structure, frequencies were compared to a number of geometrical parameters demonstrating that crosslink formation requires substantial RNA motions. In addition, the results show that the restricted pattern of crosslink formation in E. coli 16S rRNA is due to the overall rigidity of the 30S subunit outside of the active site. One consequence of these conclusions is that potocrosslinking rates depend on the ease of inter-nucleotide conformational movements. This was exploited in a study that used the temperature response of the rate constant for the UVA-induced photo-crosslink between s4U8 X C13 in E. coli tRNA to determine tRNA geometry and internal energy. The rate constants followed Arrhenius behavior in their dependence on temperature, and this allowed calculation of the activation energy associated with the conformational rearrangement necessary to bring the photoreactive bonds together. The experiments show that changes in the tRNA on ribosomes can be uncovered by photocrosslinking, that RNA mobility occurs by transient conformational changes, and describe a new technique than can quantitatively measure the internal energy associated with these conformational movements.

The Sec complex is the major translocase to mediate membrane protein insertion in bacteria, but there exists some proteins whose insertion is independent of the Sec complex. In year 2000, YidC was discovered that is essential for insertion of proteins into membranes in bacteria, especially for the Sec-independent proteins. Chapter 1 is an in-depth review about YidC in membrane protein assembly. In Chapter 2, we found YidC interacts with leader peptidase during its membrane insertion using crosslinking techniques. These data combined with the in vivo depletion study of YidC (James Samuelson’s dissertation, 2000) provide strong evidence to support YidC is a translocase component in bacteria. In Chapter 3, I report that the Sec-independent Pf3 coat protein requires YidC for insertion into the membrane. Using photocrosslinking techniques, we find that Pf3 coat interacts strongly with YidC only after its transmembrane segment is fully exposed outside the ribosome tunnel. Interaction between Pf3 coat and YidC occurs even when the proton motive force does not function on Pf3 coat. Our study demonstrates that YidC can directly interact with a Sec-independent membrane protein and its role is to fold the Pf3 protein into a transmembrane configuration. In Chapter 4, we found incorporation of site-specific protease sites into YidC results in YidC temperature-sensitive (ts) or cold-sensitive (cs) mutants. The YidC ts and cs strains were then constructed. The membrane insertion of the Sec-independent M13 procoat protein is inhibited in YidC ts strains when the cells were grown at 42 °C for 20 minutes. This provides the strongest evidence thus far that YidC plays a direct role in membrane protein insertion. Using the cs YidC strain, we find the insertion of the Sec-dependent leader peptidase is inhibited at 25 °C, whereas the insertion of M13 procoat is nearly normal. The cs YidC mutant shows a reduced interaction with the Sec machinery. These data suggest that the cold-sensitive YidC mutant is blocked in the Sec-related function, while its activity for inserting procoat is functioning almost normally. These properties of the cold-sensitive mutant strongly support the idea that YidC can function alone or with the Sec machinery.