A majority of biological microscopy investigations involve the focusing of visible light with conventional lenses. Fluorescence microscopy is one of the most widely used tools in biology but its resolution has historically suffered from the diffraction limit to about 200 nm laterally and 800 nm axially. In the past decade, this resolution problem has been overcome by the rapidly emerging field of super-resolution microscopy. The first demonstrated super-resolution technique, STimulated Emission Depletion (STED) Microscopy, is the topic of this Dissertation. This Dissertation has two primary areas of focus: the design optimization of a STED microscope, covered in Chapters 2-4, and its application to super-resolution imaging in cells and tissues, covered in Chapters 4-6. Chapter 2 describes the STED apparatus and experimental methods used. This chapter covers the guiding principles behind the design of a STED microscope, which forms a basis for understanding the logic underlying the homebuilt STED microscope which was constructed for this research. This STED microscope has a typical resolution of approximately 60 nm (full-width-at-half-maximum) or 25 nm (sigma) and has the sensitivity to image single fluorophores. In Chapter 3, a framework for evaluating and optimizing STED performance in the presence of several key tradeoffs is presented. Chapter 4 describes both developments in STED Microscopy required to utilize far-red-emitting dyes and the challenges associated with performing super-resolution imaging in intact Drosophila tissue. In Chapter 5, the optimization of labeling density revealed the 9-fold symmetry of a centriole protein structure, an important organelle in cell development. In Chapter 6, Huntingtin protein aggregates are resolved beyond the diffraction limit in a cell model of the neurodegenerative Huntington's disease.

The ability of organic chemists to design and synthesize functional molecules has revolutionized the ways in which we can probe biological systems. From catalysts capable of releasing bioactive molecules from bioinactive precursors to oligomers that enable or enhance the uptake of molecules across cell membrane and cell wall barriers, the work described herein emphasizes the power of designing for function. These new tools allow for control over the location of many biologically relevant molecules, including drugs, probes, imaging agents, metabolic modulators, and pesticides, with ramifications for a variety of biochemical, agricultural, and medicinal applications. Chapter 1 reviews the development of selective catalysts designed for use in biological systems. The catalysts have primarily been used for bioconjugations, expanding the classical repertoire of bioconjugation techniques to allow for selective chemistries at many more functional groups than was traditionally possible. Emerging strategies for imaging in biological systems using transition metal catalysis are also reviewed, as are transition metal-based catalytic therapeutics. The synthesis and evaluation of a novel bioorthogonal ruthenium catalyst designed for release of biologically active molecules from inactive precursors is the focus of Chapter 2. Although the challenges associated with using transition metals in biological systems are apparent, creative design ultimately led to the development of a useful bioorthogonal catalyst / substrate pair. This system allows for real-time visualization of transition metal catalysis to generate a biologically active compound that releases photons when it encounters its intracellular enzyme target. Chapters 3 and 4 detail the synthesis and application of molecular transporter scaffolds. Chapter 3 introduces a novel organocatalytic ring-opening oligomerization of guanidinylated cyclic carbonates to access molecular transporter scaffolds in 1 step. This strategy allows for rapid access to molecular transporters of varying lengths. In addition, the synthesis allows for concomitant probe or drug attachment and the carbonate-based backbones of the resulting transporters are biodegradable on a timescale allowing for cellular uptake and intracellular degradation. The ability of molecular transporters to cross not only cell membranes, but also cell walls, is discussed in Chapter 4. These studies focused on the delivery of small molecules and proteins across the algal cell wall and cell membrane barriers using D-octaarginine-based molecular transporters. With this method, it was shown that fluorescein-octaarginine conjugates were able to cross these barriers in a variety of algal species from the class Chlorophyceae, although several species showed no uptake or only cell surface staining. It was also shown in the algal model organism Chlamydomonas reinhardtii that octaarginine-protein conjugates could cross the cell wall and cell membrane barriers to deliver a functional protein to the intracellular algal space.

Biological barriers prevent the uptake of many potentially harmful xenobiotics yet also limit the delivery and cellular entry of a variety of drugs to their targets. One approach to this problem selects only those compounds with appropriate properties, namely solubility in polar biological fluids yet also able to pass through the nonpolar cellular membrane. This restriction would therefore preclude the use of many polar (e.g. siRNA) and nonpolar (e.g. Taxol) compounds whose inherent physical properties cause problems with formulation, distribution or bioavailability. The research described herein utilizes an alternative strategy that involves molecular transporters, which are agents that, when attached to poorly soluble or poorly bioavailable drugs, produce conjugates that exhibit excellent water solubility and simultaneously an increased ability to cross tissue and cell barriers. Bioactivatable molecular transporter conjugates of the immunosuppressant drug Cyclosporin A incorporating disulfide linkers were developed, evaluated for their biological activity in vitro, and administered topically in an in vivo mouse model. In addition to the inherent physical properties limitation that prevents drug passage across the cell membrane, another significant contributor to the failure of many therapeutics is the overexpression of membrane proteins (e.g. P-glycoprotein or Pgp) that mediate the unidirectional efflux of drugs out of target cells, often doing so prior to the drug reaching its intracellular target. Bioactivatable oligoarginine transporter conjugates of Taxol, a substrate for Pgp, were prepared and shown to overcome efflux-mediated resistance in primary human ovarian cancer tissue ex vivo. The encapsulation of transporter conjugates inside biodegradable polymeric nanoparticles was also investigated as a way to achieve sustained release of transporter drug conjugates over extended periods of time, thereby avoiding metabolism or toxicity problems associated with a bolus administration of therapeutic agents. Nanoparticles encapsulating transporter-probe conjugates were formed using two different methods, and shown to release their encapsulated cargo over time under biologically relevant conditions.

The protein kinase C isozyme family has been implicated in a number of diseases representing the majority of the most significant challenges to global human health, including cancer, neurodegenerative diseases (Alzheimer's disease, depression, schizophrenia, etc.), cardiovascular disease, HIV/AIDS, diabetes, and chronic pain. As such, potent and selective modulation of this enzyme family using small molecule ligands designed for a particular function has tremendous therapeutic potential. A number of agents have been identified as ligands for the C1 domain of protein kinase C. Many of these compounds, including the endogenous ligand diacylglycerol and the complex bryostatin family of natural products, act by inducing an initial activation event, resulting in the translocation of protein kinase C to the plasma membrane where it can participate in the phosphorylation of downstream serine and threonine residues. The bryostatins are complex macrolides isolated from the marine organism Bugula neritina. Although a number of agents from this family of natural products have been shown to be biologically active, bryostatin 1 in particular has garnered tremendous therapeutic interest owing to its remarkable potency and activity profile. Specifically, bryostatin 1 has been shown to stimulate apoptosis, bolster the immune system, reverse multidrug resistance, synergize with other anticancer agents, enhance memory and learning in rodent models through the induction of synapse formation (synaptogenesis), reverse the effects of stroke in animal models, and induce latent HIV in vitro. As a result of this activity profile, bryostatin 1 is currently in phase I and II clinical trials for cancer treatment and is being advanced to the clinic for the treatment of Alzheimer's disease. However, despite its remarkable clinical potency (often ~ 1 mg is required for a 8 week treatment cycle in humans), the extremely low supply of bryostatin 1 prohibits its continued human clinical use and investigation for the treatment of additional therapeutic indications. In an effort to address the issues associated with the supply and unoptimized nature of a number of complex natural product protein kinase C ligands, the Wender group developed a pharmacophore model for C1 domain binding in the mid 1980s. This resulted in the design and synthesis of highly simplified, functional protein kinase C ligands based on the diacylglycerol scaffold. Additionally, structurally simplified, synthetically accessible bryostatin analogs were designed and shown to have comparable or even superior potency relative to bryostatin 1 for protein kinase C binding and in vitro anticancer activity. Described herein is the design, synthesis, and biological evaluation of a series of macrocyclic diacylglycerol analogs in an effort to improve protein kinase C affinity by reducing the entropic penalty of the binding event relative to the endogenous linear diacylglycerols. The binding affinity was found to be highly dependent on macrocycle size, with the most potent analog being up to two orders of magnitude more potent than the linear diacylglycerols (consistent with previous reports). Moreover, these analogs were prepared in a step-economical fashion (3-4 steps from commercial materials). Additionally, the design and synthesis of members of the first series of B-ring tetrahydropyran bryostatin analogs produced in the Wender group is reported. The use of a novel, high yielding Prins macrocyclization allowed for the retention of the synthetic convergency that has become a hallmark of the B-ring dioxane analogs produced previously in the Wender group. Several compounds from this B- ring tetrahydropyran class were found to be among the most potent analogs produced to date (with respect to protein kinase C affinity and in vitro anticancer activity). Despite the high potency and synthetic accessibility of the previously reported bryostatin analogs, these compounds lacked the ability to activate protein kinase C isozymes (as measured by their ability to induce the translocation of the enzyme from the cytosol to the plasma membrane) with a high degree of selectivity but were not equally non-selective either. The ability to tune isozyme selectivity has tremendous therapeutic potential and, in an effort to address this challenge, a series of A-ring functionalized, B-ring tetrahydropyran analogs was designed and synthesized using the Prins macrocyclization methodology. It was found that the C8 geminal dimethyl group on the A-ring in combination with C7 hydroxyl functionality imparts selectivity for the conventional protein kinase C [Beta]I over the novel protein kinase C [lowercase Delta}. Alternatively, C8 geminal dimethyl functionality in combination with C7 acetate functionality results in a high degree of non-selectivity for these isoforms. C13 functionalization was found to increase the potency of this already highly active analog class. Finally, several of the B-ring tetrahydropyran bryostatin analogs were shown to synergize with taxol in a human leukemia cell line. Additionally, a lead B-ring dioxane bryostatin analog was shown to be capable of inhibiting tumor growth in vivo in a transgenic mouse lymphoma model. Protein kinase C was implicated in this activity by monitoring the phosphorylation of downstream proteins as well as by performing inhibitor studies. This lead analog was also shown to induce apoptosis in a number of human B- and T-lymphocytes. The apoptotic induction observed in the murine cell line used for this pilot in vivo study was found to be independent of direct cell cycle effects. Significantly, this represents the first academic report of bryostatin analog efficacy and safety in vivo.

My graduate studies have focused on the design, synthesis, and biological evaluation of novel probe and drug delivery technologies. This research has explored the development of new molecular transporter scaffolds with a focus on step economy and translational costs as well as evaluation of their uptake and delivery properties in cells and animals. Chapter 1 provides a historical context and overview of guanidinium-rich molecular transporter technology. Chapter 2 describes the development of a new family of guanidinium-rich oligocarbonate molecular transporter which are flexibly and efficiently assembled by a one-step oligomerization strategy. These novel oligocarbonate transporters were shown to exhibit excellent uptake properties both in cells and animal models. Chapter 3 is directed at the utility of an oligomerization approach to generate molecular transporters by the design, synthesis, and evaluation of new aphipathic co-oligomers for the delivery of siRNA, an oligonucleotide cargo of intense therapeutic interest. Amphipathic carbonate co-oligomers were prepared by an oligomerization strategy and demonstrated to effectively package, deliver, and release functional siRNA in cells. Chapter 4 describes the effects of a branched guanidinium array on the transport and delivery efficiency of releasable dendrimeric guanidinium-rich transporters. These transporters were synthesized and demonstrated to deliver and release a small molecule for turnover by its intracellular target enzyme by bioluminescence assays in cells and transgenic animal models. Chapter 5 describes the design, synthesis, and preliminary biological evaluation of lipidated molecular transporter derivatives of the immunosuppressant drug rapamycin for topical delivery.

My graduate studies have focused on the development of novel drug delivery technologies of research, clinical, and industrial significance. More specifically, my research has focused on the design, synthesis, and application of guanidinium-rich molecular transporters for the delivery of siRNA into cells. My studies have also focused on using molecular transporters to develop the first molecular method to deliver cargo into algae, and on the development of new strategies to control the timing and amount of drug or probe release in cells. Though all of this research is fundamentally based on organic chemistry, these projects have broad applications in research, industrial and clinical settings. Chapter 1 reviews the strategies that have been developed for the delivery of siRNA in both cells and animals. Rather than divide the field by chemical type (e.g. peptide vs. protein) or disease indication, this review categorizes siRNA deliver agents by the identity of their cationic moiety for siRNA complexation. Guanidinium-containing delivery vectors are presented, as are vectors containing ammonium and phosphonium groups. Highlighting this field by cationic moiety reveals how little research has been done to compare the effects of the cation's identity on deliver efficacy, toxicity, and pharmacokinetics. Chapter 2 describes the design, synthesis, and evaluation of guanidinium-rich amphipathic oligocarbonate molecular transporters for the complexation, delivery, and release of siRNA in cells. The synthetic ease of the metal-free carbonate oligomerization to synthesize these transporters afforded fast access to a series of transporters that systematically probed the functionality required for effective complexation, delivery, and release of siRNA. Transporters were characterized and evaluated for biological activity in immortalized human keratinocytes. The transporters discovered in this study were highly effective, with target gene silencing of up to 90% observed. Chapter 3 focuses on efforts towards expanding the scope of both the chemical space and cell types tested in the delivery of siRNA with amphipathic oligocarbonate molecular transporters. These second generation delivery systems have improved physical properties, including smaller and more stable particle sizes, relative to their first generation counterparts described in Chapter 2. These transporters delivered siRNA to primary keratinocytes, melanoma cells, and ovarian cancer cells. Chapter 4 details the development of the first molecular method for the delivery of small molecule probes and large protein cargos into algal cells. It was shown that oligoarginine could facilitate the uptake of fluorescein, or the larger, FAM-streptavidin protein, into cells. A catalytically active protein was delivered into cells and was shown to maintain catalytic activity even after delivery. Chapter 5 describes the design of new strategies to control the timing and amount of cargo released inside cells. Efforts towards a novel linker carrying two copies of drug or probe are described, as well as the combination of microneedles and luciferin-transporter conjugates for transdermal delivery in vivo.

In this work, a function-oriented array of simplified analogs of the daphnane diterpene orthoester class of natural products bearing palmitate, phenyl, or phenylacetyl orthoesters was synthesized starting from commerically available starting materials via a key late stage common diversification intermediate. These families of novel compounds were evaluated for both selective PKC activation and growth inhibition of K562 (myleogenous leukemia) cancer cells. While these analogs showed no growth inhibitory activity in K562 cells up to concentrations of 10 micromolar, they did display varying profiles of PKC activation. One analog in particular demonstrated the ability to activate and cause translocation of conventional PKC b1 and novel PKC d to the same extent as resiniferonol, a potent natural resiniferonoid. A second analog, however, was found to activate novel PKC d selectively over conventional PKC b1; surprisingly, resiniferonol did not share this selectivity profile. Because the simplified, functional analogs synthesized in this study were shown to activate PKC while having no growth inhibitory activity, these compounds should be further investigated for their potential as therapeutic leads for the treatment of diseases like Alzheimer's or Parkinson's, in which (selective) activation of PKC could serve a therapeutic purpose without being plagued by growth inhibitory pathways.

Embryonic development is a remarkable program of cell proliferation, migration, and differentiation that transforms a single fertilized egg into a complex multicellular organism. This process depends on spatial and temporal control of gene function, and deciphering the molecular mechanisms that underlie pattern formation requires novel methods for perturbing gene expression with similar precision. Synthetic reagents can help meet this demand, and in this thesis I describe the development and application of caged morpholino (cMO) oligonucleotides for inactivating genes in zebrafish and other optically transparent organisms with spatiotemporal control simply by irradiating embryonic tissues with a focused light beam. In chapter 1 I provide an overview of the zebrafish model system of vertebrate development and survey the capabilities and limitations of various oligonucleotide-based technologies for perturbing RNA function and tracking RNA expression in zebrafish. I examine various light-gated oligonucleotide technologies that exploit the optical transparency of zebrafish embryos, including cMOs, for achieving spatiotemporal control of RNA function. In chapter 2 we describe the initial synthesis of a cMO targeting expression of the no tail a (ntla) transcription factor. By permitting spatiotemporal gene regulation in zebrafish embryos, the ntla cMO was used to make initial observations into the time-dependent role of this gene in notochord formation. In chapter 3 we report optimized methods for the design and synthesis of hairpin cMOs, incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, I have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances established the generality of cMO technologies to facilitate the application of these chemical probes in vivo for functional genomic studies. Finally, in chapter 4 we illustrate the utility of the cMO technology in isolating spatiotemporally-distinct functions of transcription factors -- genes that play diverse roles during embryonic development, with each controlling multiple cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level; however, probing in vivo gene function with comparable spatiotemporal precision has been a technological challenge. To address this need, I have integrated cMOs with similarly caged fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies. Using this approach, I have dynamically profiled the No tail-a (Ntla)-dependent transcriptome at different stages of zebrafish mesoderm development, discovering discrete sets of genes that are associated with either notochord cell fate commitment or subsequent changes in cell function. Our studies elucidated the roles of several Ntla-regulated genes in notochord development and demonstrated the activation of multiple transcriptomes within a cell lineage by a single transcription factor.

The development of live cell RNA imaging techniques will lead to the unraveling of many important biological processes. To achieve this goal, there have been three different strategies developed. They are the development of small molecule probes, nucleic acid probes, and green fluorescent protein (GFP) probes. In the following thesis, the pros and cons of each approach are discussed, followed by a proposal to resolve the limitations. In the small molecule case, a probe was developed that utilized a quenched sulforhodamine dye. It was designed so that its structure can be rationally modified from the initial lead compound. An aptamer sequence that activates the sulforhodamine probe with micro molar affinity was found by in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX), followed by fluorescence screening in E.coli. The rational modification of the structure of the initial sulforhodamine probe resulted in an overall 33-fold increase in binding affinity compared to the initial lead compound. Instead of the chemical modification of the lead compound, the small molecule's cell permeability and binding affinity to the target could be improved by linking to cell penetrating peptides (CPP). A CPP is a short peptide sequence composed of poly arginine amino acids which shows excellent cell uptake and affinity to RNA. However, the use of the CPP-linked dye in live cell imaging has been limited by strong signals in the endosome region. An attempt was made to overcome this difficulty by linking a quencher molecule to the dye-CPP via a disulfide bond, which only breaks when it enters the cytosol. For the nucleic acid probe, the major problem was its low cell permeability and low signal-to-background ratio due to the low copy number of mRNA targets within the cell. We made mutant Hammerhead ribozymes and embedded them in a non-coding region of the GFP expression vector that can be transfected to mammalian cells. This modified Hammerhead ribozyme acts as a logic gate, and the signal is amplified by the expression of GFP in the presence of the target mRNA. In vitro and in vivo results are discussed. Finally, a fragmented GFP system, the fluorescence of which could be recovered by binding to a specific RNA tag, was developed. The major problem for the GFP-mediated RNA imaging system was the low signal-to-background ratio from the GFP probe that is not bound to the RNA tag. To find the non-fluorescent GFP, the GFP was truncated from the C-terminus such that it loses its fluorescence with minimum loss of amino acids. An RNA sequence that has high affinity to this GFP was found by in vitro SELEX. The subsequent E.coli screening found an RNA sequence that reactivates the fluorescence of the GFP probe.

Fluorescence microscopy is one of the most widely used tools in cell biology due its intrinsically high detection sensitivity coupled with the ability to genetically label proteins and other cellular structures with fluorescent tags. However, the resolution of fluorescence microscopy has historically been limited to about 200 nm laterally and 800 nm axially because of the diffraction limit of visible light. In the past five years, imaging below the diffraction limit ("super-resolution imaging") by localizing single fluorophores, one at a time (1-3), has opened a wide a variety of new biological systems for study. This Dissertation is a collection of both techniques for two and three dimensional super-resolution imaging as well as applications in bacterial and yeast imaging. References 1. Betzig E, et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642-1645. 2. Hess ST, Girirajan TPK & Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91: 4258-4272. 3. Rust MJ, Bates M & Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3: 793-795.

Templated fluorescence activation is an elegant nucleic acid detection technique, which relies on a target-activated chemical reaction between two oligonucleotide probes, eliciting a fluorescence readout. This method reports on genetic markers in solution phase and in cells. The research described in this thesis was aimed to expand the reaction scope of templated fluorescence activation and to develop probes, which overcome shortcomings of previous designs. Examples of DNA template-mediated fluorogenic reactions developed during this project include an organomercury-induced cyclization of Rhodamine thiosemicarbazide and the deprotection of a 7-azidomethoxycoumarin profluorophore by a Staudinger reaction. The most promising probe design (Q-STAR probes) is based on the reductive cleavage of an [alpha]-azidoether quencher release linker conjugated to a fluorophored-labeled DNA. A triphenylphosphine DNA-probe rapidly activates Q-STAR probes in the presence of the matched DNA target strand, reporting its presence by a strong fluorescence turn-on signal. Q-STAR probes are inert to aqueous conditions and cellular components, properties that were suboptimal for previous probe designs. Q-STAR probes report the target with single nucleotide specificity and enable an amplified detection signal by harnessing the target as a catalyst for the templated reaction. Q-STAR probes efficiently detect the presence of rRNAs in bacteria and mammalian cells. The probes are responsive to single nucleotide differences, which allows discriminating bacteria species by genetic variations using fluorescence microscopy or flow cytometry. Similarly, rRNAs in mammalian cells generate a strong fluorescence turn-on signal for Q-STAR probes. Templated fluorescence activation schemes bear considerable promise for applications in clinical diagnostics and molecular biology. The probe designs described in this thesis, in particular Q-STAR probes, constitute a major advancement in the field and will help achieve these goals.

CLC chloride channels and transporters play diverse physiological roles in processes ranging from regulating bone-density, muscle excitability, and blood pressure, to facilitating extreme-acid survival of pathogenic bacteria. Defects in CLC proteins cause human disorders in these processes. Small-molecule inhibitors of the CLCs would be useful as drugs for treating a variety of CLC-related human diseases and also to investigate CLC physiology. In addition, inhibitors are powerful tools for studying molecular mechanisms of Cl-- gating. Trapping channels or transporters in particular conformational states with high-affinity ligands could potentially advance our understanding of the structural basis for CLC activity. Despite their usefulness, specific small-molecule inhibitors for CLC proteins are scarce. To address this shortfall, we have exploited the 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) scaffold to develop two novel classes of CLC inhibitors. DIDS has been used as an anion-transport inhibitor for decades and was first used to inhibit CLCs over 30 years ago. However, experiments to determine the compound's mode of inhibition led us to discover that minor contaminants in the DIDS solutions inhibit CLC proteins more effectively than DIDS itself. The contaminants were found to derive from hydrolysis of the labile isothiocyanate moieties. The structures of five major hydrolysis products were determined by 1H NMR, HRMS analysis, and chemical synthesis to be DIDS-based polythioureas. These compounds bind directly to the CLC proteins, as evidenced by the fact that they inhibit purified, reconstituted ClC-ec1 and that inhibition of ClC-Ka can be prevented by the point mutation N68D. These polythioureas are the highest affinity inhibitors known for the CLCs and provide a new class of chemical probes for dissecting the molecular mechanisms of chloride transport. The second class of identified CLC inhibitors combines the DIDS core structure with alkyl chain carboxylic acids. The most potent inhibitor identified, 4,4'-octanamidostilbene-2,2'-disulfonic acid (OADS), inhibits ClC-ec1 with an apparent affinity of 29 [Mu]M. As a means to identify the inhibitor-binding site, we synthesized photo-reactive diazirine derivatives of OADS and showed that these photo-affinity reagents specifically inhibit ClC-ec1. Experiments to identify the binding site using 'top-down' mass spectrometry, in which the protein is cleaved into peptide fragments via electron-capture dissociation, have identified an intracellular binding region encompassing 76 amino acids, or 16% of the protein. Current efforts using protease digestion procedures are focused on further refinement of the binding region. Once located, protein/inhibitor interactions gleaned from the labeling of ClC-ec1 could allow us to rationally design more potent inhibitors of CLC transporters and channels.

Vesicle fusion is a central process in transport and communication in biology. In neuronal transmission, synaptic vesicles carrying neurotransmitters dock and fuse to the plasma membrane of the neuron, a process mediated by a combination of several membrane anchored and soluble proteins. Fusion results in the merger of the two apposing lipid bilayers, leading to the exchange of both the lipidic and aqueous components. The fusion reaction is thought to proceed through several stages: first, the membranes are brought into close proximity (docking), second, the outer leaflets mix, but the inner leaflets and contents remain separate (hemi-fusion), and finally, the inner leaflet and contents exchange (full fusion). Due to the complex nature of the fusion reaction and the multitude of proteins involved, the mechanism of the fusion reaction is not well understood. Simplified model systems for vesicle fusion can bring insight into the mechanism by studying the fusion reaction in a more defined and controllable system. This thesis describes a DNA-based model for the protein fusion machinery. Previously, DNA-lipids were used to tether lipid vesicles to glass-supported lipid bilayers. These vesicles could be observed by fluorescence microscopy, and are laterally mobile along the plane parallel to the supported bilayer. DNA-mediated docking between vesicles was characterized, but fusion was not observed due to the fact that the DNA partners were both coupled at the 5' end, so antiparallel hybridization holds the membranes apart. In this work, a new synthesis of DNA-lipid conjugates is described which allows coupling at both the 3' and 5' end of the DNA. Incorporation of complementary DNA-lipids coupled at opposite ends mediates fusion between lipid vesicles. Vesicle fusion was measured in bulk fluorescence assays (Chapter 2 and 3), by both lipid mixing and content mixing assays. The rate of vesicle fusion showed a strong dependence on the number of DNA per vesicle, as well as the sequence of the DNA. Consistent with previous results measured for the docking reaction, fusion was faster for a repeating DNA sequence than for a non-repeating sequence that required full overlap of the strands for hybridization. The role of membrane proximity on the rate of vesicle fusion was investigated in Chapter 3 by insertion of a short spacer sequence at the membrane-proximal end of fusion sequences. The length of the spacer sequence was varied between two and 24 bases, corresponding to length scales of approximately 1-12 nm. Fusion, as measured in bulk assays by lipid and content mixing, decreases systematically as the membranes are held progressively further apart, demonstrating a clear dependence of the rate of the fusion reaction on membrane proximity. While the bulk vesicle fusion assays showed that DNA-lipids can mediate vesicle fusion, these ensemble measurements convolve the multiple steps (docking, hemifusion, and full fusion) of the fusion reaction, complicating any kinetic analysis. In order to image individual vesicle fusion events between tethered vesicles, a new tethering strategy was developed (Chapter 4). This strategy exploits the dependence of DNA hybridization on salt by covalently attaching lipid vesicles to a glass-supported lipid bilayer, then triggering DNA-mediated docking and fusion by spiking the salt concentration. The kinetics of individual vesicle fusion events were subsequently measured using a FRET-based lipid mixing assay for many vesicles (Chapter 6). An analysis of the distribution of waiting times from docking to fusion indicated that this transition occurs in a single step. A second model membrane architecture was used to study individual fusion events between vesicles and a planar bilayer (Chapters 5 and 6). This architecture uses a DNA-tethered planar free-standing bilayer as the target membrane. The kinetics of individual vesicle fusion events to this membrane patch were also consistent with a single step process, as for vesicle to vesicle fusion. In this system, it was also possible to observe content transfer of vesicles containing a self-quenched aqueous dye (Chapter 5). By analyzing the diffusion profile of the dye, it was shown that the dye indeed is transferred into the region below the planar membrane patch, and is not released into the solution above the patch due to vesicle rupture or leakage.

The bryostatins are a family of structurally complex natural products isolated from the marine bryozoan Bugula neritina. Bryostatin 1 is currently being investigated for cancer, Alzheimer's and HIV/AIDS indications. Despite these remarkable activities, research on the bryostatins is hampered by their low natural abundance. Efficient access by total chemical synthesis has been in large part precluded by the bryostatins' structural complexity. This dissertation describes the design, synthesis, and preliminary biological evaluation of functional bryostatin analogs that possess biological activities comparable or superior to the natural product. These fully synthetic analogs were convergently assembled in a uniquely step-economical manner using novel macrocyclization strategies, including macroacetalization and Prins-driven macrocyclization approaches. Bryostatin analogs were identified that possess unique affinities (subnanomolar) and selectivities for protein kinase C (PKC). Synthetic bryostatin analogs also exhibit subnanomolar antileukemic activity in in vitro assays. The convergent total synthesis of bryostatin 9, a highly potent congener of the natural product family, is also described.

The role of atomistic modeling of molecules and organic compounds in biology and pharmaceutical research is constantly increasing, providing insights on chemical and biological phenomena at the highest resolution. To achieve relevant results, however, computational biology has to deal with systems containing at least 1000 atoms. Such big molecules cause large computational demands and impose limitations on the level of theory used to describe molecular interactions. Classical molecular mechanics based on various empirical relationships has become a workhorse of computational biology, as a practical compromise between accuracy and computational cost. Several decades of classical force field development have seen many successes. Nevertheless, more accurate treatment of bio-molecules from first principles is highly desirable. Hartree-Fock (HF) and density functional theory (DFT) are two low-level ab initio methods that provide sufficient accuracy to interpret experimental data. They are therefore the methods of choice to study large biological systems. Recently DFT has been applied to calculate single point energy of a solvated Rubredoxin protein. The system contained 2825 atoms and required more than two hours on a supercomputer with 8196 parallel cores. This study clearly demonstrates the scale of problems one has to tackle in first principles calculations of biologically relevant systems. Dynamical simulations requiring thousands of single point energy and force evaluations therefore appear to be completely out of reach. This fact has essentially prohibited the use of first principles methods for many important biological systems. Fortunately, the computer industry is evolving quickly and novel computing architectures such as graphical processing units (GPUs) are emerging. The GPU is an indispensable part any modern desktop computer. It is special purpose hardware responsible for graphics processing. Most problems in computer graphics are embarrassingly parallel, meaning they can be split into a large number of smaller subproblems that can be solved in parallel. This fact has guided GPU development for more than a decade; and modern GPUs evolved into a massively parallel computing v architecture containing hundreds of basic computational units, which all together can perform trillions of arithmetic operations per second. The large computational performance and low price of consumer graphics cards makes it tempting to consider using them for computationally intensive general purpose computing. This fact was recognized long ago and several groups of enthusiasts attempted to use GPUs for non-graphics computing in the early 2000's. One of the few successes from these attempts is now known as Folding@Home. These early attempts were primarily stymied by three major problems: lack of adequate development frameworks, limited precision available on GPUs, and the difficulty of mapping existing algorithms onto the new architecture. The two former impediments have been recently alleviated by the introduction of efficient GPU programming toolkits such as CUDA and the latest generation of graphics cards supporting full double precision arithmetic operations in hardware. These advances led to an explosion of interest in general purpose GPU computing and led to the development of many GPU-based high performance applications in various fields such as classical molecular dynamics, magnetic resonance imaging, and computational fluid dynamics. Most of the projects, however, lie far outside of quantum chemistry which is likely caused by the complexity of quantum chemistry algorithms and the associated difficulty of mapping them onto the GPU architecture. Various specific features of the hardware require complete redesign of conventional HF and DFT algorithms in order to fully benefit from the large computational performance of GPUs. We have successfully solved this problem and implemented the new algorithms in TeraChem, a high performance general purpose quantum chemistry package designed for graphical processing units from the ground up. Using TeraChem, we performed the first ab initio molecular dynamics simulation of an entire Bovine pancreatic trypsin inhibitor (BPTI) protein for tens of picoseconds on a desktop workstation with eight GPUs operating in parallel. Coincidently, this was also the first protein ever simulated on a computer using the classical molecular mechanics approach. BPTI binds to trypsin with a binding free energy of approximately 20 kcal/mol, making BPTI one of the strongest non-covalent binders. It vi is even more remarkable that a single BPTI amino acid LYS15 contributes half of the binding free energy by forming a salt bridge with one of the trypsin's negatively charged residues inside the binding pocket. In fact, the LYS15's contribution to the overall binding energy is approximately twice as large as what would be expected based on experimental measurements of salt bridge interactions in other proteins. Our simulation of BPTI demonstrated that substantial charge transfer occurs at the proteinwater interface, where between 2.0 and 3.5 electrons are transferred from the interfacial water to the protein. This effect decreases the net protein charge from +6e as observed in gas-phase experiments to +4e or less. We demonstrate how this effect may explain the unusual binding affinity of the LYS15 amino acid.

Polyketides are a large class of structurally diverse natural products which posses a wide range of biological activities. Unfortunately, despite the potential utility of these compounds in the clinic, large scale production of many of these natural products from their native hosts remains a challenge. Additionally, due to their complexity, engineering better pharmacokinetic properties by traditional synthetic means is often challenging. A better understanding of the biosynthetic machinery which produces polyketides allows for optimization of their production and paves the way for bioactivity-based pathway reengineering. This work begins with an introduction detailing attempts to unravel the biosynthetic underpinnings of two key natural product families, the tetracyclines and the thiostreptons, with an eye toward ultimately reengineering the pathways. Then, our efforts to reconstruct and reengineer the biosynthesis and biological activity of the type II polyketides frenolicin and A-74528 are detailed. Successful reconstruction of a chimeric biosynthetic pathway to frenolicin B and subsequent reengineering of that pathway to produce novel frenolicin analogs is described. Then, the biological activity of these compounds both in vitro against the parasites Toxoplasma gondii and Plasmodium falciparum is discussed. Additional studies against Plasmodium berghi in mice show that frenolicin B is an effective antiparastic agent in vivo. Following this, engineering of a biosynthetic pathway to the novel antiviral agent A-74528 from S. sp. SANK 61196 is presented and the impact of various tailoring enzymes on metabolite production are explored.

The number of reports per year on single-molecule imaging experiments has grown roughly exponentially since the first successful efforts to optically detect a single molecule were completed over two decades ago. Single-molecule spectroscopy has developed into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of single-molecule biophysics has resulted from its benefits in probing heterogeneous populations, one molecule at a time, as well as from advances in microscopes and detectors. There is a need for new fluorophores that can be used for single-molecule imaging in biological media, because imaging in cells and in organisms require emitters that are bright and photostable, red-shifted to avoid pumping cellular autofluorescence, and chemically and photophysically tunable. To this end, we have designed and characterized fluorescent probes based on a class of nonlinear-optical chromophores termed DCDHFs. This dissertation describes various physical and optical studies on these emitters, from sensing local environment to photoactivation. Chapter 1 is a general introduction to fluorescence and single-molecule spectroscopy and imaging. Single-molecule experiments in living cells are discussed and probes used for such experiments are summarized and compared. Chapter 2 explores the basic photophysics of the DCDHF fluorophores and some general methods of measuring relevant spectroscopic parameters, including photostability. Chapter 3 discusses the various approaches we have taken to modify particular properties by changing the fluorophore's structure. We have redesigned the DCDHF fluorophore into a photoactivatable fluorogen -- a chromophore that is nonfluorescent until converted to a fluorescent form using light -- described in Chapter 4. Finally, a different, chemical route to fluorescence activation is presented in Chapter 5. The remainder of the Dissertation is the Appendix and a full Bibliography. The Appendix includes a table of photophysical parameter for DCDHF fluorophore, various protocols used in the experiments discussed, MatLab codes, and NMR spectra.

This thesis discusses a number of projects involving the use of modified nucleotides and oligonucleotides in addressing some basic science questions and some clinical and technological applications.The first chapter details our efforts at using telomere-encoding circular DNA in elongating zebrafish telomeres. We microinjected our synthetic circular DNA into zebrafish embryos and studied their telomere length 24 hrs later. Using Quantitative Fluorescence in situ Hybridization (Q-FISH) as the analytical tool to determine telomere length, we observed no significant difference in telomere length between the group injected with the synthetic DNA and the control group. In the second chapter we studied the potential of a non-polar shape mimic of iodo-uracil as an imager of tumors. We continued discussing our work with non-polar nucleotide isosteres in the third chapter where we used them in investigating a novel active site in polymerase from Pyrococcus furiosus (Pfu). From our studies we concluded that although shape was an important factor in distinguishing bases, this binding site also employed hydrogen bonding to identify nucleoabases. The lack of any recognition of the syn-oxidized bases suggested that the enzyme preferred to recognize bases in the anti conformation rather than syn. In the fourth chapter we were interested in understanding the factors determining the fidelity and selectivity observed in RNA Polymerase II mediated transcription. Once again we used non-polar shape mimics of thymidine (dF) and adenine (dQ) to study the importance of shape and hydrogen bonding. We observed that the thymidine mimic was recognized better by the RNA Polymerase II active site than the mis-match bases. The adenine isostere on the other hand, was poorly recognized. This preliminary study demonstrates the importance of both shape and hydrogen bonding. The last chapter discusses our studies using polyfluorophores on a DNA backbone to detect gases. Following a combinatorial method, a library of oligodeoxyfluorosides (ODFs) were synthesized from which sensors of gases were selected. Using this method we were able to select optical sensors for a diverse set of small molecules in vapor state.

Cell division is a major developmental event in the life cycle of a bacterial cell. Caulobacter crescentus division is asymmetric, producing daughter cells that differ in morphology and polar features: a sessile stalked cell and a motile swarmer cell that subsequently differentiates into a stalked cell. In this work we investigate the assembly of the Caulobacter cell division machinery (the divisome) using genetics, biochemistry, and microscopy. In Caulobacter, the cell division process requires a set of approximately twenty-three proteins localizing from the cytoplasm to the outer membrane. To understand divisome assembly as a function of the cell cycle, we generated fluorescent fusions to analyze the temporal regulation of 19 representative divisome and division-site localized proteins. In Chapter 2, we identified a series of stages and transitions in divisome assembly and the associated events yielding a comprehensive temporal picture of the process. The assembly interdependency for divisome formation in Caulobacter appears to involve cooperative rather than sequential recruitment, suggesting that it is a multiprotein subcomplex model. In Chapter 3, we describe our investigation of the Tol-Pal complex where we demonstrated that it plays a vital role for membrane integrity maintenance and that it is essential for viability. Cryo-electron microscope images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Thus, the Caulobacter trans-envelope Tol-Pal complex is a key component of cell envelope structure and function, mediating outer membrane constriction at the final step of cell division, as well as the positioning of a protein localization factor. In Chapter 4, we describe our examination of the FtsZ binding protein, ZapA. FtsZ is the most highly conserved divisome protein that polymerizes into a contractile ring near midcell, defining the future site of cell division. We showed that ZapA is required to maintain a normal cell length, and promotes Z ring assembly. The biochemical and functional studies suggest that Caulobacter ZapA is a positive regulator of Z-ring assembly. In summary, we have addressed three major stages in developments of the divisome in Caulobacter: Z-ring assembly, divisome maturation and outer membrane invagination. These experiments have provided a new understanding of how the Caulobacter cell temporally executes the cell division program to propagate reliably and how Caulobacter cell division is performed.