Books by Subject
- pt. A-B, 2007. Springerpt. B, 2007 SpringerFrank A. Carey and Richard J. Sundberg.pt. A. Structure and mechanisms -- pt. B. Reactions and synthesis.
- 2007 CRCnetBASEJohn L. Holmes, Christiane Aubry, and Paul M. Mayer.
- 2013 SpringerJosef Flammer, Maneli Mozaffarieh, Hans Bebie.What Is Light? -- The Interaction Between Light and Matter -- Light Sources -- Examinations with Light -- Ultrasound Diagnostics -- Further Imaging Procedures -- Interventions with Laser Light -- Some History of Chemistry -- Oxygen -- Water -- Carbon Dioxide (CO2) -- Nitric Oxide -- Redox Reactions -- DNA -- RNA -- Proteins -- Lipids -- Matter: Using Water as an Example -- If You Are Interested in More ... -- Appendix: Units and Constants.
- 2012 Wileyedited by Nathan Brown.
- Biological fluorescence imaging on the nanoscale : STED super-resolution microscopy of cells and tissues2013Lana Lau.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.
- Bioorthogonal transition metal catalysis for prodrug and proprobe release, II. Step-economical synthesis of molecular transporters and evaluation of their uptake across cell membrane and cell wall barriers2013Brian Michael Trantow.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.
- 2010 WileyAbraham, Donald J.; Burger, Alfred; Rotella, David P."... provides an established, recognized, authoritative and comprehensive source on medicinal chemistry and drug discovery and development. This flagship reference for medicinal chemists and pharmaceutical professions has been thoroughly updated and expanded across 8 volumes to incorporate the entire process of drug development (preclinical testing, clinical trials, etc.) alongside the traditional strengths in medicinal chemistry and drug discovery"--Provided by publisher.
- 2007 WileyKang Li.Chapter 1 Ceramic Membranes and Membrane Processes, p. 1-20 -- Chapter 2. Preparation of Ceramic Membranes, p. 21-57 -- Chapter 3. Characterization of Ceramic Membranes, p. 59-95 -- Chapter 4. Transport and Separation of Gases in Porous Ceramic Membranes, p. 97-134 -- Chapter 5. Ceramic Hollow Fibre Membrane Contactors for Treatment of Gases/Vapours, p. 135-168 -- Chapter 6 Mixed Conducting Ceramic Membranes for Oxygen Separation, p. 169-215 -- Chapter 7. Mixed Conducting Ceramic Membranes for Hydrogen Permeation, p. 217-243 -- Chapter 8. Ceramic Membrane Reactors, p. 245-298.
- 2013 CRCnetBASEAlan Rodgman and Thomas A. Perfetti.Ch. 1. Hydrocarbons -- ch. 2. Alcohols and phytosterols -- ch. 3. Aldehydes and ketones -- ch. 4. Carboxylic acids -- ch. 5. Esters -- ch. 6. Lactones -- ch. 7. Anhydrides -- ch. 8. Carbohydrates and their derivatives -- ch. 9. Phenols and quinones -- ch. 10. Ethers -- ch. 11. Nitriles -- ch. 12. Acyclic amines -- ch. 13. Amides -- ch. 14. Imides -- ch. 15. N-nitrosamines -- ch. 16. Nitroalkanes, nitroarenes, and nitrophenols -- ch. 17. Nitrogen heterocyclic components -- ch. 18. Miscellaneous components -- ch. 19. Fixed and variable gases -- ch. 20. Metallic and nonmetallic elements, isotopes, ions, and salts -- ch. 21. Pesticides and growth regulators -- ch. 22. Genes, nucleotides, and enzymes -- ch. 23. Hoffmann analytes -- ch. 24. Tobacco and/or tobacco smoke components used as tobacco ingredients -- ch. 25. Pyrolysis -- ch. 26. Carcinogens, tumorigens, and mutagens vs. anticarcinogens, inhibitors, and antimutagens -- ch. 27. Free radicals -- ch. 28. Summary.
- 2006 MyiLibrary2006 ProQuest Ebook Central, Limited to 1 simultaneous usersedited by Marcel Dicke and Willem Takken.
- 2011 Springer Protocolsedited by Joe Zhongxiang Zhou.Historical overview of chemical library design / Roland E. Dolle -- Chemoinformatics and library design / Joe Zongxiang Zhou -- Molecular library design using multi-objective optimization methods / Christos A. Nicolaou and Christos C. Kannas -- A scalable approach to combinatorial library design / Puneet Sharma, Srinivasa Salapaka, and Carolyn Beck -- Application of Free-Wilson selectivity analysis for combinatorial library design / Simone Sciabola ... [et al.] -- Application of QSAR and shape pharmacophore modeling approaches for targeted chemical library design / Jerry O. Ebalunode, Weifan Zheng, and Alexander Tropsha -- Combinatorial library design from reagent pharmacophore fingerprints / Hongming Chen, Ola Engkvist, and Niklas Blomberg -- Docking methods for structure-based library design / Claudio N. Cavasotto and Sharangdhar S. Phatak -- Structure-based library design in efficient discovery of novel inhibitors / Shunqi Yan and Robert Selliah -- Structure-based and property-compliant library design of 11[beta]-HSD1 adamantyl amide inhibitors / Genevieve D. Paderes ... [et al.] -- Design of screening collections for successful fragment-based lead discovery / James Na and Qiyue Hu -- Fragment-based drug design / Eric Feyfant ... [et al.] -- LEAP into the Pfizer Global Virtual Library (PGVL) space : creation of readily synthesizable design ideas automatically / Qiyue Hu ... [et al.] -- The design, annotation, and application of a kinase-targeted library / Hualin Xi and Elizabeth A. Lunney -- PGVL hub : an integrated desktop tool for medicinal chemists to streamline design and synthesis of chemical libraries and singleton compounds / Zhengwei Peng ... [et al.] -- Design of targeted libraries against the human Chk1 kinase using PGVL hub / Xengwei Peng and Qiyue Hu -- GLARE : a tool for product-oriented design of combinatorial libraries / Jean-François Truchon -- CLEVER : a general design tool for combinatorial libraries / Tze Hau Lam ... [et al.].
- 2008 CRCnetBASEedited by James A. Romano, Jr., Brian J. Lukey, Harry Salem.Brief history and use of chemical warfare agents in warfare and terrorism / Harry Salem, Andrew L. Ternay, Jr., and Jeffery K. Smart -- The chemistry of chemical warfare agents / Peter Kikilo, Vitaly Fedorenko, and Andrew L. Ternay, Jr. -- Chemical warfare agent threat to drinking water / Harry Salem ... [et al.] -- Health effects of low-level exposure to nerve agents / John H. McDonough and James A. Romano, Jr. -- Toxicokinetics of nerve agents / Marcel J. van der Schans, Hendrik P. Benschop, and Christopher E. Whalley -- Application of genomic, proteomic, and metabolomic technologies to the development of countermeasures against chemical warfare agents / Jennifer Sekowski and James Dillman -- Novel approaches to medical protection against chemical warfare nerve agents / Ashima Saxena ... [et al.] -- Nerve agent bioscavengers : progress in development of a new mode of protection against organophosphorus exposure / David E. Lenz ... [et al.] -- Butyrylcholinesterase and its synthetic C-terminal peptide confer in-vitro suppression of amyloid fibrils formation / Erez Podoly ... [et al.] -- A novel medical countermeasure for organophosphorus intoxication : connection to Alzheimer's disease and dementia / Edna F. R. Pereira ... [et al.] -- Inhalation toxicology of nerve agents / Paul A. Dabisch ...[et al.] -- Vesicants and oxidative stress / Milton G. Smith ...[et al.] -- Health effects of exposure to vesicant agents / Charles G. Hurst and William J. Smith -- Cyanides : toxicology, clinical presentation, and medical management / Bryan Ballantyne and Harry Salem -- Chemicals used for riot control and personal protection / Harry Salem, Bryan Ballantyne, and Sydney Katz -- Mechanism of action of botulinum neurotoxin and overview of medical countermeasure development for intoxication / Michael Adler ... [et al.] -- Ricin and related toxins : review and perspective / Charles B. Millard and Ross D. LeClaire -- Screening smokes : applications, toxicology, clinical considerations and medical management / Bryan Ballantyne and Harry Salem -- Clinical detection of exposure to chemical warfare agents / Benedict R. Capacio ... [et al.] -- Personal protective equipment (ppe) : practical and theoretical considerations / Michael R. Jones -- Chemical warfare agent decontamination from skin / Brian J. Lukey ... [et al.] -- Chemical warfare, chemical terrorism, and traumatic stress responses : an assessment of psychological impact / James A. Romano, Jr. ... [et al.] -- Emergency response to a chemical warfare agent incident : domestic preparedness, first response, and public health considerations / David H. Moore and Barbara Saunders-Price -- Emergency medical response to a chemical terrorist attack / Stephen A. Pulley and Michael R. Jones.
- 2002 CRCnetBASEselected and arranged by Carl C. Gaither and Alma E. Cavazos-Gaither ; illustrated by Andrew Slocombe.Abstraction -- Accident -- Accuracy -- Acid -- Adsorption -- Aesthetic -- Affinity -- Age -- Air -- Alchemy -- Ambition -- Analogy -- Analysis -- Analyst -- Answer -- Apparatus -- Approximate -- Atom -- Atomic weight -- Authority -- Average -- Balance -- Beauty -- Biochemistry -- Book -- Bubble -- Calculation -- Candle -- Cause and effect -- Central limit theorem -- Chance -- Chaos -- Chemical -- Chemical affinities -- Chemical engineer -- Chemical limericks -- Chemical mnemonics -- Chemist -- Chemistry -- Chemistry and life -- Chemistry and medicine -- Chemistry songs -- Classroom emanations -- Commandments -- Common sense -- Communication -- Compound -- Concept -- Confusion -- Cosmochemistry -- Creativity -- Criticism -- Crystal -- Crystallography -- Curiosity -- Data -- Definition -- Demonstration -- Difference -- Discovery -- Disorder -- Distill -- Electron -- Element -- Energy -- Energy state -- Enzyme -- Error -- Ethics -- Experience -- Experiment -- Experimenter -- Explain -- Fact -- Faith -- Fermentation -- Filter -- Fire -- Fluorochemistry -- Force -- Forecast -- Formula -- Fractal -- Gas -- Generality -- Genius -- Geochemistry -- Glassware -- God -- Graph -- Guess -- Heat -- History -- Hypothesis -- Idea -- Ignorance -- Imagination -- Impossible -- Improbable -- Independence -- Inference -- Information -- Inorganic -- Instrument -- Ion -- Knowledge -- Laboratory -- Language -- Law -- Learn -- Life -- Literature -- Little Willie -- Magic -- Mathematics -- Matter -- Measurement -- Mechanics -- Metal -- Metaphor -- Metaphysics -- Method -- Microscope -- Model -- Molecule -- Motion -- Mystery -- Naivete -- Name -- Nature -- Nomenclature -- Notation -- Null hypothesis -- Numbers -- Observation -- Occam's razor -- Opinion -- Order -- Organic -- Outlier -- Paradox -- Periodic law -- Philosophy -- Physical science -- Plagerism -- Pollution -- Postulate -- Prayer -- Precision -- Prediction -- Probability -- Problem -- Progress -- Proposition -- Publication -- Purity -- Purpose -- Question -- Radical -- Random -- Reaction -- Reason -- Research -- Results -- Rust -- Salt -- Science -- Scientific -- Scientist -- Silver trees -- Simplicity -- Solid -- Soluble -- Solution -- Speculation -- Statistical -- Statistician -- Statistics -- Symbol -- Symmetry -- Synthesis -- Teaching -- Terminology -- Theorist -- Theory -- Thermodynamics -- Thought -- Trial and error -- Truth -- Uncertainty -- Understand -- Unexpected -- Unknown -- Vacuum -- Vision -- Volume -- Water -- Wisdom -- Word -- Work -- Writing -- X-rays.
- 2013 CRCnetBASEedited by Goutam Brahmachari."Natural products play crucial roles in modern drug development and constitute a prolific source of novel lead compounds or pharmacophores for ongoing drug discovery programs. Chemistry and Pharmacology of Naturally Occurring Bioactive Compounds presents cutting-edge research in the chemistry of bioactive natural products and demonstrates how natural product research continues to make significant contributions in the discovery and development of new medicinal entities."--Page  of cover.
- 2014 Wileyedited by Dr. Ravin Narain.General methods of bioconjugation -- Covalent and noncovalent bioconjugation strategies -- Polymer bioconjugates -- Bioconjugates based on poly(ethylene glycol)s and polyglycerols -- Synthetic polymer bioconjugate systems -- Natural polymer bioconjugate systems -- Dendrimer bioconjugates: synthesis and applications -- Organic nanoparticles based bioconjugates -- Bioconjugation strategies: lipids, liposomes, polymersomes, and microbubbles -- Organic nanoparticle bioconjugate: micelles, cross-linked micelles, and nanogels -- Carbon nanotubes and fullerene C60 bioconjugates -- Inorganic nanomaterials bioconjugates (metals, metal oxides--quantum dots, iron-oxide) -- Gold nanomaterials bioconjugates -- Methods for magnetic nanoparticle synthesis and functionalization -- Quantum dots bioconjugates -- Silica nanoparticle bioconjugates -- Polyhedral oligomeric silsesquioxanes (POSS) bioconjugates -- Cell-based, hydrogels/microgels and glyco-bioconjugates -- Cell-based bioconjugates -- Bioresponsive hydrogels and microgels -- Conjugation strategies used for the preparation of carbohydrate-conjugate vaccines -- Characterization, physico-(bio)chemical properties, and applications of bioconjugates -- Properties and characterization of bioconjugates -- Physico-chemical and biochemical properties of bioconjugates -- Applications of bioconjugates.
- 2006 CRCnetBASEN. Leo Benoiton.Chapter 1. Fundamentals of Peptide Synthesis -- Chapter 2. Methods for the Formation of Peptide Bonds -- Chapter 3. Protectors and Methods of Deprotection -- Chapter 4. Chirality in Peptide Synthesis -- Chapter 5. Solid-Phase Synthesis -- Chapter 6. Reactivity, Protection and Side Reactions -- Chapter 7. Ventilation of Activated Forms and Coupling Methods -- Chapter 8. Miscellaneous.
- 2007 SpringerB.A. Bunin, B. Siesel, G.A. Morales, J. Bajorath.
- 2008 CRCnetBASEeditors, Gordon Rees Jones, Anthony G. Deakin, Joseph W. Spencer.
- Lide, David R.; Weast, Robert C.Also available: Print – 2002/03.
- 2008 CRCnetBASEedited by Anil K. Bhowmick.
- 2011Christina Barnes Cooley.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.
- Design, synthesis, and evaluation of molecular transporters for the delivery of siRNA, and the application of molecular transporters to new cell types and new release strategies2013Erika Isabella Geihe Stanzl.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.
- 2011Minsub Chung.Many cellular processes including cell-cell communications and regulated membrane transport are mediated by membrane proteins and depend upon the ability of lipid membranes to be a differentially permeable barrier. However, the roles and function of membrane proteins are often difficult to study due to the complexity of the native membranes and lack of reliable and flexible artificial model lipid membranes. Supported lipid bilayers (SLB) have been used as a model system to study biological membrane behavior and the structure and function of membrane proteins and receptors in a simpler context apart from the complex cellular environment. Although SLBs have the advantages of simple formation, easy handling and are well-suited for investigation by a suite of surface sensitive methods due to their planar geometry, the close proximity of the lower leaflet to the solid support often leads to unfavorable interactions with integral membrane proteins. This causes distortion of the protein conformation and possible loss of its reactivity and function. Moreover, this interaction with the substrate often traps proteins and reduces their mobility in the membranes. Recognizing this limitation, we have developed a new model membrane architecture in which the DNA-tethered lipid bilayer is either to fixed DNA on a surface or to laterally mobile DNA displayed on a supported bilayer. This separates the lipid membranes from surface interactions and provides a more favorable environment for integral membrane protein with large globular domains. With mobile DNA hybrid tethers, stable tethered bilayers were made with specific lipid composition, while those with fixed tethers are stable regardless of membrane composition. The mobile tethers between a tethered and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions. By careful choice of composition, improved stability was obtained and we can investigate the lateral segregation of DNA hybrids when different lengths are present. Based on a theoretical model, the effects of population, length and affinity of DNA complexes are simulated and described. This model system captures some of the essential physics of synapse formation and is a step toward understanding lipid membrane behavior in a cell-to-cell junction. To demonstrate the excellent environment provided by DNA-tethered membranes for studying transmembrane proteins free from any surface interactions, the behavior of a transmembrane protein, the photosynthetic reaction center, reconstituted in the DNA-tethered membranes is investigated. Inspired by DNA-mediated membrane fusion studies of our group, we applied the DNA-machinery to achieve fusion of small (~ 100 nm) proteoliposomes for delivery of membrane proteins to either giant vesicles or DNA-tethered planar lipid membrane patches. The diffusion behavior of delivered proteins is measured and compared with those in supported bilayers. Also, the protein activity and orientation before and after fusion is analyzed. This will offer a feasible method to incorporate intact membrane proteins to already formed model membranes. In addition, the behavior of proteins during the fusion event will provide insight into the mechanism of DNA-mediated lipid membrane fusion. The geometry of our model membrane system directly mimics that of a neuronal synapse. We expect that this architecture will be readily transferable to other model membrane fusion systems, including systems using reconstituted SNARE proteins. Consequently, it will be of considerable interest to a wide range of researchers.
- 2012Tracy Curtis Holmes, II.Developing novel therapies for gram-negative bacterial infections and glioblastoma multiforme I. cloning and characterization of the guadinomine biosynthetic gene cluster II. developing a novel chemo-sensitizing agent to treat glioblastoma. This thesis explores the development of novel therapies for the treatment of two complicated problems: Gram-negative bacterial infections and glioblastoma multiforme, the most aggressive form of brain cancer. Part I of the thesis summarizes the current body of knowledge regarding guadinomines, their biosynthesis and implications for developing novel anti-infective agents. Part II of the thesis summarizes the development of the small molecule, ERW1227B, and its ability to sensitize glioblastoma cells to standard therapies. Part I. Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509. They are the first microbial metabolites shown to inhibit the Type III Secretion System (TTSS) of Gram-negative bacteria. The TTSS is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli, Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. Inhibition of the TTSS can mitigate virulence which is important considering that Gram-negative bacteria infect millions each year, leading to considerable morbidity and mortality. The guadinomine (gdn) biosynthetic gene cluster has been sequenced, and encodes a chimeric multimodular polyketide synthase -- nonribosomal peptide synthetase spanning 26 open reading frames and 51.2 kb. It also encodes enzymes responsible for the biosynthesis of the unusual aminomalonyl-ACP extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by genetic inactivation of the cluster, as well as heterologous expression and analysis of enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these biologically important compounds. Part II. Glioblastomas display variable phenotypes that include increased drug-resistance associated with enhanced migratory and anti-apoptotic characteristics. These shared characteristics contribute to failure of clinical treatment regimens. Identification of novel compounds that both promote cell death and impair cellular motility is a logical strategy to develop more effective clinical protocols. Previously, we described the ability of the small molecule, KCC009, a tissue transglutaminase inhibitor, to sensitize glioblastoma cells to chemotherapy. In the current study, we synthesized a series of related compounds that show variable ability to promote cell death and impair motility in glioblastomas, irrespective of their ability to inhibit TG2. Each compound has a 3-bromo-4,5-dihydroisoxazole component that presumably reacts with a nucleophilic cysteine thiol residue in one (or more) target protein(s) that have affinity for the small molecule. Our studies focused on the effects of the compound, ERW1227B. Treatment of glioblastoma cells with ERW1227B was associated with both down-regulation of the PI-3 kinase/Akt pathway, which enhanced cell death; as well as disruption of focal adhesions and intracellular actin fibers, which impaired cellular mobility. Bioassays as well as time-lapse photography of glioblastoma cells treated with ERW1227B showed cell death and rapid loss of cellular motility. Mice studies with in vivo glioblastoma models demonstrated the ability of ERW1227B to sensitize tumor cells to cell death after treatment with either chemotherapy or radiation. The above findings identify ERW1227B as a potential novel therapeutic agent in the treatment of glioblastomas.
- 2011Ilya Anatoliy Shestopalov.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.
- Development of genetic tools and techniques for controlling and quantitatively assessing gene-dosage profiles2013Joshua P. Ferreira.Cancer arises from the alteration of genes and the deregulation of the inherent control mechanisms existing with a cell. Cancer progression is the result of several of these genes or pathways being altered. Unfortunately, its analysis is not as straightforward as identifying a handful of discrete, independent mutational events occurring within a cell. Rather, these genes, pathways, and other regulatory elements are interconnected. Altering the expression level (dose) of one gene can have direct and indirect effects on many additional genes/pathways. Furthermore, combinations of genes can interact to have collaborative or antagonistic effects that are greater than or less than the sum of their individual contributions. It is not enough to study the effects of single genes/pathways at a few discrete expression levels. To this end, we have developed genetic tools that allow for controlling gene expression over a full range. Controlling gene expression at the level of transcription allowed for a 40-fold range of expression to be investigated. However, the range in expression of the transcriptionally controlled system varied across cell lines. Expanding beyond this system, we have turned to controlling gene expression at the level of translation. Using translational control elements, we were able to varying gene expression over a 200 -- 300-fold range. Furthermore, the translational control system was shown to be consistent across six different cell lines and with every transgene that has been tested to date. To address the fact that cancer progression is a multi-faceted event, we developed a system that would allow observation of the effects resulting from the interactions of multiple genes. By using retroviral vectors equipped with fluorescent protein fusions, we successfully derived a system that has the capacity to interrogate up to three genes of interest within a single culture of cells. This single culture makes it logistically feasible to study such large combinations of gene dosage levels. This single culture is heterogeneous in expression for each of the transgenes introduced; and by utilizing flow cytometry, the precise dosage level of each transgene can be correlated to measureable phenotypes at the single-cell level. To demonstrate how the tools we have developed can be utilized to quantitatively assess gene-dosage profiles, we ectopically controlled the expression of various mutant forms of the oncogenic version of H-Ras (H-RasG12V) in both murine fibroblast and pre-B cells. We chose to study proliferation as a measurable phenotypic read-out. In NIH/3T3 fibroblasts we observed a maximum in proliferation at low levels of expression of H-RasG12V. A mutant version, H-RasG12V T35S, which is only able to signal down the MAPK pathway, exhibited maximal proliferation at intermediate expression levels. Other H-Ras mutants did not exhibit any proliferation optima when expressed by themselves. In contrast, when the mutants were investigated in pairwise fashion, some cooperation could be observed between particular mutant pairs. Finally, the effect of these H-Ras mutants on proliferation was investigated in a murine pre-B cell line. By adding a reference population of cells to a culture of cells expressing H-Ras mutant oncogenes over a range of expression levels, we were able to track the population dynamics between these two subsets of cells. A simple mathematical approach will be detailed to demonstrate how we can calculate the net proliferation rate as a function of H-Ras expression level by tracking the distribution of these two cell populations over time.
- 2011Jungjoon Kempthorne Lee.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.
- Development of techniques for three-dimensional super-resolution fluorescence microscopy and their application to biological systems2011Michael Anthony Thompson.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.
- 2006 CRCnetBASEedited by Peter M. Collins.
- 2011Andrew Everett Howery.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.
- Dissertation]. Part I, Unnatural thymidine analogs and shape mimics as substrates for human thymidine kinases. Part II, Fluorescent size-expanded DNA analogs as efficient substrates for a template-independent DNA polymerase2011Sarah King Jarchow-Choy.This thesis is composed of two separate studies involving unnatural nucleoside (DNA) analogs in two different types of enzymes: human thymidine kinases 1 and 2 and terminal deoxynucleotidyl transferase (TdT). The ability of two types of unnatural DNA analogs, nonpolar nucleoside analogs and expanded nucleoside analogs, to act as efficient substrates in enzymes will be described. Nonpolar nucleoside analogs lacking the ability to hydrogen bond were synthesized to systematically vary in size and shape, and then used to probe the ability of two types of human thymidine kinases (TK1 and TK2) to recognize and phosphorylate these analogs. The results establish that nucleoside recognition mechanisms for these two classes of thymidine kinase are very different. On the basis of this data, nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells, suggesting new designs for bioactive molecules. Another class of nucleoside analogs, expanded nucleoside (xDNA) analogs, maintain the ability to hydrogen-bond to their respective natural bases, but have enhanced pi-stacking due to their larger size, allowing the molecule to have greater stability in a DNA duplex, and unique fluorescent properties. It was found that terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, can accept multiple xDNA nucleotide analogs as substrates with efficiencies close to that of natural nucleotides. In addition, the expanded adenine (xA) and cytosine (xC) analogs show a visible and spectral change in fluorescence when TdT incorporates multiple analogs. The ease of enzymatic synthesis of these analogs and their inherent fluorescence suggest their use in nucleic acid labeling and hybridization studies. The comparable efficiencies which nonpolar nucleoside analogs and xDNA nucleotide analogs have to natural bases in thymidine kinases and TdT give new information about the steric and electronic requirements of these enzymes, and will be useful for potential therapeutic and biotechnological applications.
- 2012Bettina Van Lengerich.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.
- 2008 HighWireInternational Commission on Radiation Units and Measurements.
- 2001- WileyContains a database of approximately 70,000 reactions and 4000 of the most frequently consulted reagents. Fully searchable by structure and sub-structure, reagent, reaction type, experimental conditions, and keyword. Also includes a searchable interface: Acronym finder (an interface for Scientific and technical acronyms, symbols, and abbreviations).
- Effects of matrix anisotropy and shear flow on endothelial cells : developing a small-diameter vascular graft to regulate endothelial function2012Edwina S. Lai.The development of atherosclerosis, a chronic inflammatory disease of the arteries, can usually be attributed to specific regions of the blood vessel. In the straight segments of an artery, endothelial cells (ECs) align with the unidirectional blood flow which commonly occurs in these simple geometries. The elongated and aligned ECs are generally found to have a healthy, athero-resistant phenotype. In contrast, branches or curved vessel geometries have regions of disturbed flow, characterized by low shear stress and high shear stress gradients. In these regions of complicated flow patterns, ECs are non-aligned and have a cobblestone cellular morphology. The non-aligned ECs elicit biological properties that promote atherosclerosis, as the location of atherosclerotic fatty plaque is often found at these bends, branches, or bifurcations. Therefore, this correlation highly suggests that the morphology and biological function are inextricably linked in ECs. The ability to regulate both EC morphology and motility, with the aim to influence EC biology, might be highly beneficial in the prevention or treatment of vascular disease. In this dissertation, anisotropic matrices of collagen nanofibrils were fabricated with a simple flow processing technique and used to investigate fundamental cell-matrix interactions with ECs. The aligned fibrils were able to regulate both the morphology and biology of ECs, thereby suggesting the nanofibrillar collagen can be a useful tool to maintain vascular homeostasis. The ECs elongated and organized their actin cytoskeleton along the direction of the aligned collagen fibrils, as demonstrated by organized actin, microtubule networks, and focal adhesions. The nanofibrillar collagen also promoted increased cellular migration along the direction of the nanofibrils. The quantification of monocyte adhesion and expression level of adhesion molecules, known testing indicators of atherosclerosis development, suggested the aligned nanofibrils also promoted an athero-resistant phenotype in the ECs. ECs are subject to biophysical cues in vivo, either in the form of surface topography (provided by the basement membrane of the ECM) or the hemodynamic effects of blood flow. The combination of these cues regulate the organization and immunogenicity of ECs and is representative of the in vivo environment. Therefore, we also investigated the endothelial behavior when both types of cues (topography and flow) were simultaneously present. At physiological levels of high shear stress (14-17 dynes/cm2), the matrix-aligned ECs were able to resist reorientation despite shear flow perpendicular to the matrix direction. The anisotropic collagen matrix could preserve the alignment and elongation of ECs as well as promote an athero-resistant phenotype after exposure to antagonistic perpendicular flow. The ability of the anisotropic nanofibrillar collagen to regulate cell morphology and especially EC immunogenicity highlights its potential in the treatment of vascular diseases. Therefore, an aligned conduit of collagen nanofibrils was fabricated to address the need for a small-diameter vascular graft capable of regulating cellular function. The vascular graft was designed to have a mechanical integrity comparable to that of native vessels and was able to regulate EC attachment, morphology, and phenotype. In addition, the aligned collagen grafts could support an anti-thrombogenic surface modification, providing short-term patency in the carotid artery model of Sprague-Dawley rats.
- Efficient access to bryostatin and functional bryostatin analogs : design, synthesis and evaluation of potent bryostatin analogs and the total synthesis of bryostatin 9 using B-ring annulative macrocyclization strategies2011Adam James Schrier.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.
- 2006 CRCnetBASEedited by Johan Sjoblom.
- 1998 Wileyeditor-in-chief, Paul von Ragué Schleyer.
- 2007 CRCnetBASEAlan E. Comyns.
- 2012Jay Thomas Fitzgerald.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.
- [v.3], 2014Jonathan Orsay.
- Fluorophores for single-molecule imaging in living cells : characterizing and optimizing DCDHF photophysics2010Samuel Joseph Lord.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.
- 2007 CRCnetBASEeditor, Carmen Socaciu.
- Handbook for DNA-encoded chemistry : theory and applications for exploring chemical space and drug discovery2014 Wileyedited by Robert A. Goodnow, Jr.Just enough knowledge? -- A brief history of the development of combinatorial chemistry and the emerging need for DNA-encoded chemistry -- A history of DNA-encoding -- DNA-compatible chemistry -- Foundations of a DNA-encoded library (DEL) -- Practices for synthesizing DNA-encoded libraries -- Chemical gene design for DNA-encoded libraries -- Analytical challenges for DNA-encoded library systems -- Information technology: functionality and architectures for DNA-encoding -- Theoretical considerations of the application of DNA-encoded libraries to drug discovery -- Begin with the end in mind : the hit-to-lead process -- Enumeration and visualization of large combinatorial chemical libraries -- Screening large compound collections -- Reported applications of DNA-encoded library chemistry -- Dual-pharmacophore DNA-encoded chemical libraries -- Hit identification and hit follow-up -- Using DNA to program chemical synthesis, discover new reactions, and detect ligand binding -- An outlook and the changing feasibility and economics of chemical diversity exploration with DNA-encoded combinatorial approaches -- Keeping the promise? an outlook on dna chemical library technology.
- 2008 CRCnetBASER.W. Sabnis.
- 2008 CRCnetBASEedited by James P. Landers.
- 2007 CRCnetBASEedited by John Regalbuto.
- 2008 CRCnetBASED. Hank Ellison.
- 2010 CRCnetBASEedited by Jean-Loup Faulon, Andreas Bender.Chapter 1. Representing Two-Dimensional (2D) Chemical Structures with Molecular Graphs / Ovidiu Ivanciuc -- Chapter 2. Algorithms to Store and Retrieve Two-Dimensional (2D) Chemical Structures / Milind Misra, Jean-Loup Faulon -- Chapter 3. Three-Dimensional (3D) Molecular Representations / Egon L. Willighagen -- Chapter 4. Molecular Descriptors / Nikolas Fechner, Georg Hinselmann, Jor̈g Kurt Wegner -- Chapter 5. Ligand- and Structure-Based Virtual Screening / Robert D. Clark, Diana C. Roe -- Chapter 6. Predictive Quantitative Structure-Activity Relationships Modeling: Data Preparation and the General Modeling Workflow / Alexander Tropsha, Alexander Golbraikh -- Chapter 7. Predictive Quantitative Structure-Activity Relationships Modeling: Development and Validation of QSAR Models / Alexander Tropsha, Alexander Golbraikh -- Chapter 8. Structure Enumeration and Sampling / Markus Meringer -- Chapter 9. Computer-Aided Molecular Design / Donald P. "Visco, Jr." -- Chapter 10. Computer-Aided Molecular Design / Diana C. Roe -- Chapter 11. Reaction Network Generation / Jean-Loup Faulon, Pablo Carbonell -- Chapter 12. Open Source Chemoinformatics Software and Database Technologies / Rajarshi Guha -- Chapter 13. Sequence Alignment Algorithms / Tatsuya Akutsu -- Chapter 14. Machine Learning-Based Bioinformatics Algorithms / Shawn Martin -- Chapter 15. Using Systems Biology Techniques to Determine Metabolic Fluxes and Metabolite Pool Sizes / Fangping Mu, Amy L. Bauer, James R. Faeder, William S. Hlavacek.
- 2008 CRCnetBASEedited by Donald A. Burns, Emil W. Ciurczak.
- 2008 CRCnetBASERobert C. Klingender.Chapter 1. Polychloroprene Rubber (CR) / by Rudiger Musch & Hans Magg -- Chapter 2. Acrylonitrile Butadiene Rubber (NBR) / by Robert Klingender -- Chapter 3. Hydrogenated Nitrile Rubber (HNBR) / by Dr. Robert Keller -- Chapter 4. Fluoroelastomers, FKM, and FEPM / by Pascal Ferrandez -- Chapter 5. Polyacrylate Elastomers Properties and Applications / by Robert Klingender -- Chapter 6. Ethylene Acrylic (AEM) Elastomer Formulation Design / by Lawrence C. Muschiatti, Yun-Tai Yu, Edward McBride and Klaus Kammerer -- Chapter 7. Polyepichlorohydrin Elastomer / by Robert Klingender -- Chapter 8. Compounding with Chlorinated Polyethylene / by Ray Laakso -- Chapter 9. Chlorosulfonated Polyethylene (CSM) and Aklylated Chlorosulfonated Polyethylene (ACSM) / by Robert Klingender -- Chapter 10. Ethylene Vinyl Acetate Elastomer (EVM) (ASTM designation AEM) / by Drs. H. Meisenheimer & A. Zens -- Chapter 11. Polysulfide Elastomers / by Stephen K. Flanders & Robert Klingender - Submitted -- Chapter 12. Plasticizers, Process Oils, Vulcanized Vegetable Oils / by Peter Rand -- Chapter 13. Vulcanization Agents for Specialty Elastomers / by Robert Ohm -- Chapter 14. Antioxidants for Specialty Elastomers by Russell Mazzeo -- Chapter 15. Processing Aids for Specialty Elastomers / by Jerry M. Sherritt -- Chapter 16. Considerations in the design of a rubber formulation / by Robert Klingender -- 16A. Oil Field elastomeric Products / by Robert C. Klingender -- 16B. Life Prediction / by John Vicic -- 16C. Compression, Transfer, and Injections Molding of Specialty Elastomers / by Robert W. Keller.
- 2003 Wileyedited by G. Gauglitz and T. Vo-Dinh.v. 1. -- Part I. Sample preparation and sample pretreatment -- ch. 1. Collection and preparation of gaseous samples / Douglas A. Lane -- ch. 2. Sample collection and preparation of liquid and solids / Brian M. Cullum, Tuan Vo-Dinh -- Part II. Methods 1: Optical spectroscopy -- ch. 3. Basics of optical spectroscopy / Martin Hof -- ch. 4. Instrumentation / Valdas Sablinskas -- ch. 5. Measurement techniques / Gerald Steiner -- ch. 6. Applications / Valdas Sablinskas, Gerald Steiner, Martin Hof -- Part III. Methods 2: Nuclear magnetic resonance spectroscopy -- ch. 7. An Introduction to solution, solid-state, and imaging NMR spectroscopy / Leslie G. Butler -- ch. 8. Solution NMR spectroscopy / Gary E. Martin, Chad E. Hadden, David J. Russell -- ch. 9. Solid-state NMR / Steven P. Brown, Lyndon Emsley -- Part IV. Methods 3: Mass spectrometry -- ch. 10. Mass spectrometry / Michael Przybylski, Wolfgang Weinmann, Thilo A. Fligge -- Part V. Methods 4: Elemental analysis -- ch. 11. X-ray fluorescence analysis / K. Janssens -- ch. 12. Atomic absorption spectrometry (AAS) and atomic emission spectrometry (AES) / Erwin Rosenberg, Ulrich Panne -- Part VI. Methods 5: Surface analysis techniques -- ch. 13. Surface analysis techniques / A. Macková, S.A. Morton, C.G.H. Walker, K. Volka -- v. 2. -- Part VII. Applications 1: Bioanalysis -- ch. 14. Bioanalysis / Willem M. Albers, Arto Annila, Nicholas J. Goddard, Gabor Patonay, Erkki Soini -- Part VIII. Applications 2: Environmental analysis -- ch. 15. LC-MS in environmental analysis / H.Fr. Schröder -- ch. 16. Gas chromatography/ion trap mass spectrometry (GC/ITMS) for environmental analysis / Michel Sablier, Toshihiro Fujii -- Part IX. Application 3: Process control -- ch. 17. Optical spectroscopy / John Green -- ch. 18. NMR / Loring A. Weisenberger -- ch. 19. Process mass spectrometry / Christian Hassell -- ch. 20. Elemental analysis / J.S. Crighton -- Part X. Hyphenated techniques -- ch. 21. Hyphenated techniques for chromatographic detection / John C. Fetzer -- Part XI. General data treatment: data bases/spectral libraries -- ch. 22. Optical spectroscopy / Steffen Thiele, Reiner Salzer -- ch. 23. Nuclear magnetic resonance spectroscopy / Wolfgang Robien -- ch. 24. Mass spectrometry / Antony N. Davies.
- 2007 CRCnetBASEedited by Leo M.L. Nollet.
- 2002 Knovelrevised by Richard J. Lewis, Sr.Abbreviations -- Condensed chemical dictionary -- Appendix I: Origin of some chemical terms -- Appendix II: Highlights in the history of chemistry -- Appendix III: Manufacturers of trademarked products (alphabetical list).
- 2009 CRCnetBASEedited by Perry G. Wang.High throughput sample preparation techniques and their application to bioanalytical protocols and purification of combinatorial libraries / Krishna Kallury -- High-throughput quantitative bioanalysis / Katty X. Wan -- Optimizing LCMS equipment to increase throughput in pharmaceutical analysis / Michael G. Frank and Douglas E. Mcintyre -- Throughput improvement of bioanalytical LC-MS/MS by sharing of detector between HPLC systems / Min Shuan Chang and Tawakol El-Shourbagy -- High throughput strategies for metabolite identification in drug discovery / Patrick J. Rudewicz, Qin Yue, and Young Shin -- Utilizing micro parallel liquid chromatography for high-throughput analyses in the pharmaceutical industry / Sergio A. Guazzotti -- Strategies and techniques for higher throughput ADME/PK assays / Walter Korfmacher -- High-throughput analysis in drug metabolism during early drug discovery / Yau Yi Lau -- High-throughput analysis in the support of process chemistry and formulation research & development in the pharmaceutical industry / Zhong Li -- On-line SPE LC/MS/MS for high throughput bioanalytical analysis / Dong Wei and Liyu Yang -- Applications of high-throughput analysis in therapeutic drug monitoring / Quanyun A. Xu and Timothy L. Madden -- High-throughput quantitative pharmaceutical analysis in drug metabolism and pharmacokinetics (DMPK) using liquid crhomatrography-mass spectrometry / Xiaohui Xu -- Designing high throughput HPLC assays for small and biological molecules / Roger K. Gilpin and Wanlong Zhou -- The advances in capillary and nano-HPLC technology for drug discovery and development / Frank J. Yang and Richard Yu -- High-throughput analysis of complex protein mixtures by mass spectrometry / Kojo S.J. Elenitoba-Johnson.
- 2005 CRCnetBASESangeeta, D.; LaGraff, John R.
- 2006 SpringerPaul Lecoq ... [et al.].
- 2012 WileyT.D.H. Bugg.From Jack Beans to designer genes -- All enzymes are proteins -- Enzymes are wonderful catalysts -- Methods for studying enzymatic reactions -- Enzymatic hydrolysis and group transfer reactions -- Enzymatic redox chemistry -- Enzymatic carbon-carbon bond formation -- Enzymatic addition/elimination reactions -- Enzymatic transformations of amino acids -- Isomerases -- Radicals in enzyme catalysis -- Non-enzymatic biological catalysis.
- 2012 WileySteen Hansen, Stig Pedersen-Bjergaard, Knut Rasmussen.Introduction to pharmaceutical analysis -- International pharmacopoeias, regulations and guidelines -- Fundamental chemical properties, buffers and pH -- Fundamentals of pharmaceutical analysis -- Titrimetric methods -- Introduction to spectroscopic methods -- UV-spectrophotometry -- IR-spectrophotometry -- Atomic spectrometry -- Fundamentals of chromatography -- Chromatographic separation principles -- Thin-layer chromatography -- High-performance liquid chromatography -- Gas chromatography -- Capillary electrophoresis -- Mass spectrometry -- Miscellaneous chemical techniques -- Sample preparation -- Analytical chemical characteristics of selected drug substances -- Quantification and quality of analytical data -- Chemical analysis of drug substances -- Chemical analysis of final pharmaceutical products -- Bioanalysis.
- 2007 NLMprepared for publication by John H. Duffus, Monica Nordberg, Douglas M. Templeton.
- Jokichi Takamine : the man who gave "adrenaline" to the world : English translation of the panels exhibited at the National Science Museum, Tokyo, Japan, December 10, 2004-January 10, 2005 in commemoration of the 150th anniversary of Takamine's birth.2007Ishida, Mitsuo; Nakatsugawa, Tsutomu.
- 2005- WileyKirk, Raymond E.; Othmer, Donald F.Includes risk management, enterprise resource planning, outsourcing, combinatorial synthesis and technology, functional foods, process automation, electronic chemicals, specialty silicones, mergers and acquisitions, nanoparticles, bioinformatics, ISO 14000, micron-scale chemical analysis, medical applications of biodegradable materials, product development, strategies, drug discovery strategies, chemistry of aging, single-site catalysis, custom manufacturing, and global chemical market analysis.
- Lead optimization for medicinal chemists : pharmacokinetic properties of functional groups and organic compounds2012 WileyFlorencio Zaragoza Dorwald.Part I : Introduction. The drug discovery process ; lead optimization -- Part II : The pharmacokinetic properties of compound classes. Alkanes ; Alkenes and alkynes ; Arenes ; Halides ; Azides ; Nitro compounds ; Azo compounds ; Triazenes ; Nitrates and nitrites ; N-nitroso compounds ; N-oxides ; Alcohols ; Phenols ; Ethers ; Epoxides ; Peroxides ; Thiols ; Thioethers ; Sulfoxides ; Sulfones ; Aliphatic amines ; Quaternary ammonium salts ; Amidines ; Guanidines, acylguanidines, and biguanides ; Anilines ; Hydrazines, acylhydrazines, and hydrazones ; Aldehydes ; Ketones ; Carboxylic acids ; Carboxylic esters ; Amides ; Lactams and imides ; Nitriles ; Carbonates ; Carbamates ; Ureas ; Thiocarbonyl compounds ; Sulfonic acids ; Sulfonic esters ; Sulfates And sulfamic acids ; Phosphonic acids ; Phosphoric acid derivatives ; N-(aminoalkyl)benzamides, -benzoates, and related compounds ; Arylalkylamines ; Phenethylamines (2-phenylethylamines) ; Aminoalkylindoles and indole alkaloids ; Phenothiazines ; Dibenzazepines and related tricyclic compounds ; 3-aryloxy-2-hydroxypropylamines (b-adrenergic antagonists; 'b-blockers') ; Opiates ; N-(carboxyalkyl)-a-amino acid amides (prils) ; Anilides and amides of glycine ; Peptides, peptidomimetics, and related oligoamides ; Oligoarylamines, oligoarylamides, oligoarylcarbamates, and oligoarylureas ; Imidazoles ; Triazoles ; Pyridines, pyrimidines, and related compounds ; Quinolines ; Nucleoside analogs ; Dihydropyridines ; Arenesulfonamides ; Sulfonylureas ; Benzodiazepines ; Steroids ; Anthracyclines ; Arylacetic, benzoic, and related carboxylic acids (NSAIDS) ; Quinolonecarboxylic acids (gyrase inhibitors) ; B-lactams ; Prostaglandin analogs ; Sartans ; Statins ; Folic acid analogs (antifolates) ; Taxanes ; Macrocyclic compounds.
- 2014 Wileyeditor, Tim Storr.Introduction to ligand design in medicinal inorganic chemistry -- Platinum-based anticancer agents -- Coordination chemistry and ligand design in the development of metal based radiopharmaceuticals -- Ligand design in D-block optical imaging agents and sensors -- Luminescent lanthanoid probes -- Metal complexes of carbohydrate-targeted ligands in medicinal inorganic chemistry -- Design of Schiff base-derived ligands: applications in therapeutics and medical diagnosis -- Metal-based antimalarial agents -- Therapeutic gold compounds -- Ligand design to target and modulate metal-protein interactions in neurodegenerative diseases -- Rational design of copper and iron chelators to treat Wilson's disease and hemochromatosis -- MRI contrast agents -- Photoactivatable metal complexes and their use in biology and medicine -- Metalloprotein inhibitors -- Ruthenium anticancer compounds with biologically-derived ligands.
- 2007 CRCnetBASEedited by Frank D. Gunstone, John L. Harwood, Albert J. Dijkstra.
- [v. 5], 2016edited by Alexander Stone Macnow, MD.Atomic Structure -- The Periodic Table -- Bonding and Chemical Interactions -- Compounds and Stoichiometry -- Chemical Kinetics -- Equilibrium -- Thermochemistry -- The Gas Phase -- Solutions -- Acids and Bases -- Oxidation-Reduction Reactions -- Electrochemistry.
- [v. 6], 2016edited by Alexander Stone Macnow, MD.Nomenclature -- Isomers -- Bonding -- Analyzing Organic Reactions -- Alcohols -- Aldehydes and Keotones I: Electrophilicity and Oxidation--Reduction -- Aldehyde and Keo tones II: Enolates -- Carboxylic Acids -- Carboxylic Acid Derivatives -- Nitrogen- and Phosphorus-Containing Compounds -- Spectroscopy -- Separations and Purifications.
- MCR 2009 : proceedings of the 4th International Conference on Multi-Component Reactions and Related Chemistry, Ekaterinburg, Russia2011 SpringerMaxim A. Mironov, editor.Catalysis and multi-component reactions -- Multi-component reactions in heterocyclic chemistry -- Multi-component reactions in drug discovery -- Novel reagents for multi-component reactions -- Design of multi-component reactions -- Multi-component reactions in supramolecular chemistry and material science.Also available: Print – 2011
- 2013Maryadele J. O'Neil, editor-in-chief ; Patricia E. Heckelman, senior associate editor ; Peter H. Dobbelaar, associate editor ; Kristin J. Roman, assistant editor ; Catherine M. Kenny, senior editorial assistant ; Linda S. Karaffa, technical assistant.
- 2007 CRCnetBASEMohammad Niyaz Khan.
- Modulated temperature differential scanning calorimetry : theoretical and practical applications in polymer characterisation2006 Springeredited by Mike Reading and Douglas J. Hourston.
- Molecular design : part I, synthesis of cyclooctatetraenes as ligands for catalysis; part II, studies on the C1B binding domain of protein kinase C2012Adam B. Lesser.The concepts of molecular analysis and design bring together structure, mechanism, and synthesis into a useful whole by allowing them to work toward a common goal. This thesis explores these concepts in two major subject areas: New Reactions and Protein/Ligand Interactions. In the first subject area, this work explores a major goal of organic synthesis, the realization of functional value of a molecular target via step economy. Indeed step economy dictates all other economies and costs including time, waste stream, and environmental goals. Improving known reactions is an important part of advancing synthesis. Of even greater consequence is the introduction of new reactions as they change how we think about bond construction. New reactions offer new ways to reduce step count and more effective and environmentally sound process options. This thesis work focuses on new reactions, new scaffolds, novel ligands for catalysis, and computational studies on these to advance this first subject area. In the second subject area, this work investigates protein/ligand interactions in the context of a cooperative project involving protein kinase C ligands. Molecular design and analysis have been fundamental in the development and implementation of this new project. This exciting area of impactful research is expected to provide numerous opportunities for further exploration. In Chapter 1, cyclooctatetraenes (COTs) are introduced as a fascinating class of molecules with great potential utility as building blocks for synthesis, scaffolds for drug discovery, ligands for catalysis, components for molecular detection devices, and novel materials. After discussing the synthesis, properties and applications of COTs, a discussion on metal-bound COTs is presented. This discussion is followed by a comprehensive critical review of the literature involving 1,2,5,6-4 COT ligands in transition metal complexes. This chapter provides background and justification for the further studies on the synthesis and use of COTs explored in Chapters 2-4. Chapter 2 describes the development of the Ni(0)-catalyzed [2+2+2+2] cycloaddition from tethered diynes that has made possible the synthesis of functionalized COTs. The initial design and development work is addressed, including the impact of important reaction variables. This is followed by an exploration of the substrate scope, including the syntheses of highly substituted COTs from non-terminal diynes to give highly substituted COTs. Studies on the physical properties and structure of these molecules are also addressed in the form of NMR experiments and X-ray crystallographic studies. Lastly, an approach toward the COT-containing natural product caulerpin utilizing the methodology is presented. This chapter provides the foundation for the COTs utilized in Chapters 3 and 4. Chapter 3 explores the use of the method developed in Chapter 2 toward metal binding ligands. A rational analysis of the COT scaffold is presented, with an emphasis on the synthesis of C2-symmetric ligands for metals. This is followed by the discussion of the synthesis and evaluation of two different ligand families encompassing 4 ligands in total. Important structural aspects of these ligands are investigated including advanced NMR and crystallographic studies. These ligands represent the first examples of topologically chiral racemic COTs used to coordinate metals through appended functionality. Initial successes and modeling studies in this area of metal-ligand binding led to the inspiration for the COTs and strategy utilized in Chapter 4. Chapter 4 is focused on the development of novel dinaphthocyclooctatetraene (dnCOT) Rh catalysts, and the resulting [5+2] cycloaddition studies. Initial unsuccessful results with COTs from Chapter 2 prompted a detailed analysis of factors involved in COT-metal bonding, including the development of a theoretical method for the prediction of COT geometries. This design-related work in turn guided the efficient synthesis of the dnCOT ligand. In addition, an expedient route to diversified dnCOTs was developed via a common bromide intermediate. The further development of dnCOT as a ligand for Rh included extensive characterization of the ligand and resulting Rh complexes in the form of crystallographic and NMR studies. Following these studies, the use of the Rh/dnCOT complex as a catalyst is examined in the context of the [5+2] cycloaddition. It was found that this species is an excellent catalyst for the reaction featuring superior reactivity in several cases when compared with other catalysts. The behavior of this catalyst was also investigated with respect to regioselectivities. The use of the Rh/dnCOT complex has also been briefly explored as a catalyst for other reactions. Finally, Chapter 5 examines the design and early implementation of a collaborative project involving protein kinase C (PKC) C1b domain ligands and REDOR NMR techniques. An introduction to PKC, bryostatin and REDOR NMR is given, followed by the overall project strategy. The design of NMR-labeled analogs of bryostatin is discussed in detailed, including computational studies used to help guide synthetic efforts. Preliminary work on elucidating the structure of the PKC/bryostatin complex is discussed in the form of docking studies to be integrated with experimental NMR data. The successful synthesis of one of the key labeled analogs designed in the previous sections, labellog 8, is described. Lastly, future directions for this promising project are explored.
- 2006 Springeredited by Richard G. Weiss and Pierre Terech.
- 2011Sarah Paige Sherlock.In recent years there has been a growing interest in utilizing nanomaterials for drug delivery and biomedical imaging applications. This work focuses on the development of two multifunctional graphitic-carbon based nanomaterials capable of acting as both drug delivery agents and as contrast agents for either magnetic resonance imaging or near-infrared fluorescence imaging. Both of these agents heat under near-infrared light and are capable of loading chemotherapy drugs making them multifunctional in nature. The first material discussed is a FeCo-graphitic carbon nanocrystal loaded with doxorubicin. Addition of near-infrared photothermal therapy significantly increases the cellular toxicity of these nanocrystals in vitro. Treatment of breast cancer tumors in mice using combined nanocrystal drug delivery and photothermal therapy resulted in complete tumor regression in 45% of mice. The imaging capability of these nanocrystals is demonstrated through high-resolution magnetic resonance imaging of microvessels in rabbits. The potential long-term biodistribution and safety of this material is evaluated. The second graphitic-carbon nanomaterial used in this work is single-walled carbon nanotubes. This material is developed as a deep-tissue fluorescent imaging agent due to their inherit photoluminescence beyond 1 micron. This light emission is demonstrated to be particularly useful for in vivo imaging by minimizing light scattering by tissues leading to crisp anatomical resolution.
- New cell-free platform for effective site-specific incorporation of non-natural amino acids and screening for orthogonal components2012Cem Albayrak.Proteins in all living systems are composed predominantly of 20 natural amino acids, each of which is incorporated by a processive mechanism involving dedicated transfer RNAs and enzymes called aminoacyl-tRNA synthetases. Despite the diversity in protein structure and function, these natural amino acids provide side chains with only limited reactivity. Site-specific incorporation of non-natural amino acids (nnAAs) expands the chemical reactivity of proteins and enables their precise post-translational modification. These nnAAs are incorporated in a manner analogous to natural amino acids. An orthogonal synthetase specific to the nnAA first couples the nnAA to an orthogonal amber suppressor tRNA (o-tRNA). The aminoacylated o-tRNA then forms a three-molecule complex with Ef-Tu and GTP, enters the ribosome, and at an amber stop codon (UAG) in the mRNA, adds the nnAA to the nascent polypeptide chain. Of the more than 30 nnAAs that have been site-specifically incorporated using Escherichia coli-based protein synthesis systems, the Swartz laboratory has been particularly interested in the nnAAs p-azido-L-phenylalanine (pAzF) and p-propargyloxy-L-phenylalanine (pPaF), since proteins containing these nnAAs can be directly coupled using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In our laboratory, this technology is used to design unique and effective vaccines and diagnostic agents. Despite the improvements in the versatility and productivity of the cell-free protein synthesis (CFPS) platform, the yields of proteins containing nnAAs (i.e. modified proteins) have been substantially lower than their natural counterparts. The first part of this dissertation summarizes our efforts in improving the current methods for site-specific nnAA incorporation. Supplementing the initial CFPS system with additional o-tRNA doubled the modified protein yields. Titration experiments and turnover calculations suggested that, despite the apparent o-tRNA limitation, the synthetase is the more inefficient orthogonal component. Nonetheless, a convenient and modular method for adequate o-tRNA supply was demonstrated to further improve the efficiency of nnAA incorporation. Using this new method, in which the o-tRNA and the modified protein are produced simultaneously during the CFPS reaction, as much as 1.67 mg/ml full-length and soluble modified super-folder GFP (sfGFP) was obtained. This represents a 6-fold improvement over previous cell-free yields of modified sfGFP. This new method was used then to study nnAA incorporation at twelve different sites in sfGFP using two different o-tRNA sequences. Our results did not confirm the previous trends for the position effect (i.e. variation of modified protein yield with nnAA incorporation position). The use of the o-tRNA that was developed to better recognize the endogenous Ef-Tu decreased the size of the position effect. In addition, the method employing in situ o-tRNA synthesis was extended to the incorporation of the nnAAs into sfGFP at 2 or 3 positions. These proteins were subsequently purified and coupled to synthesize linear and branched protein polymers. After polymerization and removal of the catalyst, the specific activity of the proteins was fully retained. The second part of this dissertation describes our efforts towards developing a new set of orthogonal components from the endogenous tyrosyl tRNA/synthetase pair. A cell-free method was first developed to screen for an orthogonal variant of the E. coli tyrosyl tRNA. 201 potential orthogonal tRNAs were identified out of a library containing 384 tRNA mutants. Eight were more carefully analyzed for orthogonality (i.e. absence of cross-reactivity with E. coli synthetases) and one was identified to be both sufficiently orthogonal and not inhibitory to CFPS. Finally, the in vitro compartmentalization (IVC) method was adapted for the production of sfGFP in emulsion CFPS reactions. A method was also developed to quantify the number of adsorbed genes on a bead by fluorescence using FACS. This protocol promises to be a powerful high-throughput method for screening orthogonal synthetases.
- 2007 ScienceDirectedited by Bruno Tota and Barry Trimmer.
- 2015 SpringerChristopher Ahern, Stephan Pless, editors.Introduction -- Engineered ionized side chains -- Cysteine modification: probing channel structure, function, and conformational change -- Functional site-directed fluorometry -- Bioreactive tethers -- Flipping the photoswitch: ion channels under light control -- Incorporation of non-canonical amino acids -- Index.Also available: Print – 2015
- 2010Kyung-sun Son.My Ph.D. work primarily involves the synthesis of ethylene-based oligomers and ethylene-styrene copolymers using organometallic catalysis. Chapter 1 reviews selective ethylene oligomerization that produces 1-hexene and 1-octene, with particular emphasis on the chromium-based catalytic systems and the mechanism by which they operate. Its application to the preparation of value-added chemicals is also covered. Chapter 2 and Chapter 3 present investigations on selective ethylene oligomerization with a Cr(PNP)Cl3/MAO catalyst system (PNP = Ph2PN(iPr)PPh2) in the presence of dialkyl zinc as an effective strategy for the co-generation of 1-octene and functionalized ethylene oligomers. Transmetallation with ZnMe2 during Cr-catalyzed ethylene tetramerization generated end-labeled 1-alkenes in Cn> 10 along with 1-octene, while that with ZnEt2 or ZnBu2 produced a mixture of end-labeled linear alkanes and 1-alkenes in Cn> 10 as well as 1-octene. Labeling studies with D2O provided a mechanistic test for metallacycle intermediates. Mechanistic proposals are presented to explain the formation of end-labeled products in the presence of various types of zinc alkyls. Chapter 4 and Chapter 5 examine a series of titanocenes [CpTiCl3, CpTiCl2TEMPO, CpNTiCl3, CpNTiCl2TEMPO, where Cp = C5H5, CpN = C5H4CH2CH2N(CH3)2, and TEMPO = 2,2,6,6-tetramethylpiperidine-N-oxyl] for styrene homopolymerization and ethylene-styrene (ES) copolymerization at 70 °C and 120 °C to determine the influence of the pendant amine group and the hydroxylaminato ligand on comonomer incorporation and distribution. Titanocenes bearing the pendant amine ligand were active for ES copolymerization, whereas complexes lacking the pendant group afforded only mixtures of homopolymers, revealing the critical role of the pendant amine donor on the copolymerization behavior. At 120 °C, the titanocene complexes with the amine group generated high molecular weight ES copolymers along with an atactic polystyrene (aPS) byproduct. The molecular weight of the ES copolymers produced by a coordination mechanism was found to coincide with that of the aPS produced by a radical polymerization mechanism. A method to separate these two components was developed by the addition of a catalytic chain-transfer agent, cobalt tetraphenylporphyrin, successfully decreasing the molecular weight of the radically-produced aPS and offering expedient separation of the ES copolymer from the aPS. Chapter 6 describes a series of new mono-, bi-, and trimetallic complexes based on the dinucleating ligand, N, N'-bis[2-(diphenylphosphino)phenyl] formamidine (PNNP), which provides two binding sites suitable for accommodating Pd, Ni, Co, Fe, and Cu. Definitive evidence for the structures of all complexes were given by X-ray crystal structures. The synthesis, characterization and crystal structure of each complex are discussed.
- edited by Lester Packer.Also available: Print – 1994
- 2006 Springerby Wilfred Vermerris and Ralph Nicholson.
- 2006 Springervolume editors, Claudia Steinem, Andreas Janshoff ; with contributions by M.A. Cooper ... [et al.].
- 2012 Springeredited by Zdenko Machala, Karol Hensel, and Yuri Akishev.
- 2013 WileyAlexander Fridman and Gary Friedman.Introduction to fundamental and applied aspects of plasma medicine -- Fundamentals of plasma physics and plasma chemistry relevant to biological and medical applications -- Selected concepts in biology and medicine for physical scientists -- Major plasma disharges and their applicability for plasma medicine -- Mechanisms of plasma interactions with cells -- Plasma sterilization of different surfaces and living tissues -- Plasma decontamination of water and air streams -- Plasma treatment of blood : non-thermal plasma-assisted blood coagulation and plasma control of blood properties -- Plasma assisted healing and treatment of diseases -- Plasma pharmacology -- Fundamentals of plasma-assisted tissue engineering and plasma processing of polymers.
- 2014 SpringerTheodore Eliades, George Eliades, editors.The polycarbonate products, adhesives, and composite resins used in dentistry may have the potential to release bisphenol-A (BPA). BPA is known to exert effects at very low doses and presents a risk to reproductive, neurological, behavioural, and metabolic development, however, the actual effects induced by dental materials have not been sufficiently covered and critically analyzed. Nevertheless, many practicing dentists will be confused by the occasionally contradictory and often misinterpreted evidence in the literature. This book therefore represents a timely and comprehensive review of our current knowledge of BPA release from dental polymers and the potential presence of endocrinological consequences. After a review of the history and evolution of the issue within the broader biomedical context, the estrogenicity of BPA is explained. The basic chemistry of the polymers used in dentistry is then presented in a simplified and clinically relevant manner. Key chapters in the book carefully evaluate the release of BPA from dental polymets and the estrogenicity of these materials. Currently available evidence on the potential estrogenic action of dental composites, sealants, and adhesives is presented, and the exaggerated conclusions of various methodological protocols are assessed. The entire dental community will find this book to be an invaluable aid to safe practice.
- 2007 SpringerJoachim Koetz, Sabine Kosmella.
- 2008 CRCnetBASEJerry Workman, Jr., Lois Weyer.Introduction to near-infrared spectra -- Alkanes and cycloalkanes -- Alkenes and alkynes -- Aromatic compounds -- Hydroxyl containing compounds -- Water -- Carbonyls -- Amines and amides -- P-H and S-H -- Carbohydrates -- Amino acids, peptides, and proteins -- Synthetic polymers and rubbers -- History of near-infrared (NIR) applications.
- 2012Colin James Bell Harvey.Macrolides have long been among the most widely used antibiotics. Despite this utility, development of new macrolides through traditional synthetic and semisynthetic approaches has been greatly hindered by the inherent structural complexity of these compounds. Precursor-directed biosynthesis is a technique which circumvents this difficulty by incorporating simple synthetic precursors into a biosynthetic pathway, allowing the bulk of the molecule to be constructed enzymatically. This dissertation describes the evolution and application of a system for the facile production of new macrolides through precursor-directed biosynthesis. The results of this work are the discovery of an unexpected macrolide structure-activity relationship and the ultimate discovery of a promising new lead for macrolide development.
- 2006 CRCnetBASEedited by Teresa Kowalska, Joseph Sherma.
- Production of complex heterologous proteins and protein assemblies using E. coli-based cell-free protein synthesis2011John Patrick Welsh.The Swartz lab has put much effort into understanding the underlying principles of E. coli-based cell-free protein synthesis (CFPS), and the technology has developed into a scalable, affordable platform for producing a wide range of protein targets. Key breakthroughs have included activating central metabolism, stabilization of critical amino acids, controlling the redox environment to produce proteins containing disulfide bonds, and using scale-up technologies to produce proteins at milligram quantities. My work has sought to expand this CFPS technology for producing valuable and complex eukaryotic protein targets by manipulating and optimizing the folding of these proteins in the heterologous CFPS environment. Furthermore, I have sought to apply these advances to specific applications of interest. By modifying a key molecular chaperone native to the eukaryotic endoplasmic reticulum (ER), the Hsp70-family chaperone, BiP, soluble production was increased in CFPS reactions for specific proteins normally secreted through this organelle, namely those from the immunoglobulin superfamily which includes antibodies, T-cell receptors, and many membrane receptors. First, the functional properties of BiP were compared to that of the E. coli Hsp70, DnaK. A fusion protein was then constructed between BiP and the ribosome-binding portion of the E. coli protein, trigger factor, to localize BiP to translating ribosomes. This replicated the native function of BiP, which provides co-translational folding assistance to nascent polypeptides. After verifying its bioactivity, this fusion protein was utilized in CFPS reactions to indicate that the chaperone functions of BiP are specific to proteins normally secreted through the eukaryotic ER, whereas DnaK demonstrates a more general chaperone mechanism. Since the discovery that somatic cells could be reprogrammed back to a pluripotent state through the viral expression of a specific set of transcription factors, there has been great interest in reprogramming using a safer and more clinically relevant protein-based approach. Production of these transcription factor proteins was greatly increased by fusing them to the C-terminus of the solubility partner, IF2 domain 1 (IF2D1). While the fusions provided marginal benefit in molar yields using a CFPS approach, in vivo E. coli expression produced the transcription factors in soluble form. The fusion proteins could be purified in milligram quantities from liter shake-flask cultures, whereas essentially no soluble protein accumulated without the fusion partner. The transcription factor fusions bound specifically to their consensus DNA sequences and partially activated some of their downstream gene targets. Another application utilizing CFPS technology is an enhanced luciferase mutant from the marine copepod, Gaussia princeps (GLuc). GLuc is both the smallest and brightest known luciferase, and previous work from our lab demonstrated that this protein could be produced at higher volumetric yields and specific activities in CFPS compared to conventional protein expression systems. By mutating key residues in the Gaussia luciferase sequence, the luminescence half-life was shown to increase over ten-fold while maintaining the initial specific activity of the wild-type. This improved mutant provides a valuable imaging agent to use in fusions and bioconjugates with other proteins such as those that recognize cell surface markers on cancer cells. In a final application, influenza vaccines were produced using CFPS by isolating specific fragments of the protein hemagglutinin (HA), a viral surface protein. Specific mutations as well as a C-terminal trimerization domain were critical for producing this protein fragment in both its monomeric and native trimeric forms. By using the CFPS platform to incorporate non-natural amino acids (nnAAs) with alkyne functional groups, the HA proteins were covalently 'clicked' to virus-like particles (VLPs) that had surface exposed nnAAs with azide functionality. The VLPs provide an immunogenic delivery platform that efficiently traffics to lymph nodes and allows for co-attachment of other adjuvants in addition to the primary HA antigen. This vaccine platform was characterized and tested in mouse models and compared to both a standard influenza vaccine as well as free HA protein fragments. In summary, CFPS is a valuable and robust method of protein production for a variety of targets. My thesis has sought to use this platform as a means to better understand folding pathways of complex, eukaryotic proteins and improve production of these proteins. To this end, CFPS has been shown to be a valuable method for elucidating and manipulating chaperone function, producing difficult proteins with enhanced function, and as a platform to produce novel vaccines.
- 2012Debanti Sengupta.There is a tremendous need for muscle regeneration therapies to replace tissue that is lost due to disease or injury. This work is focused on developing engineered replacement therapies for lost or damaged skeletal and cardiovascular muscle tissue. In order to create viable, clinically relevant regenerative therapies, I have used recombinant protein technology to synthesize a family of protein-engineered biomaterials that incorporate critical cues for the recapitulation of in vivo muscle tissue conditions. Crucially, these biomaterials allow the independent tuning of multiple biomaterial properties including cell adhesivity, biomaterial stiffness, and topographical cues. Each of these biomaterial properties impacts muscle cell behavior independently as well as in concert. I have exploited the range of biomaterial tunability available to us to identify biomaterial conditions that enable production of striated muscle tissue using primary human skeletal muscle myoblasts (hMBs) that may be electrically paced. I have further demonstrated that mouse model skeletal muscle cell lines do not accurately recapitulate primary human myoblast behavior in response to biomaterial properties, particularly with respect to integrin-matrix interactions. Importantly, the expression of mature muscle markers that indicate muscle cell differentiation could be accelerated simply by changing biomaterial stiffness and introducing biomaterial microtopography. Finally, as demonstration that this protein-engineered biomaterial strategy may have application in the regeneration of myocardial as well as skeletal muscle tissue, cardiomyocytes derived from human embryonic stem cells exhibited good cell viability and improved contractility on these biomaterials. This thesis represents important advancements in the development of regenerative muscle tissue engineered constructs.
- Ruthenium-catalyzed redox- and cycloisomerizations : atom economic syntheses of carbo- and heterocycles2010Alicia Carolina Gutierrez.A facile and 100% regioselective cycloisomerization--6-pi-cyclization method for obtaining pyridines is described. The unsaturated ketones and aldehydes derived from the cycloisomerization of primary and secondary propargyl diynols in the presence of CpRu(MeCN)3PF6 are converted to 1-azatrienes which in turn undergo a subsequent electrocyclization--dehydration to provide pyridines with excellent regiocontrol. The ruthenium-catalyzed cycloisomerization is a mild, atom-economical method for obtaining the requisite dienone and dienal substrates. The cycloisomerization--6-pi-cyclization sequence may be carried out in one pot or in two independent steps. Additionally, the azatriene cyclization may be carried out in 1-3 hours under microwave irradiation or, for more sensitive substrates, at lower temperatures for longer periods of time (6--24 hours at 90 °C). An atom-economical method for the convenient synthesis of tetrahydropyrans and tetrahydrofurans is also described. Enones and enals derived from the [IndRu(PPh3)2Cl]-catalyzed redox-isomerization of primary and secondary propargyl alcohols undergo a subsequent intramolecular conjugate addition to provide cyclic ethers in excellent yields. Lastly, we have developed complementary methods for the transition metal-catalyzed enyne cycloisomerizations of cyclic olefins. By using distinct ruthenium and palladium catalysts, decalins and 7,6-bicycles can be obtained with dichotomous stereochemical outcomes. The change in mechanism that accompanies the change in metal affords trans-fused 1,4-dienes with ruthenium and their cis-fused diastereomers under palladium catalysis. In the reactions under ruthenium catalysis, a coordinating group is required, and acts to direct the metal to the same side of the carbocycle, resulting in the observed trans diastereoselectivity. Subtle changes in the carbocyclic substrate led to the discovery of a heretofore-unobserved mechanistic pathway, providing bicyclic cycloisomerization products under palladium catalysis, tricyclic products under ruthenium catalysis in DMA, and a 1:1 mixture of the two under ruthenium catalysis in acetone. The different coordination requirements of the two paths allow for the reaction to be shuttled through the metallacycle pathway (generating tricyclic products) when DMA is used as a solvent. This method of obtaining these cyclobutene products complements the existing methods for catalyzing the [2+2] cycloaddition of alkynes. While other methods have been demonstrated for larger ring sizes with palladium, and for specific alkyne termini with platinum and gold, this method is unique in its substrate scope. To the best of our knowledge, this is the first example of a [2+2] cycloaddition catalyzed by CpRu(MeCN)3PF6.
- 2009 CRCnetBASET.S.S. Dikshith.
- 2013William Hazen Parsons.Voltage-gated sodium channels (NaV) serve an essential role in physiology. Accordingly, their dysfunction is associated with a number of human diseases and disorders, including epilepsy, cardiac arrhythmias, and chronic pain conditions. Designed by Nature as a chemical weapon, the naturally occurring channel blocker (+)-saxitoxin (STX) can be repurposed as a tool for studying these large integral membrane proteins. Access to new toxin derivatives through de novo synthesis offers a unique strategy to probe NaV structure and function, circumventing key limitations associated with existing methods for studying these proteins. The preparation and electrophysiological evaluation of an expanded library of N21-modified STX analogues is described herein. Characterization of the binding properties of these nanomolar NaV inhibitors has contributed to the development of an enhanced model for the structure of the inner pore of the channel. A select set of STX-fluorophore conjugates that bind reversibly to NaV with submicromolar potency serve as fluorescent markers of channels in living cells to study channel motility in the cell membrane. By contrast, maleimide-conjugated STX derivatives can be engineered to act as irreversible inhibitors of ion conductance when applied to wild-type NaV isoforms. The unique binding behavior of these derivatives has been leveraged to develop a new class of NaV probes for use in live cell imaging experiments and protein profiling studies. Maleimide-toxin conjugates with bioorthogonal reactive groups have been synthesized and can be employed for ligation of visualization and isolation tags to covalently modified channels. Appendage of fluorine-18 to a modified STX affords a probe for studying NaV expression in living subjects. An efficient synthetic route yields a derivative that binds with nanomolar affinity to several NaV isoforms. Biodistribution, autoradiography, and PET-MRI imaging studies demonstrate accumulation of the radiotracer at the site of injury in a rat model of neuropathic pain. This uptake correlates with the previously reported upregulation of NaV isoforms at this site, validating the utility of this probe as a NaV imaging agent. Collectively, these STX derivatives, uniquely available through chemical synthesis, represent a novel set of molecular probes for studying NaV function in vitro and in vivo.
- Serial cycloadditions for the rapid assembly of synthetic building blocks and therapeutic leads, part II: New strategies for HIV eradication : potent prostratin analogs induce latent HIV expression2013Dennis Nick Fournogerakis.My graduate studies have been focused on two separate projects that are unified by the theme of step economy and exemplify two major concepts within step economy. The first of these is the development of new reactions or serial reactions aimed at creating ways to achieve significant molecular complexity increases in a short synthetic sequence to access targets of value. Complementary to this is the concept of function-oriented synthesis, which involves the design and synthesis of novel structures of value that contribute to our understanding of the biological effects of the compound and results in the refinement of lead structures to produce clinically more optimal candidates. Chapter 1 reviews the development of serial cycloadditions and their application in organic synthesis. Serial cycloadditions are valuable reactions for organic chemists because of the significant increase in molecular complexity observed in a single step. The review separates the collective body of work into non-catalyzed serial cycloadditions and transition metal catalyzed cycloadditions. The non-catalyzed variants can involve the combining of simple starting materials into polycyclic ring systems, but are typically limited to more activated dienes or dienophiles. The transition metal mediated processes developed more recently have utilized unactivated reaction partners but, typically, rely on complex starting substrates. This review highlights the potential opportunity to develop serial cycloadditions that combine simple unactivated reaction partners to produce scaffolds of interest. Chapter 2 discusses the development of 4-trimethylsilyl-but-2-yn-1-ol as a regioselective butatriene equivalent for serial [5+2]/vinylogous Peterson olefination/[4+2] cycloadditions to produce linear polycyclic ring systems. 4-trimethylsilyl-but-2-yn-1-ol behaves as initial 2-carbon component in the Rh-catalyzed [5+2] cycloaddition with vinylcyclopropane and then subsequently eliminates to a diene that can be utilized for further elaboration. A diverse set of dienophiles was evaluated for the second cycloaddition and both alkynes and alkenes were found to be suitable reaction partners, with up to nearly quantitative yields obtained. The reaction was utilized to access a key intermediate for proposed simplified analogs of staurosporine. Chapter 3 begins by describing the current state of the HIV pandemic and the limitations of the current therapeutic options (Highly Active Antiretroviral Therapy - HAART). The principal barrier to developing a cure for HIV is the latent reservoir that is insusceptible to HAART. The natural product prostratin is a clinical lead for a promising HIV eradication strategy that involves the deliberate activation of these latent reservoirs. Novel analogs of prostratin were synthesized and found to exhibit activity that greatly exceeded the natural product in multiple biological assays. Most impressively, a lead analog was found to potently induce HIV expression in patient samples from HIV-infected individuals on suppressive HAART. Chapter 4 delves into the design, synthesis, and evaluation of 2nd-generation analogs of prostratin. These analogs were inspired by the realization of a key intramolecular hydrogen bond in prostratin and can be accessed in a more step-economical fashion than the original analogs because they circumvented a problematic deoxygenation step in the original synthesis. The new analogs that incorporated ether functionality at C12 of prostratin were found to have higher affinities for PKC, the presumed biological target for prostratin, and more potent induction of latent HIV in vitro and ex vivo. Finally, Chapter 5 describes the further manipulation of the prostratin scaffold in hopes of obtaining properties of a clinically optimized agent. Novel transformations on the scaffold are reported and preliminary biological evaluation of the new analogs is discussed. Based on these results, new information was obtained about the tolerability of the protein binding pocket for certain modifications and this will facilitate the future development of novel PKC modulators.
- 2011Hsiao-lu Lee.Since the first successful detection single molecules over two decades ago, single-molecule spectroscopy has developed into a burgeoning field with a wealth of experiments at room temperature and inside living cells. Probing asynchronous and heterogeneous populations in situ, one molecule at a time, is not only desirable, but critical for many biological questions. Further, super-resolution imaging based on sequential imaging of sparse subsets of single molecules, has seen explosive growth within the last five years. This dissertation describes both the application of live-cell single-molecule imaging as an answer to important biological questions, and development and validation of fluorescent probes for targeted super-resolution imaging.
- Single-molecule fluorescence and super-resolution imaging of Huntington's disease protein aggregates2012Whitney Clara Duim.Single-molecule, super-resolution fluorescence microscopy is a powerful technique for studying biological systems because it reveals details beyond the optical diffraction limit (on the 20-100 nm scale) such as structural and conformational heterogeneity. Further, single-molecule imaging measures distributions of behaviors directly through the interrogation of many individual molecules and reports on the nanoscale environment of molecules. Sub-diffraction imaging adds increased resolution to the advantages of fluorescence imaging over the techniques of atomic force microscopy and electron microscopy for studying biological structures, which include imaging of large fields of view in aqueous environments, specific identification of protein(s) of interest by fluorescent labeling, low perturbation of the system, and the ability to image living systems in near real-time (limited by the time required for super-resolution sequential imaging). This dissertation describes the application of single-molecule and super-resolution fluorescence imaging to studying the huntingtin (Htt) protein aggregates that are a hallmark of Huntington's disease and that have been implicated in the pathogenesis of the disease. The intricate nanostructures formed by fibrillar Htt aggregates in vitro and the sub-diffraction widths of individual fibers mark the amyloids as important targets for high-resolution optical imaging. The characterization of Htt aggregate species is critical for understanding the mechanism of Huntington's disease and identifying potential therapies. Following an introduction to single-molecule, super-resolution imaging and Huntington's disease in Chapter 1, Chapter 2 describes the single-molecule methods, experimental techniques, Htt protein sample preparations, and data analysis performed in this dissertation. Chapter 3 discusses the development of super-resolution imaging of Htt protein aggregates and the validation of the images by atomic force microscopy. Chapter 4 continues the study of Htt by one- and two-color super-resolution with imaging of Htt protein aggregates over time from the initial protein monomers to the large aggregate assemblies of amyloid fibers. In Chapter 5, I detail our progress to-date in studying the earliest stages of Htt aggregation using zero-mode waveguide technology. Chapter 6 concludes the dissertation with a discussion of the results from additional projects comprising the effect of chaperonin proteins on Htt aggregation, extension of super-resolution Htt imaging to three dimensions, and cellular imaging of Htt aggregates. The future directions for these exciting projects are summarized with the expectation that research efforts directed in these areas will contribute to our understanding of Htt aggregation and Huntington's disease.
- Size-expanded ribonucleic acids (xRNAs) : a novel component in siRNAs for investigating the steric restrictions of RISC in RNAi2012Armando Ricardo Hernández.This thesis project describes the synthesis and use of size-expanded ribonucleic acids (xRNAs) as unnatural components in small interfering RNAs (siRNAs), which serve as a novel set of steric probes for investigating and identifying size restrictions that exist in the active site of the RNA-induced silencing complex (RISC), a critical protein complex in the RNA interference (RNAi) mechanism. xRNAs are analogous to natural RNAs, in which they retain canonical Watson Crick base-pairing groups, except their nucleobases are expanded linearly by 2.4 Å via benzo-fusion. In this report, the synthesis of the entire set of four xRNA nucleosides and their photophysical properties are described. Two of the xRNA nucleosides (i.e. rxA and rxU) are then converted to their corresponding phosphoramidites for use in automated RNA synthesis to synthesize various sets of xRNA-substituted siRNA strands for biophysical and biological studies. RNAi activity studies in HeLa cells using a firefly/Renilla luciferase dual reporter assay showed that xRNA substitutions in the antisense strand displayed poor RNAi activity in the middle of the sequence (positions 7-14) but were generally well tolerated near the 5'- and 3'-ends. Most significantly, single xRNA substitutions at the 3'-end showed RNAi activity that was more potent than that of unmodified siRNAs. Thermal denaturation studies revealed small changes in melting temperature (+1.4 to --5.0 °C); xRNA substitutions in the middle were found to be more thermodynamically destabilizing whereas the ends were shown to be stabilizing. Serum stability studies showed that xRNA-containing siRNAs have longer half-lives in human serum than unmodified siRNAs. Finally, xRNA-substituted siRNA strands and duplexes showed interesting position-dependent fluorescent properties, while CD spectroscopy experiments revealed that xRNA-substituted siRNAs does not greatly distort the native A-form helical structure of the duplexes. Taken together, the data demonstrate the utility of xRNA nucleobases as size-expanded mechanistic probes for RNAi research.
- Small changes big impact, ligand influence on dioxygen, semiquinone, and phenoxyl radical copper complexes2012Pratik Verma.Reductive activation of dioxygen by copper to generate potent oxidants for multi- electron organic transformations is exploited extensively in biological systems. This thesis focuses on two types of multi-electron oxidants: Cu2O2 complexes and copper complexes with redox-active ligands. The goal of this work is to identify the influence of nitrogen containing ancillary ligands on properties of dioxygen, semiquinone and phenoxyl radical complexes of copper. This work is aimed primarily at synthetic chemists interested in rational design of ligands for creating bio-inspired oxidants and oxidation catalysts. Chapter 1 of this thesis reports the identification of a Density Functional Theory (DFT) protocol for deriving structure-property relationships in Cu2O2 complexes. Chapter 2 of this thesis applies towards modeling electronic spectra, the DFT protocols that were validated in Chapter 1 for modeling thermodynamics. Chapter 3 of this thesis describes the properties and reactivity of Cu2O2 complex generated from a new hybrid permethylated-amine-guanidine ligand based on a 1,3- propanediamine backbone (2L). Chapter 4 of this thesis describes the characterization of an intermediate (C) that is observed in both phenol hydroxylation and catechol oxidation with the SP core supported by N1, N2-di-t-butylethane-1,2-diamine (DBED). Chapter 5 of this thesis describes the influence of sulfanyl substituents on the optical and redox properties of copper-bonded phenoxyls.
- 2006 SpringerK.-H. Hellwich, C.D. Siebert ; translated by Allan D. Dunn.
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