Theoretical investigations of experimental gravitation

<p>This thesis has two basic themes: the investigation of new experiments which can be used to test relativistic gravity, and the investigation of new technologies and new experimental techniques which can be applied to make gravitational wave astronomy a reality.p> <p>Advancing technology will soon make possible a new class of gravitation experiments: pure laboratory experiments with laboratory sources of non-Newtonian gravity and laboratory detectors. The key advance in techno1ogy is the development of resonant sensing systems with very low levels of dissipation. Chapter 1 considers three such systems (torque balances, dielectric monocrystals, and superconducting microwave resonators), and it proposes eight laboratory experiments which use these systems as detectors. For each experiment it describes the dominant sources of noise and the technology required.p> <p>The coupled electro-mechanical system consisting of a microwave cavity and its walls can serve as a gravitational radiation detector. A gravitational wave interacts with the walls, and the resulting motion induces transitions from a highly excited cavity mode to a nearly unexcited mode. Chapter 2 describes briefly a formalism for analyzing such a detector, and it proposes a particular design.p> <p>The monitoring of a quantum mechanical harmonic oscillator on which a classical force acts is important in a variety of high-precision experiments, such as the attempt to detect gravitational radiation. Chapter 3 reviews the standard techniques for monitoring the oscillator; and it introduces a new technique which, in principle, can determine the details of the force with arbitrary accuracy, despite the quantum properties of the oscillator.p> <p>The standard method for monitoring the oscillator is the "amplitude- and-phase" method (position or momentum transducer with output fed through a linear amplifier). The accuracy obtainable by this method is limited by the uncertainty principle. To do better requires a measurement of the type which Braginsky has called "quantum nondemolition." A well-known quantum nondemolition technique is "quantum counting," which can detect an arbitrarily weak force, but which cannot provide good accuracy in determining its precise time-dependence. Chapter 3 considers extensively a new type of quantum nondemolition measurement - a "back-action-evading" measurement of the real part X₁ (or the imaginary part X₂) of the oscillator's complex amplitude. In principle X₁ can be measured arbitrarily quickly and arbitrarily accurately, and a sequence of such measurements can lead to an arbitrarily accurate monitoring of the classical force.p> <p>Chapter 3 describes explicit gedanken experiments which demonstrate that X₁ can be measured arbitrarily quickly and arbitrarily accurately, it considers approximate back-action-evading measurements, and it develops a theory of quantum nondemolition measurement for arbitrary quantum mechanical systems.p> <p>In Rosen's "bimetric" theory of gravity the (local) speed of gravitational radiation v_g is determined by the combined effects of cosmological boundary values and nearby concentrations of matter. It is possible for v_g to be less than the speed of light. Chapter 4 shows that emission of gravitational radiation prevents particles of nonzero rest mass from exceeding the speed of gravitational radiation. Observations of relativistic particles place limits on v_g and the cosmological boundary values today, and observations of synchrotron radiation from compact radio sources place limits on the cosmological boundary values in the past.p>

Theoretical studies of novel organic systems : biradicals, polyradicals, and conducting polymers

<p>Chapter 1p> <p>Cyclobutanediyl has been studied in both its singlet and triplet states by ab initio electronic structure theory. The triplet, which is the ground state of the molecule, exists in both C_(2h) and C_(2v) forms, which interconvert via a C_s transition state. For the singlet, only a C_(2h) form is found. It passes, via a C_s transition state, onto the C_(2v) surface on which bicyclobutane is the only minimum. The ring-flipping (inversion) process in bicyclobutane includes the singlet biradical as an intermediate, and involves a novel, nonleast motion pathway. Semiclassical periodic orbit theory indicates that the various minima on both the singlet and triplet surfaces can interconvert via quantum mechanical tunneling.p> <p>Chapter 2p> <p>The dimethylenepolycyclobutadienes (n) are the non-Kekulé analogues of the classical acenes. Application of a variety of theoretical methods reveals several novel features of such structures. Most interesting is the emergence of a parity rule. When n is even, n is predicted to be a singlet, with n disjoint NBMOs. When n is odd, theory predicts a triplet ground state with (n+1) NBMOs that are not fully disjoint.p> <p>Chapter 3p> <p>Bi(cyclobutadienyl) (2), the cyclobutadiene analogue of biphenyl, and its homologues tri- (3) and tetra(cyclobutadienyl) (4) have been studied using electronic structure theory. Ab initio calculations on 2 reveal that the central bond is a true double bond, and that the structure is best thought of as two allyl radicals plus an ethylene. The singlet and triplet states are essentially degenerate. Trimer 3 is two allyls plus a dimethylenecyclobutanediyl, while 4 is two coplanar bi(cyclobutadienyl) units connected by a single bond. For both 3 and 4, the quintet, triplet, and singlet states are essentially degenerate, indicating that they are tetraradicals. The infinite polymer, polycyclobutadiene, has been studied by HMO, EHCO, and VEH methods. Several geometries based on the structures of 3 and 4 have been studied, and the band structures are quite intriguing. A novel crossing between the valence and conduction bands produces a small band gap and a high density of states at the Fermi level.p> <p>Chapter 4p> <p>At the level of Hückel theory, polyfulvene has a HOCO-LUCO degeneracy much like that seen in polyacetylene. Higher levels of theory remove the degeneracy, but the band gap (E_g) is predicted to be significantly smaller than analogous structures such as polythiophene and polypyrrole at the fulvenoid geometry. An alternative geometry, which we have termed quinoid, is also conceivable for polyfulvene, and it is predicted to have a much larger E_g. The effects of benzannelation to produce analogues of polyisothianaphthene have been evaluated. We propose a new model for such structures based on conventional orbital mixing arguments. Several of the proposed structures have quite interesting properties, which suggest that they are excellent candidates for conducting polymers.p> <p>Chapter 5p> <p>Theoretical studies of polydimethylenecyclobutene and polydiisopropylidene- cyclobutene reveal that, because of steric crowding, they cannot achieve a planar, fully conjugated structure in either their undoped or doped states. Rather, the structure consists of essentially orthogonal hexatriene units. Such a structure is incompatible with conventional conduction mechanisms involving polarons and bipolarons.p>

Expression of eph-family receptor tyrosine kinases and ephrins in the tadpole of the frog Xenopus laevis, and possible roles in the development of retinotectal topography

<p>Assembling a nervous system requires exquisite specificity in the construction of neuronal connectivity. One method by which such specificity is implemented is the presence of chemical cues within the tissues, differentiating one region from another, and the presence of receptors for those cues on the surface of neurons and their axons that are navigating within this cellular environment.p> <p>Connections from one part of the nervous system to another often take the form of a topographic mapping. One widely studied model system that involves such a mapping is the vertebrate retinotectal projection-the set of connections between the eye and the optic tectum of the midbrain, which is the primary visual center in non-mammals and is homologous to the superior colliculus in mammals. In this projection the two-dimensional surface of the retina is mapped smoothly onto the two-dimensional surface of the tectum, such that light from neighboring points in visual space excites neighboring cells in the brain. This mapping is implemented at least in part via differential chemical cues in different regions of the tectum.p> <p>The Eph family of receptor tyrosine kinases and their cell-surface ligands, the ephrins, have been implicated in a wide variety of processes, generally involving cellular movement in response to extracellular cues. In particular, they possess expression patterns-i.e., complementary gradients of receptor in retina and ligand in tectum- and in vitro and in vivo activities and phenotypes-i.e., repulsive guidance of axons and defective mapping in mutants, respectively-consistent with the long-sought retinotectal chemical mapping cues.p> <p>The tadpole of Xenopus laevis, the South African clawed frog, is advantageous for in vivo retinotectal studies because of its transparency and manipulability. However, neither the expression patterns nor the retinotectal roles of these proteins have been well characterized in this system. We report here comprehensive descriptions in swimming stage tadpoles of the messenger RNA expression patterns of eleven known Xenopus Eph and ephrin genes, including xephrin-A3, which is novel, and xEphB2, whose expression pattern has not previously been published in detail. We also report the results of in vivo protein injection perturbation studies on Xenopus retinotectal topography, which were negative, and of in vitro axonal guidance assays, which suggest a previously unrecognized attractive activity of ephrins at low concentrations on retinal ganglion cell axons. This raises the possibility that these axons find their correct targets in part by seeking out a preferred concentration of ligands appropriate to their individual receptor expression levels, rather than by being repelled to greater or lesser degrees by the ephrins but attracted by some as-yet-unknown cue(s).p>

The development of an asymmetric palladium-catalyzed conjugate addition and its application toward the total syntheses of taiwaniaquinoid natural products

<p>The asymmetric synthesis of quaternary stereocenters remains a challenging problem in organic synthesis. Past work from the Stoltz laboratory has resulted in methodology to install quaternary stereocenters α- or γ- to carbonyl compounds. Thus, the asymmetric synthesis of β-quaternary stereocenters was a desirable objective, and was accomplished by engineering the palladium-catalyzed addition of arylmetal organometallic reagents to α,β-unsaturated conjugate acceptors.p> <p>Herein, we described the rational design of a palladium-catalyzed conjugate addition reactions utilizing a catalyst derived from palladium(II) trifluoroacetate and pyridinooxazole ligands. This reaction is highly tolerant of protic solvents and oxygen atmosphere, making it a practical and operationally simple reaction. The mild conditions facilitate a remarkably high functional group tolerance, including carbonyls, halogens, and fluorinated functional groups. Furthermore, the reaction catalyzed conjugate additions with high enantioselectivity with conjugate acceptors of 5-, 6-, and 7-membered ring sizes. Extension of the methodology toward the asymmetric synthesis of flavanone products is presented, as well.p> <p>A computational and experimental investigation into the reaction mechanism provided a stereochemical model for enantioinduction, whereby the α-methylene protons adjacent the enone carbonyl clashes with the tert-butyl groups of the chiral ligand. Additionally, it was found that the addition of water and ammonium hexafluorophosphate significantly increases the reaction rate without sacrificing enantioselectivity. The synergistic effects of these additives allowed for the reaction to proceed at a lower temperature, and thus facilitated expansion of the substrate scope to sensitive functional groups such as protic amides and aryl bromides. Investigations into a scale-up synthesis of the chiral ligand (S)-tert-butylPyOx are also presented. This three-step synthetic route allowed for synthesis of the target compound of greater than 10 g scale. p> <p>Finally, the application of the newly developed conjugate addition reaction toward the synthesis of the taiwaniaquinoid class of terpenoid natural products is discussed. The conjugate addition reaction formed the key benzylic quaternary stereocenter in high enantioselectivity, joining together the majority of the carbons in the taiwaniaquinoid scaffold. Efforts toward the synthesis of the B-ring are presented. p>

Development of protein-catalyzed capture (PCC) agents with application to the specific targeting of the E17K Point mutation of Akt1

<p>This thesis describes the expansion and improvement of the iterative in situ click chemistry OBOC peptide library screening technology. Previous work provided a proof-of-concept demonstration that this technique was advantageous for the production of protein-catalyzed capture (PCC) agents that could be used as drop-in replacements for antibodies in a variety of applications. Chapter 2 describes the technology development that was undertaken to optimize this screening process and make it readily available for a wide variety of targets. This optimization is what has allowed for the explosive growth of the PCC agent project over the past few years.p> <p>These technology improvements were applied to the discovery of PCC agents specific for single amino acid point mutations in proteins, which have many applications in cancer detection and treatment. Chapter 3 describes the use of a general all-chemical epitope-targeting strategy that can focus PCC agent development directly to a site of interest on a protein surface. This technique utilizes a chemically-synthesized chunk of the protein, called an epitope, substituted with a click handle in combination with the OBOC in situ click chemistry libraries in order to focus ligand development at a site of interest. Specifically, Chapter 3 discusses the use of this technique in developing a PCC agent specific for the E17K mutation of Akt1. Chapter 4 details the expansion of this ligand into a mutation-specific inhibitor, with applications in therapeutics.p>

Development of Asymmetric Protonation Reactions for the Synthesis of Indoline Alkaloids

<p>Nitrogen-containing heterocycles, such as indolines and pyrroloindolines, are prevalent in a variety of diverse natural products, many of which exhibit remarkable biological activities. These frameworks have inspired innovative research aimed at discovering novel methods for their stereoselective preparation.p> <p>We have developed an enantioselective synthesis of pyrroloindolines based on a formal (3 + 2) cycloaddition of indoles and 2-amidoacrylates. This reaction is promoted by (R)-BINOL•SnCl₄; this complex is a Lewis acid-assisted Brønsted acid that effects a highly face-selective catalyst-controlled protonation of an enolate. Mechanistic studies also determined that the initial product of this reaction is an indolinium ion, which upon aqueous workup undergoes cyclization to the pyrroloindoline.p> <p>Based on this result, we investigated alternative nucleophiles to trap the indolinium ion. First, addition of sodium borohydride to the optimized reaction conditions yields indoline-containing amino acid derivatives.p> <p>Next, carbon nucleophiles were explored. Indole substrates incorporating a tethered alkene were exposed to the conditions for the formal (3 + 2) cycloaddition, resulting in a conjugate addition/asymmetric protonation/Prins cyclization cascade. In this transformation, the indolinium ion is attacked by the olefin, and the resulting carbocation is quenched by a chloride ion. Zirconium tetrachloride was found to be the optimal Lewis acid. Stoichiometric proton and chloride sources were also found to be crucial for reactivity.p>

Development of Metalloenzyme Dioxygen Reduction Cathodes

<p>The prime thrust of this dissertation is to advance the development of fuel cell dioxygen reduction cathodes that employ some variant of multicopper oxidase enzymes as the catalyst. The low earth-abundance of platinum metal and its correspondingly high market cost has prompted a general search amongst chemists and materials scientists for reasonable alternatives to this metal for facilitating catalytic dioxygen reduction chemistry. The multicopper oxidases (MCOs), which constitute a class of enzyme that naturally catalyze the reaction O₂ + 4Hp>+p> + 4ep>-p> → 2H₂O, provide a promising set of biochemical contenders for fuel cell cathode catalysts. In MCOs, a substrate reduces a copper atom at the type 1 site, where charge is then transferred to a trinuclear copper cluster consisting of a mononuclear type 2 or “normal copper” site and a binuclear type 3 copper site. Following the reduction of all four copper atoms in the enzyme, dioxygen is then reduced to water in two two-electron steps, upon binding to the trinuclear copper cluster. We identified an MCO, a laccase from the hyperthermophilic bacterium Thermus thermophilus strain HB27, as a promising candidate for cathodic fuel cell catalysis. This protein demonstrates resilience at high temperatures, exhibiting no denaturing transition at temperatures high as 95°C, conditions relevant to typical polymer electrolyte fuel cell operation.p> <p>In Chapter I of this thesis, we discuss initial efforts to physically characterize the enzyme when operating as a heterogeneous cathode catalyst. Following this, in Chapter II we then outline the development of a model capable of describing the observed electrochemical behavior of this enzyme when operating on porous carbon electrodes. Developing a rigorous mathematical framework with which to describe this system had the potential to improve our understanding of MCO electrokinetics, while also providing a level of predictive power that might guide any future efforts to fabricate MCO cathodes with optimized electrochemical performance. In Chapter III we detail efforts to reduce electrode overpotentials through site-directed mutagenesis of the inner and outer-sphere ligands of the Cu sites in laccase, using electrochemical methods and electronic spectroscopy to try and understand the resultant behavior of our mutant constructs. Finally, in Chapter IV, we examine future work concerning the fabrication of enhanced MCO cathodes, exploring the possibility of new cathode materials and advanced enzyme deposition techniques.p>

A search for fractionally charged particles in cosmic rays and a theoretical interpretation of the results

<p>An array of two spark chambers and six trays of plastic scintillation counters was used to search for unaccompanied fractionally charged particles in cosmic rays near sea level. No acceptable events were found with energy losses by ionization between 0.04 and 0.7 that of unit-charged minimum-ionizing particles. New 90%-confidence upper limits were thereby established for the fluxes of fractionally charged particles in cosmic rays, namely, (1.04 ± 0.07)x10p>-10p> and (2.03 ± 0.16)x10p>-10p> cmp>-2p>srp>-1p>secp>-1p> for minimum-ionizing particles with charges 1/3 and 2/3, respectively.p> <p>In order to be certain that the spark chambers could have functioned for the low levels of ionization expected from particles with small fractional charges, tests were conducted to estimate the efficiency of the chambers as they had been used in this experiment. These tests showed that the spark-chamber system with the track-selection criteria used might have been over 99% efficient for the entire range of energy losses considered.p> <p>Lower limits were then obtained for the mass of a quark by considering the above flux limits and a particular model for the production of quarks in cosmic rays. In this model, which is one involving the multi-peripheral Regge hypothesis, the production cross section and a corresponding mass limit are critically dependent on the Regge trajectory assigned to a quark. If quarks are "elementary'' with a flat trajectory, the mass of a quark can be expected to be at least 6 ± 2 BeV/cp>2p>. If quarks have a trajectory with unit slope, just as the existing hadrons do, the mass of a quark might be as small as 1.3 ± 0.2 BeV/cp>2p>. For a trajectory with unit slope and a mass larger than a couple of BeV/cp>2p>, the production cross section may be so low that quarks might never be observed in nature.p>

Mitochondria and the development of sea urchin embryos

<p>After artificial activation or fertilization of non-nucleate fragments or eggs of the sea urchin, the mitochondria actively synthesize RNA. The RNA made in non-nucleate fragments is shown to be mostly single stranded and to be associated primarily with the low speed pellet of centrifuged cellular homogenates.p> <p>Protein synthesis is observed in non-nucleate fragments in the presence or absence of the mitochondrial RNA synthesis: it is found to be qualitatively similar but quantitatively less in the absence of the RNA synthesis. The continued syntheses of proteins in the non-nucleate fragments in the absence of mitochondrial RNA synthesis provides additional evidence for the presence of a stable messenger RNA component in the unfertilized sea urchin egg.p> <p>Since the uptake or actinomycin D was found to be inhibited by the presence of a fertilization membrane, ethidium bromide, at 10 μgs/ml, is used as an effective inhibitor of RNA synthesis in non-nucleate fragments and in early cleavage stage embryos. However, this same concentration of ethidium bromide is found to be only partially effective in blocking RNA synthesis at the mesenchyme blastula stage of development.p> <p>Low concentrations of ethidium bromide (2 and 5 μgs/ml) are found not to be lethal but to be capable of producing moderate developmental defects. In the presence of concentrations of ethidium bromide adequate to inhibit all the mitochondrial RNA synthesis (10 μgs/ml of ethidium bromide), from fertilization on, the embryos do not cleave beyond the 4-8 cell stages. When similar concentrations of ethidium bromide are added at an early mesenchyme blastula stage, the embryos do not gastrulate but continue to swim for more than 24 additional hours (adequate for control embryos to develop to a late prism stage). These results lead to the conclusion that mitochondrial RNA synthesis may be very essential for normal development to occur. p> <p>DNA is synthesized in the non-nucleate fragments of sea urchin eggs. None of the newly synthesized DNA is found in the closed circular form. When phenol extracted directly from the fragments, the DNA is found to sediment at approximately 38 and 27s in sucrose gradients but neither of these size classes could be found associated with the isolated mitochondria. The template for the synthesis of DNA in non-nucleate fragments remains unknown.p>

Theoretical investigation of turbulent boundary layer over a wavy surface

<p>The important features of the two-dimensional incompressible turbulent flow over a wavy surface of wavelength comparable with the boundary layer thickness are analyzed.p> <p>A turbulent field method using model equation for turbulent shear stress similar to the scheme of Bradshaw, Ferriss and Atwell (1967) is employed with suitable modification to cover the viscous sublayer. The governing differential equations are linearized based on the small but finite amplitude to wavelength ratio. An orthogonal wavy coordinate system, accurate to the second order in the amplitude ratio, is adopted to avoid the severe restriction to the validity of linearization due to the large mean velocity gradient near the wall. Analytic solution up to the second order is obtained by using the method of matched-asymptotic-expansion based on the large Reynolds number and hence the small skin friction coefficient.p> <p>In the outer part of the layer, the perturbed flow is practically "inviscid." Solutions for the velocity, Reynolds stress and also the wall pressure distributions agree well with the experimental measurement. In the wall region where the perturbed Reynolds stress plays an important role in the process of momentum transport, only a qualitative agreement is obtained. The results also show that the nonlinear second-order effect is negligible for amplitude ratio of 0.03. The discrepancies in the detailed structure of the velocity, shear stress, and skin friction distributions near the wall suggest modifications to the model are required to describe the present problem.p>

Development of roll waves in open channels

<p>This study is concerned with some of the properties of roll waves that develop naturally from a turbulent uniform flow in a wide rectangular channel on a constant steep slope . The wave properties considered were depth at the wave crest, depth at the wave trough, wave period, and wave velocity . The primary focus was on the mean values and standard deviations of the crest depths and wave periods at a given station and how these quantities varied with distance along the channel.p> <p>The wave properties were measured in a laboratory channel in which roll waves developed naturally from a uniform flow . The Froude number F (F = u_n/√gh_n, u_n = normal velocity , h_n = normal depth, g =acceleration of gravity) ranged from 3. 4 to 6. 0 for channel slopes S_o of . 05 and . 12 respectively . In the initial phase of their development the roll waves appeared as small amplitude waves with a continuous water surface profile . These small amplitude waves subsequently developed into large amplitude shock waves. Shock waves were found to overtake and combine with other shock waves with the result that the crest depth of the combined wave was larger than the crest depths before the overtake. Once roll waves began to develop, the mean value of the crest depths h_nmax increased with distance . Once the shock waves began to overtake, the mean wave period T_av increased approximately linearly with distance.p> <p>For a given Froude number and channel slope the observed quantities hp>-p>_max/h_n , T' (T' = S_o T_av √g/h_n), and the standard deviations of hp>-p>_max/h_n and T', could be expressed as unique functions of l/h_n (l = distance from beginning of channel) for the two-fold change in h_n occurring in the observed flows . A given value of hp>-p>_max/h_n occurred at smaller values of l/h_n as the Froude number was increased. For a given value of h /hhp>-p>_max/h_n the growth rate of δhp>-p>_max/hp>-p>_maxδl of the shock waves increased as the Froude number was increased.p> <p>A laboratory channel was also used to measure the wave properties of periodic permanent roll waves. For a given Froude number and channel slope the hp>-p>_max/h_n vs. T' relation did not agree with a theory in which the weight of the shock front was neglected. After the theory was modified to include this weight, the observed values of hp>-p>_max/h_n were within an average of 6.5 percent of the predicted values, and the maximum discrepancy was 13.5 percent.p> <p>For hp>-p>_max/h_n sufficiently large (hp>-p>_max/h_n > approximately 1.5) it was found that the hp>-p>_max/h_n vs. T' relation for natural roll waves was practically identical to the hp>-p>_max/h_n vs. T' relation for periodic permanent roll waves at the same Froude number and slope. As a result of this correspondence between periodic and natural roll waves, the growth rate δhp>-p>_max/hp>-p>_maxδl of shock waves was predicted to depend on the channel slope, and this slope dependence was observed in the experiments.p>

Experimental and theoretical studies of the electrothermal instability in a nonequilibrium plasma

<p>Experimental and theoretical studies have been made of the electrothermal waves occurring in a nonequilibrium MHD plasma. These waves are caused by an instability that occurs when a plasma having a dependence of conductivity on current density is subjected to crossed electric and magnetic fields. Theoretically, these waves were studied by developing and solving the equations of a steady, one-dimensional nonuniformity in electron density. From these nonlinear equations, predictions of the maximum amplitude and of the half width of steady waves could be obtained. Experimentally, the waves were studied in a nonequilibrium discharge produced in a potassium-seeded argon plasma at 2000°K and 1 atm. pressure. The behavior of such a discharge with four different configurations of electrodes was determined from photographs, photomultiplier measurements, and voltage probes. These four configurations were chosen to produce steady waves, to check the stability of steady waves, and to observe the manifestation of the waves in a MHD generator or accelerator configuration.p> <p>Steady, one-dimensional waves were found to exist in a number of situations, and where they existed, their characteristics agreed with the predictions of the steady theory. Some extensions of this theory were necessary, however, to describe the transient phenomena occurring in the inlet region of a discharge transverse to the gas flow. It was also found that in a discharge away from the stabilizing effect of the electrodes, steady waves became unstable for large Hall parameters. Methods of prediction of the effective electrical conductivity and Hall parameter of a plasma with nonuniformities caused by the electrothermal waves were also studied. Using these methods and the values of amplitude predicted by the steady theory, it was found that the measured decrease in transverse conductivity of a MHD device, 50 per cent at a Hall parameter of 5, could be accounted for in terms of the electrothermal instability. p>

Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

<p>Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively. p> <p>The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10p>9p> daltons and that of the mitochondrial DNA is 35-40 x 10p>6p> daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.p> <p>RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.p> <p>Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-p>3p>H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-p>3p>H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.p> <p>RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.p> <p>In the early cleavage stages the light polysomes are mostly affected by actinomycin.p>

Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System

<p>Cancer chemotherapy has advanced from highly toxic drugs to more targeted treatments in the last 70 years. Chapter 1 opens with an introduction to targeted therapy for cancer. The benefits of using a nanoparticle to deliver therapeutics are discussed. We move on to siRNA in particular, and why it would be advantageous as a therapy. Specific to siRNA delivery are some challenges, such as nuclease degradation, quick clearance from circulation, needing to enter cells, and getting to the cytosol. We propose the development of a nanoparticle delivery system to tackle these challenges so that siRNA can be effective.p> <p>Chapter 2 of this thesis discusses the synthesis and analysis of a cationic mucic acid polymer (cMAP) which condenses siRNA to form a nanoparticle. Various methods to add polyethylene glycol (PEG) for stabilizing the nanoparticle in physiologic solutions, including using a boronic acid binding to diols on mucic acid, forming a copolymer of cMAP with PEG, and creating a triblock with mPEG on both ends of cMAP. The goal of these various pegylation strategies was to increase the circulation time of the siRNA nanoparticle in the bloodstream to allow more of the nanoparticle to reach tumor tissue by the enhanced permeation and retention effect. We found that the triblock mPEG-cMAP-PEGm polymer condensed siRNA to form very stable 30-40 nm particles that circulated for the longest time – almost 10% of the formulation remained in the bloodstream of mice 1 h after intravenous injection.p> <p>Chapter 3 explores the use of an antibody as a targeting agent for nanoparticles. Some antibodies of the IgG1 subtype are able to recruit natural killer cells that effect antibody dependent cellular cytotoxicity (ADCC) to kill the targeted cell to which the antibody is bound. There is evidence that the ADCC effect remains in antibody-drug conjugates, so we wanted to know whether the ADCC effect is preserved when the antibody is bound to a nanoparticle, which is a much larger and complex entity. We utilized antibodies against epidermal growth factor receptor with similar binding and pharmacokinetics, cetuximab and panitumumab, which differ in that cetuximab is an IgG1 and panitumumab is an IgG2 (which does not cause ADCC). Although a natural killer cell culture model showed that gold nanoparticles with a full antibody targeting agent can elicit target cell lysis, we found that this effect was not preserved in vivo. Whether this is due to the antibody not being accessible to immune cells or whether the natural killer cells are inactivated in a tumor xenograft remains unknown. It is possible that using a full antibody still has value if there are immune functions which are altered in a complex in vivo environment that are intact in an in vitro system, so the value of using a full antibody as a targeting agent versus using an antibody fragment or a protein such as transferrin is still open to further exploration.p> <p>In chapter 4, nanoparticle targeting and endosomal escape are further discussed with respect to the cMAP nanoparticle system. A diboronic acid entity, which gives an order of magnitude greater binding (than boronic acid) to cMAP due to the vicinal diols in mucic acid, was synthesized, attached to 5kD or 10kD PEG, and conjugated to either transferrin or cetuximab. A histidine was incorporated into the triblock polymer between cMAP and the PEG blocks to allow for siRNA endosomal escape. Nanoparticle size remained 30-40 nm with a slightly negative ca. -3 mV zeta potential with the triblock polymer containing histidine and when targeting agents were added. Greater mRNA knockdown was seen with the endosomal escape mechanism than without. The nanoparticle formulations were able to knock down the targeted mRNA in vitro. Mixed effects suggesting function were seen in vivo.p> <p>Chapter 5 summarizes the project and provides an outlook on siRNA delivery as well as targeted combination therapies for the future of personalized medicine in cancer treatment.p>