Proteolytic processing of the nonstructural proteins of dengue 2 virus

The genomes of many positive stranded RNA viruses and of all retroviruses are translated as large polyproteins which are proteolytically processed by cellular and viral proteases. Viral proteases are structurally related to two families of cellular proteases, the pepsin-like and trypsin-like proteases. This thesis describes the proteolytic processing of several nonstructural proteins of dengue 2 virus, a representative member of the Flaviviridae, and describes methods for transcribing full-length genomic RNA of dengue 2 virus. Chapter 1 describes the in vitro processing of the nonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a system that allows identification of residues within the protease that are directly or indirectly involved with substrate recognition. Chapter 3 describes methods to produce genome length dengue 2 RNA from cDNA templates.

The nonstructural protein NS3 is structurally related to viral trypsinlike proteases from the alpha-, picorna-, poty-, and pestiviruses. The hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins was tested using an efficient in vitro expression system and antisera specific for the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed using T7 RNA polymerase and the RNA translated in reticulocyte lysates. Proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain necessary and sufficient for correct cleavage to the first 184 amino acids of NS3. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.

Biochemical and genetic experiments using viral proteinases have defined the sequence requirements for cleavage site recognition, but have not identified residues within proteinases that interact with substrates. A biochemical assay was developed that could identify residues which were important for substrate recognition. Chimeric proteases between yellow fever and dengue 2 were constructed that allowed mapping of regions involved in substrate recognition, and site directed mutagenesis was used to modulate processing efficiency.

Expression in vitro revealed that the dengue protease domain efficiently processes the yellow fever polyprotein between NS2A and NS2B and between NS2B and NS3, but that the reciprocal construct is inactive. The dengue protease processes yellow fever cleavage sites more efficiently than dengue cleavage sites, suggesting that suboptimal cleavage efficiency may be used to increase levels of processing intermediates in vivo. By mutagenizing the putative substrate binding pocket it was possible to change the substrate specificity of the yellow fever protease; changing a minimum of three amino acids in the yellow fever protease enabled it to recognize dengue cleavage sites. This system allows identification of residues which are directly or indirectly involved with enzyme-substrate interaction, does not require a crystal structure, and can define the substrate preferences of individual members of a viral proteinase family.

Full-length cDNA clones, from which infectious RNA can be transcribed, have been developed for a number of positive strand RNA viruses, including the flavivirus type virus, yellow fever. The technology necessary to transcribe genomic RNA of dengue 2 virus was developed in order to better understand the molecular biology of the dengue subgroup. A 5' structural region clone was engineered to transcribe authentic dengue RNA that contains an additional 1 or 2 residues at the 5' end. A 3' nonstructural region clone was engineered to allow production of run off transcripts, and to allow directional ligation with the 5' structural region clone. In vitro ligation and transcription produces full-length genomic RNA which is noninfectious when transfected into mammalian tissue culture cells. Alternative methods for constructing cDNA clones and recovering live dengue virus are discussed.

Electron tunneling in proteins and water

The subject of this thesis is electronic coupling in donor-bridge-acceptor systems. In Chapter 2, ET properties of cyanide-bridged dinuclear ruthenium complexes were investigated. The strong interaction between the mixed-valent ruthenium centers leads to intense metal-to-metal charge transfer bands (MMCT). Hush analysis of the MMCT absorption bands yields the electronic-coupling strength between the metal centers (H_(AB)) and the total reorganization energy (λ). Comparison of ET kinetics to calculated rates shows that classical ET models fail to account for the observed kinetics and nuclear tunneling must be considered.

In Chapter 3, ET rates were measured in four ruthenium-modified highpotential iron-sulfur proteins (HiPIP), which were modified at position His50, His81, His42 and His18, respectively. ET kinetics for the His50 and His81 mutants are a factor of 300 different, while the donor-acceptor separation is nearly identical. PATHWAY calculations corroborate these measurements and highlight the importance of structural detail of the intervening protein matrix.

In Chapter 4, the distance dependence of ET through water bridges was measured. Photoinduced ET measurements in aqueous glasses at 77 K show that water is a poor medium for ET. Luminescence decay and quantum yield data were analyzed in the context of a quenching model that accounts for the exponential distance dependence of ET, the distance distribution of donors and acceptors embedded in the glass and the excluded volumes generated by the finite sizes of the donors and acceptors.

In Chapter 5, the pH-dependent excited state dynamics of ruthenium-modified amino acids were measured. The [Ru(bpy)_(3)] ^(2+) chromophore was linked to amino acids via an amide linkage. Protonation of the amide oxygen effectively quenches the excited state. In addition. time-resolved and steady-state luminescence data reveal that nonradiative rates are very sensitive to the protonation state and the structure of the amino acid moiety.

Computationally Guided Monomerization of Red Fluorescent Proteins of the Class Anthozoa

Red fluorescent proteins (RFPs) have attracted significant engineering focus because of the promise of near infrared fluorescent proteins, whose light penetrates biological tissue, and which would allow imaging inside of vertebrate animals. The RFP landscape, which numbers ~200 members, is mostly populated by engineered variants of four native RFPs, leaving the vast majority of native RFP biodiversity untouched. This is largely due to the fact that native RFPs are obligate tetramers, limiting their usefulness as fusion proteins. Monomerization has imposed critical costs on these evolved tetramers, however, as it has invariably led to loss of brightness, and often to many other adverse effects on the fluorescent properties of the derived monomeric variants. Here we have attempted to understand why monomerization has taken such a large toll on Anthozoa class RFPs, and to outline a clear strategy for their monomerization. We begin with a structural study of the far-red fluorescence of AQ143, one of the furthest red emitting RFPs. We then try to separate the problem of stable and bright fluorescence from the design of a soluble monomeric β-barrel surface by engineering a hybrid protein (DsRmCh) with an oligomeric parent that had been previously monomerized, DsRed, and a pre-stabilized monomeric core from mCherry. This allows us to use computational design to successfully design a stable, soluble, fluorescent monomer. Next we took HcRed, which is a previously unmonomerized RFP that has far-red fluorescence (λemission = 633 nm) and attempted to monomerize it making use of lessons learned from DsRmCh. We engineered two monomeric proteins by pre-stabilizing HcRed’s core, then monomerizing in stages, making use of computational design and directed evolution techniques such as error-prone mutagenesis and DNA shuffling. We call these proteins mGinger0.1 (λem = 637 nm / Φ = 0.02) and mGinger0.2 (λem = 631 nm Φ = 0.04). They are the furthest red first generation monomeric RFPs ever developed, are significantly thermostabilized, and add diversity to a small field of far-red monomeric FPs. We anticipate that the techniques we describe will be facilitate future RFP monomerization, and that further core optimization of the mGingers may allow significant improvements in brightness.

Part I - The radiation of elastic waves from a spherical cavity in a half space. Part II - Precision determination of focal depths and epicenters of earthquakes.

Part I: The dynamic response of an elastic half space to an explosion in a buried spherical cavity is investigated by two methods. The first is implicit, and the final expressions for the displacements at the free surface are given as a series of spherical wave functions whose coefficients are solutions of an infinite set of linear equations. The second method is based on Schwarz's technique to solve boundary value problems, and leads to an iterative solution, starting with the known expression for the point source in a half space as first term. The iterative series is transformed into a system of two integral equations, and into an equivalent set of linear equations. In this way, a dual interpretation of the physical phenomena is achieved. The systems are treated numerically and the Rayleigh wave part of the displacements is given in the frequency domain. Several comparisons with simpler cases are analyzed to show the effect of the cavity radius-depth ratio on the spectra of the displacements.

Part II: A high speed, large capacity, hypocenter location program has been written for an IBM 7094 computer. Important modifications to the standard method of least squares have been incorporated in it. Among them are a new way to obtain the depth of shocks from the normal equations, and the computation of variable travel times for the local shocks in order to account automatically for crustal variations. The multiregional travel times, largely based upon the investigations of the United States Geological Survey, are confronted with actual traverses to test their validity.

It is shown that several crustal phases provide control enough to obtain good solutions in depth for nuclear explosions, though not all the recording stations are in the region where crustal corrections are considered. The use of the European travel times, to locate the French nuclear explosion of May 1962 in the Sahara, proved to be more adequate than previous work.

A simpler program, with manual crustal corrections, is used to process the Kern County series of aftershocks, and a clearer picture of tectonic mechanism of the White Wolf fault is obtained.

Shocks in the California region are processed automatically and statistical frequency-depth and energy depth curves are discussed in relation to the tectonics of the area.

Mechanisms of Transposable Element Repression by Piwi Proteins in the piRNA Pathway of Drosophila Germ Cells

The ability to reproduce is a defining characteristic of all living organisms. During reproduction, the integrity of genetic material transferred from one generation to the next is of utmost importance. Organisms have diverse strategies to ensure the fidelity of genomic information inherited between generations of individuals. In sexually reproducing animals, the piRNA pathway is an RNA-interference (RNAi) mechanism that protects the genomes of germ cells from the replication of ‘selfish’ genetic sequences called transposable elements (TE). When left unabated, the replication of TE sequences can cause gene disruption, double-stranded DNA breaks, and germ cell death that results in sterility of the organism. In Drosophila, the piRNA pathway is divided into a cytoplasmic and nuclear branch that involves the functions of three Piwi-clade Argonaute proteins—Piwi, Aubergine (Aub) and Argonaute-3 (Ago3)—which bind piwi-interacting RNA (piRNA) to form the effector complexes that represses deleterious TE sequences.

The work presented in this thesis examines the function and regulation of Piwi proteins in Drosophila germ cells. Chapter 1 presents an introduction to piRNA biogenesis and to the essential roles occupied by each Piwi protein in the repression of TE. We discuss the architecture and function of germ granules as the cellular compartments where much of the piRNA pathway operates. In Chapter 2, we present how Piwi in the nucleus co-transcriptionally targets genomic loci expressing TE sequences to direct the deposition of repressive chromatin marks. Chapter 3 examines the cytoplasmic function of the piRNA pathway, where we find that the protein Krimper coordinates Aub and Ago3 in the piRNA ping-pong pathway to adaptively target and destroy TE transcripts. Chapter 4 explores how interactions of Piwis with associated proteins are modulated by arginine methylation modifications. Lastly, in Chapter 5 I present evidence that the cytoplasmic branch of the piRNA pathway can potentially ‘cross-talk’ with the nuclear branch to transfer sequence information to better target and co-transcriptionally silence the genomic loci coding active TE sequences. Overall, the work presented in this thesis constitutes a part of the first steps in understanding the molecular mechanisms that protect germ cells from invasion by TE sequences.

Structural Characterization of Pro-inflammatory and Anti-inflammatory Immunoglobulin G Fc Proteins

Immunoglobulin G (IgG) is central in mediating host defense due to its ability to target and eliminate invading pathogens. The fragment antigen binding (Fab) regions are responsible for antigen recognition; however the effector responses are encoded on the Fc region of IgG. IgG Fc displays considerable glycan heterogeneity, accounting for its complex effector functions of inflammation, modulation and immune suppression. Intravenous immunoglobulin G (IVIG) is pooled serum IgG from multiple donors and is used to treat individuals with autoimmune and inflammatory disorders such as rheumatoid arthritis and Kawasaki’s disease, respectively. It contains all the subtypes of IgG (IgG1-4) and over 120 glycovariants due to variation of an Asparagine 297-linked glycan on the Fc. The species identified as the activating component of IVIG is sialylated IgG Fc. Comparisons of wild type Fc and sialylated Fc X-ray crystal structures suggests that sialylation causes an increase in conformational flexibility, which may be important for its anti-inflammatory properties.

Although glycan modifications can promote the anti-inflammatory properties of the Fc, there are amino acid substitutions that cause Fcs to initiate an enhanced immune response. Mutations in the Fc can cause up to a 100-fold increase in binding affinity to activating Fc gamma receptors located on immune cells, and have been shown to enhance antibody dependent cell-mediated cytotoxicity. This is important in developing therapeutic antibodies against cancer and infectious diseases. Structural studies of mutant Fcs in complex with activating receptors gave insight into new protein-protein interactions that lead to an enhanced binding affinity.

Together these studies show how dynamic and diverse the Fc region is and how both protein and carbohydrate modifications can alter structure, leading to IgG Fc’s switch from a pro-inflammatory to an anti-inflammatory protein.

The isolation and partial biochemical analysis of Sindbis virus proteins

Recently, the amino acid sequences have been reported for several proteins, including the envelope glycoproteins of Sindbis virus, which all probably span the plasma membrane with a common topology: a large N-terminal, extracellular portion, a short region buried in the bilayer, and a short C-terminal intracellular segment. The regions of these proteins buried in the bilayer correspond to portions of the protein sequences which contain a stretch of hydrophobic amino acids and which have other common characteristics, as discussed. Reasons are also described for uncertainty, in some proteins more than others, as to the precise location of some parts of the sequence relative to the membrane.

The signal hypothesis for the transmembrane translocation of proteins is briefly described and its general applicability is reviewed. There are many proteins whose translocation is accurately described by this hypothesis, but some proteins are translocated in a different manner.

The transmembraneous glycoproteins E1 and E2 of Sindbis virus, as well as the only other virion protein, the capsid protein, were purified in amounts sufficient for biochemical analysis using sensitive techniques. The amino acid composition of each protein was determined, and extensive N-terminal sequences were obtained for E1 and E2. By these techniques E1 and E2 are indistinguishable from most water soluble proteins, as they do not contain an obvious excess of hydrophobic amino acids in their N-terminal regions or in the intact molecule.

The capsid protein was found to be blocked, and so its N-terminus could not be sequenced by the usual methods. However, with the use of a special labeling technique, it was possible to incorporate tritiated acetate into the N-terminus of the protein with good specificity, which was useful in the purification of peptides from which the first amino acids in the N-terminal sequence could be identified.

Nanomole amounts of PE2, the intracellular precursor of E2, were purified by an immuno-affinity technique, and its N-terminus was analyzed. Together with other work, these results showed that PE2 is not synthesized with an N-terminal extension, and the signal sequence for translocation is probably the N-terminal amino acid sequence of the protein. This N-terminus was found to be 80-90% blocked, also by Nacetylation, and this acetylation did not affect its function as a signal sequence. The putative signal sequence was also found to contain a glycosylated asparagine residue, but the inhibition of this glycosylation did not lead to the cleavage of the sequence.

PP backward elastic scattering between 0.70 and 2.37 GeV/c

̄pp backward elastic scattering has been measured for the cos θ_cm region between – 1.00 and – 0.88 and for the incident ̄p laboratory momentum region between 0.70 and 2.37 GeV/c. These measurements, done in intervals of approximately 0.1 GeV/c, have been performed at the Alternating Gradient Synchrotron at Brookhaven National Laboratory during the winter of 1968. The measured differential cross sections, binned in cos θ_cm intervals of 0.02, have statistical errors of about 10%. Backward dipping exists below 0.95 GeV/c and backward peaking above 0.95 GeV/c. The 180˚ differential cross section extrapolated from our data shows a sharp dip centered at 0.95 GeV/c and a broad hump centered near 1.4 GeV/c. Our data have been interpreted in terms of resonance effects and in terms of diffraction dominance effects.

The interactions of basic proteins and DNA

I. ELECTROPHORESIS OF THE NUCLEIC ACIDS

A zone electrophoresis apparatus using ultraviolet optics has been constructed to study nucleic acids at concentrations less than 0.004%. Native DNA has a mobility about 15% higher than denatured DNA over a range of conditions. Otherwise, the electrophoretic mobility is independent of molecular weight, base composition or source. DNA mobilities change in the expected way with pH but the fractional change in mobility is less than the calculated change in charge. A small decrease in mobility accompanies an increase in ionic strength. RNA’s from various sources have mobilities slightly lower than denatured DNA except for s-RNA which travels slightly faster. The important considerations governing the mobility of nucleic acids appear to be the nature of the hydrodynamic segment, and the binding of counterions. The differences between electrophoresis and sedimentation stem from the fact that all random coil polyelectrolytes are fundamentally free draining in electrophoresis.

II. THE CYTOCHROME C/DNA COMPLEX

The basic protein, cytochrome c, has been complexed to DNA. Up to a cytochrome:DNA mass ratio of 2, a single type of complex is formed. Dissociation of this complex occurs between 0.05F and 0.1F NaCl. The complexing of cytochrome to DNA causes a slight increase in the melting temperature of the DNA, and a reduction of the electrophoretic mobility proportional to the decrease in net charge. Above a cytochrome:DNA mass ratio of 2.5, a different type of complex is formed. The results suggest that complexes such as are formed in the Kleinschmidt technique of electron microscopy would not exist in bulk solution and are exclusively film phenomena.

III. STUDIES OF THE ELECTROPHORESIS AND MELTING BEHAVIOUR OF NUCLEOHISTONES

Electrophoresis studies on reconstituted nucleohistones indicate that the electrophoretic mobility for these complexes is a function of the net charge of the complex. The mobility is therefore dependent on the charge density of the histone complexing the DNA, as well as on the histone/DNA ratio. It is found that the different histones affect the transition from native to denatured DNA in different ways. It appears that histone I is exchanging quite rapidly between DNA molecules in 0.01 F salt, while histone II is irreversibly bound. Histone III-IV enhances the capacity of non-strand separated denatured DNA to reanneal. Studies on native nucleoproteins indicate that there are no gene-sized uncomplexed DNA regions in any preparations studied.

IV. THE DISSOCIATION OF HISTONE FROM CALF THYMUS CROMATIN

Calf thymus nucleoprotein was treated with varying concentrations of NaCl. The identity of the histones associated and dissociated from the DNA at each salt concentration was determined by gel electrophoresis. It was found that there is no appreciable histone dissociation below 0.4 F NaCl. The lysine rich histones dissociate between 0.4 and 0.5 F NaCl. Their dissociation is accompanies by a marked increase in the solubility of the chromatin. The moderately lysine rich histones dissociate mainly between 0.8 and 1.1 F NaCl. There are two arginine rich histone components: the first dissociates between 0.8 F and 1.1 F NaCl, but the second class is the very last to be dissociated from the DNA (dissociation beginning at 1.0 F NaCl). By 2.0 F NaCl, essentially all the histones are dissociated.

The properties of the extracted nucleoprotein were studied. The electrophoretic mobility increases and the melting temperature decreases as more histones are dissociated from the DNA. A comparison with the dissociation of histones from DNA in NaClO₄ shows that to dissociate the same class of histones, the concentration of NaCl required is twice that of NaClO₄.

The influence of the NN* channel on elastic NN scattering

The problem of two channels NN and NN*, coupled through unitarity, is studied to see whether sizable peaks can be produced in elastic nucleon-nucleon scattering due to the opening of a strongly coupled inelastic channel. One-pion-exchange (OPE) interactions are calculated to estimate the NN*→NN* and NN→NN* amplitudes. The OPE production amplitudes are used as the sole dynamical input to drive the multichannel ND^-1 equations in the determinental approximation, and the effect on the J = 2+ (¹D₂) elastic NN scattering amplitude is studied as the width of the unstable N* and strength of coupling to the inelastic channel are varied. A cusp-type enhancement appears in the NN channel near the NN* threshold but for the known value of the N* width the cusp is so “wooly” that any resulting elastic peak is likely to be too broad and diminished in height to be experimentally prominent. A brief survey of current experimental knowledge of the real part of the ¹D₂ NN phase shift near the NN* threshold is given, and the values are found to be much smaller than the nearly “resonant” phase shifts predicted by the coupled channel model.

Energy inequalities and error estimates for axisymmetric torsion of thin elastic shells of revolution

The problem motivating this investigation is that of pure axisymmetric torsion of an elastic shell of revolution. The analysis is carried out within the framework of the three-dimensional linear theory of elastic equilibrium for homogeneous, isotropic solids. The objective is the rigorous estimation of errors involved in the use of approximations based on thin shell theory.

The underlying boundary value problem is one of Neumann type for a second order elliptic operator. A systematic procedure for constructing pointwise estimates for the solution and its first derivatives is given for a general class of second-order elliptic boundary-value problems which includes the torsion problem as a special case.

The method used here rests on the construction of “energy inequalities” and on the subsequent deduction of pointwise estimates from the energy inequalities. This method removes certain drawbacks characteristic of pointwise estimates derived in some investigations of related areas.

Special interest is directed towards thin shells of constant thickness. The method enables us to estimate the error involved in a stress analysis in which the exact solution is replaced by an approximate one, and thus provides us with a means of assessing the quality of approximate solutions for axisymmetric torsion of thin shells.

Finally, the results of the present study are applied to the stress analysis of a circular cylindrical shell, and the quality of stress estimates derived here and those from a previous related publication are discussed.

Part I. Synchronization of the cell division cycle of HeLa cells in suspension cultures. Part II. Studies on chromosomal proteins of HeLa cells during the cell division cycle

Part I

These studies investigate the potential of single and double treatments with either 5-fluorodeoxyuridine of excess thymidine to induce cell division synchrony in suspension cultures of HeLa cells. The patterns of nucleic acid synthesis and cell proliferation have been analyzed in cultures thus synchronized. Several changes in cell population during long incubation with 5-fluorodeoxyuridine or excess thymidine are also described. These results are subjected to detailed evaluation in terms of the degree and quality of synchrony finally achieved.

Part II

Histones and non-histone proteins associated with interphase and metaphase chromosomes of HeLa cells have been qualitatively and quantitatively analyzed. Histones were fractionated by chromatography on Amberlite CG-50 and further characterized by analytical disc electrophoresis and amino acid analysis of each chromatographic fraction. It is concluded that histones of HeLa cells are comprised of only a small number of major components and that these components are homologous to those of other higher organisms. Of all the histones, arginine-rich histone III alone contains cysteine and can polymerize through formation of intermolecular disulfide bridges between histone III monomers.

A detailed comparison by chromatography and disc electrophoresis established that interphase and metaphase histones are made up of similar components. However, certain quantitative differences in proportions of different histones of interphase and metaphase cells are reported. Indirect evidence indicates that a certain proportion of metaphase histone III is polymerized through intermolecular disulfide links, whereas interphase histone III occurs mainly in the monomeric form.

Metaphase chromosomes are associated with an additional acid-soluble protein fraction which is absent from interphase chromosomes. All of these additional acid-soluble proteins of metaphase chromosomes are shown to be non-histones and it is concluded that the histone/DNA ratio is identical in interphase and metaphase chromosomes. The bulk of acid-soluble non-histone proteins of metaphase chromosomes were found to be polymerized through disulfide bridges; corresponding interphase non-histone proteins displayed no evidence of similar polymerization.

The factors responsible for the condensed configuration and metabolic inactivity of metaphase chromosomes are discussed in light of these findings.

The relationship between histone and DNA synthesis in nondividing differentiated chicken erythrocyte cells and in rapidly dividing undifferentiated HeLa cells is also investigated. Of all the histones, only arginine-rich histones are synthesized in mature erythrocytes. Histone synthesis in HeLa cells was studied in both unsynchronized and synchronized cultures. In HeLa cells, only part of the synthesis of all histone fractions is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis.

Hydrothermal experiments on the thermal stability of amino substances in sediments

Aspartic acid, threonine, serine and other thermally unstable amino acids have been found in fine-grained elastic sediments of advanced geologic age. The presence of these compounds in ancient sediments conflicts with experimental data determined for their simple thermal decomposition.

Recent and Late Miocene sediments and their humic acid extracts, known to contain essentially complete suites of amino acids, were heated with H₂O in a bomb at temperatures up to 500°C in order to compare the thermal decomposition characteristics of the sedimentary amino compounds.

Most of the amino acids found in protein hydrolyzates are obtained from the Miocene rock in amounts 10 to 100 times less than from the Recent sediment. The two unheated humic acids are rather similar despite their great age difference. The Miocene rock appears uncontaminated by Recent carbon.

Yields of amino acids generally decline in the heated Recent sediment. Some amino compounds apparently increase with heating time in the Miocene rock.

Relative thermal stabilities of the amino acids in sediments are generally similar to those determined using pure aqueous solutions. The relative thermal stabilities of glutamic acid, glycine, and phenylalanine vary in the Recent sediment but are uniform in the Miocene rock.

Amino acids may occur in both proteins and humic complexes in the Recent sediment, while they are probably only present in stabilized organic substances in the Miocene rock. Thermal decomposition of protein amino acids may be affected by surface catalysis in the Recent sediment. The apparent activation energy for the decomposition of alanine in this sediment is 8400 calories per mole. Yields of amino compounds from the heated sediments are not affected by thermal decomposition only.

Amino acids in sediments may only be useful for geothermometry in a very general way.

A better picture of the amino acid content of older sedimentary rocks may be obtained if these sediments are heated in a bomb with H₂O at temperatures around 150°C prior to HCl hydrolysis.

Leucine-isoleucine ratios may prove to be useful as indicators of amino acid sources or for evaluating the fractionation of these substances during diagenesis. Leucine-isoleucine ratios of the Recent and Miocene sediments and humic acids are identical. The humic acids may have a continental source.

The carbon-nitrogen and carbon-hydrogen ratios of sediments and humic acids increase with heating time and temperature. Ratios comparable to those in some kerogens are found in the severely heated Miocene sediment and humic acid.

Large plane deformations of thin elastic sheets of neo-hookean material

Large plane deformations of thin elastic sheets of neo-Hookean material are considered and a method of successive substitutions is developed to solve problems within the two-dimensional theory of finite plane stress. The first approximation is determined by linear boundary value problems on two harmonic functions, and it is approached asymptotically at very large extensions in the plane of the sheet. The second and higher approximations are obtained by solving Poisson equations. The method requires modification when the membrane has a traction-free edge.

Several problems are treated involving infinite sheets under uniform biaxial stretching at infinity. First approximations are obtained when a circular or elliptic inclusion is present and when the sheet has a circular or elliptic hole, including the limiting cases of a line inclusion and a straight crack or slit. Good agreement with exact solutions is found for circularly symmetric deformations. Other examples discuss the stretching of a short wide strip, the deformation near a boundary corner which is traction-free, and the application of a concentrated load to a boundary point.

Using Earth Deformation Caused by Surface Mass Loading to Constrain the Elastic Structure of the Crust and Mantle

Surface mass loads come in many different varieties, including the oceans, atmosphere, rivers, lakes, glaciers, ice caps, and snow fields. The loads migrate over Earth's surface on time scales that range from less than a day to many thousand years. The weights of the shifting loads exert normal forces on Earth's surface. Since the Earth is not perfectly rigid, the applied pressure deforms the shape of the solid Earth in a manner controlled by the material properties of Earth's interior. One of the most prominent types of surface mass loading, ocean tidal loading (OTL), comes from the periodic rise and fall in sea-surface height due to the gravitational influence of celestial objects, such as the moon and sun. Depending on geographic location, the surface displacements induced by OTL typically range from millimeters to several centimeters in amplitude, which may be inferred from Global Navigation and Satellite System (GNSS) measurements with sub-millimeter precision. Spatiotemporal characteristics of observed OTL-induced surface displacements may therefore be exploited to probe Earth structure. In this thesis, I present descriptions of contemporary observational and modeling techniques used to explore Earth's deformation response to OTL and other varieties of surface mass loading. With the aim to extract information about Earth's density and elastic structure from observations of the response to OTL, I investigate the sensitivity of OTL-induced surface displacements to perturbations in the material structure. As a case study, I compute and compare the observed and predicted OTL-induced surface displacements for a network of GNSS receivers across South America. The residuals in three distinct and dominant tidal bands are sub-millimeter in amplitude, indicating that modern ocean-tide and elastic-Earth models well predict the observed displacement response in that region. Nevertheless, the sub-millimeter residuals exhibit regional spatial coherency that cannot be explained entirely by random observational uncertainties and that suggests deficiencies in the forward-model assumptions. In particular, the discrepancies may reveal sensitivities to deviations from spherically symmetric, non-rotating, elastic, and isotropic (SNREI) Earth structure due to the presence of the South American craton.