Brain type II calcium and calmodulin-dependent protein kinase: characterization of a brain-region specific isozyme and regulation by autophosphorylation

A variety of molecular approaches have been used to investigate the structural and enzymatic properties of rat brain type ll Ca^(2+) and calmodulin-dependent protein kinase (type ll CaM kinase). This thesis describes the isolation and biochemical characterization of a brain-region specific isozyme of the kinase and also the regulation the kinase activity by autophosphorylation.

The cerebellar isozyme of the type ll CaM kinase was purified and its biochemical properties were compared to the forebrain isozyme. The cerebellar isozyme is a large (500-kDa) multimeric enzyme composed of multiple copies of 50-kDa α subunits and 60/58-kDa β/β’ subunits. The holoenzyme contains approximately 2 α subunits and 8 β subunits. This contrasts to the forebrain isozyme, which is also composed of and β/β'subunits, but they are assembled into a holoenzyme of approximately 9 α subunits and 3 β/β ' subunits. The biochemical and enzymatic properties of the two isozymes are similar. The two isozymes differ in their association with subcellular structures. Approximately 85% of the cerebellar isozyme, but only 50% of the forebrain isozyme, remains associated with the particulate fraction after homogenization under standard conditions. Postsynaptic densities purified from forebrain contain the forebrain isozyme, and the kinase subunits make up about 16% of their total protein. Postsynaptic densities purified from cerebellum contain the cerebellar isozyme, but the kinase subunits make up only 1-2% of their total protein.

The enzymatic activity of both isozymes of the type II CaM kinase is regulated by autophosphorylation in a complex manner. The kinase is initially completely dependent on Ca^(2+)/calmodulin for phosphorylation of exogenous substrates as well as for autophosphorylation. Kinase activity becomes partially Ca^(2+) independent after autophosphorylation in the presence of Ca^(2+)/calmodulin. Phosphorylation of only a few subunits in the dodecameric holoenzyme is sufficient to cause this change, suggesting an allosteric interaction between subunits. At the same time, autophosphorylation itself becomes independent of Ca^(2+) These observations suggest that the kinase may be able to exist in at least two stable states, which differ in their requirements for Ca^(2+)/calmodulin.

The autophosphorylation sites that are involved in the regulation of kinase activity have been identified within the primary structure of the α and β subunits. We used the method of reverse phase-HPLC tryptic phosphopeptide mapping to isolate individual phosphorylation sites. The phosphopeptides were then sequenced by gas phase microsequencing. Phosphorylation of a single homologous threonine residue in the α and β subunits is correlated with the production of the Ca^(2+) -independent activity state of the kinase. In addition we have identified several sites that are phosphorylated only during autophosphorylation in the absence of Ca^(2+)/ calmodulin.

Detailed properties of high redshift galaxies

Galaxies evolve throughout the history of the universe from the first star-forming sources, through gas-rich asymmetric structures with rapid star formation rates, to the massive symmetrical stellar systems observed at the present day. Determining the physical processes which drive galaxy formation and evolution is one of the most important questions in observational astrophysics. This thesis presents four projects aimed at improving our understanding of galaxy evolution from detailed measurements of star forming galaxies at high redshift.

We use resolved spectroscopy of gravitationally lensed z ≃ 2 - 3 star forming galaxies to measure their kinematic and star formation properties. The combination of lensing with adaptive optics yields physical resolution of ≃ 100 pc, sufficient to resolve giant Hii regions. We find that ~ 70 % of galaxies in our sample display ordered rotation with high local velocity dispersion indicating turbulent thick disks. The rotating galaxies are gravitationally unstable and are expected to fragment into giant clumps. The size and dynamical mass of giant Hii regions are in agreement with predictions for such clumps indicating that gravitational instability drives the rapid star formation. The remainder of our sample is comprised of ongoing major mergers. Merging galaxies display similar star formation rate, morphology, and local velocity dispersion as isolated sources, but their velocity fields are more chaotic with no coherent rotation.

We measure resolved metallicity in four lensed galaxies at z = 2.0 − 2.4 from optical emission line diagnostics. Three rotating galaxies display radial gradients with higher metallicity at smaller radii, while the fourth is undergoing a merger and has an inverted gradient with lower metallicity at the center. Strong gradients in the rotating galaxies indicate that they are growing inside-out with star formation fueled by accretion of metal-poor gas at large radii. By comparing measured gradients with an appropriate comparison sample at z = 0, we demonstrate that metallicity gradients in isolated galaxies must flatten at later times. The amount of size growth inferred by the gradients is in rough agreement with direct measurements of massive galaxies. We develop a chemical evolution model to interpret these data and conclude that metallicity gradients are established by a gradient in the outflow mass loading factor, combined with radial inflow of metal-enriched gas.

We present the first rest-frame optical spectroscopic survey of a large sample of low-luminosity galaxies at high redshift (L < L*, 1.5 < z < 3.5). This population dominates the star formation density of the universe at high redshifts, yet such galaxies are normally too faint to be studied spectroscopically. We take advantage of strong gravitational lensing magnification to compile observations for a sample of 29 galaxies using modest integration times with the Keck and Palomar telescopes. Balmer emission lines confirm that the sample has a median SFR ∼ 10 M_sun yr^−1 and extends to lower SFR than has been probed by other surveys at similar redshift. We derive the metallicity, dust extinction, SFR, ionization parameter, and dynamical mass from the spectroscopic data, providing the first accurate characterization of the star-forming environment in low-luminosity galaxies at high redshift. For the first time, we directly test the proposal that the relation between galaxy stellar mass, star formation rate, and gas phase metallicity does not evolve. We find lower gas phase metallicity in the high redshift galaxies than in local sources with equivalent stellar mass and star formation rate, arguing against a time-invariant relation. While our result is preliminary and may be biased by measurement errors, this represents an important first measurement that will be further constrained by ongoing analysis of the full data set and by future observations.

We present a study of composite rest-frame ultraviolet spectra of Lyman break galaxies at z = 4 and discuss implications for the distribution of neutral outflowing gas in the circumgalactic medium. In general we find similar spectroscopic trends to those found at z = 3 by earlier surveys. In particular, absorption lines which trace neutral gas are weaker in less evolved galaxies with lower stellar masses, smaller radii, lower luminosity, less dust, and stronger Lyα emission. Typical galaxies are thus expected to have stronger Lyα emission and weaker low-ionization absorption at earlier times, and we indeed find somewhat weaker low-ionization absorption at higher redshifts. In conjunction with earlier results, we argue that the reduced low-ionization absorption is likely caused by lower covering fraction and/or velocity range of outflowing neutral gas at earlier epochs. This result has important implications for the hypothesis that early galaxies were responsible for cosmic reionization. We additionally show that fine structure emission lines are sensitive to the spatial extent of neutral gas, and demonstrate that neutral gas is concentrated at smaller galactocentric radii in higher redshift galaxies.

The results of this thesis present a coherent picture of galaxy evolution at high redshifts 2 ≲ z ≲ 4. Roughly 1/3 of massive star forming galaxies at this period are undergoing major mergers, while the rest are growing inside-out with star formation occurring in gravitationally unstable thick disks. Star formation, stellar mass, and metallicity are limited by outflows which create a circumgalactic medium of metal-enriched material. We conclude by describing some remaining open questions and prospects for improving our understanding of galaxy evolution with future observations of gravitationally lensed galaxies.

Growth and characterization of novel, III-V semiconductor heterostructures

The material presented in this thesis concerns the growth and characterization of III-V semiconductor heterostructures. Studies of the interactions between bound states in coupled quantum wells and between well and barrier bound states in AlAs/GaAs heterostructures are presented. We also demonstrate the broad array of novel tunnel structures realizable in the InAs/GaSb/AlSb material system. Because of the unique broken-gap band alignment of InAs/GaSb these structures involve transport between the conduction- and valence-bands of adjacent layers. These devices possess a wide range of electrical properties and are fundamentally different from conventional AlAs/GaAs tunnel devices. We report on the fabrication of a novel tunnel transistor with the largest reported room temperature current gains. We also present time-resolved studies of the growth fronts of InAs/GainSb strained layer superlattices and investigations of surface anion exchange reactions.

Chapter 2 covers tunneling studies of conventional AlAs/GaAs RTD's. The results of two studies are presented: (i) A test of coherent vs. sequential tunneling in triple barrier heterostructures, (ii) An optical measurement of the effect of barrier X-point states on Γ-point well states. In the first it was found if two quantum wells are separated by a sufficiently thin barrier, then the eigenstates of the system extend coherently across both wells and the central barriers. For thicker barriers between the wells, the electrons become localized in the individual wells and transport is best described by the electrons hopping between the wells. In the second, it was found that Γ-point well states and X-point barrier states interact strongly. The barrier X-point states modify the energies of the well states and increase the escape rate for carriers in the quantum well.

The results of several experimental studies of a novel class of tunnel devices realized in the InAs/GaSb/AlSb material system are presented in Chapter 3. These interband tunnel structures involve transport between conduction- and valence-band states in adjacent material layers. These devices are compared and contrasted with the conventional AlAs/GaAs structures discussed in Chapter 2 and experimental results are presented for both resonant and nonresonant devices. These results are compared with theoretical simulations and necessary extensions to the theoretical models are discussed.

In chapter 4 experimental results from a novel tunnel transistor are reported. The measured current gains in this transistor exceed 100 at room temperature. This is the highest reported gain at room temperature for any tunnel transistor. The device is analyzed and the current conduction and gain mechanisms are discussed.

Chapters 5 and 6 are studies of the growth of structures involving layers with different anions. Chapter 5 covers the growth of InAs/GainSb superlattices for far infrared detectors and time resolved, in-situ studies of their growth fronts. It was found that the bandgap of superlattices with identical layer thicknesses and compositions varied by as much as 40 meV depending on how their internal interfaces are formed. The absorption lengths in superlattices with identical bandgaps but whose interfaces were formed in different ways varied by as much as a factor of two. First the superlattice is discussed including an explanation of the device and the complications involved in its growth. The experimental technique of reflection high energy electron diffraction (RHEED) is reviewed, and the results of RHEED studies of the growth of these complicated structures are presented. The development of a time resolved, in-situ characterization of the internal interfaces of these superlattices is described. Chapter 6 describes the result of a detailed study of some of the phenomena described in chapter 5. X-ray photoelectron spectroscopy (XPS) studies of anion exchange reactions on the growth fronts of these superlattices are reported. Concurrent RHEED studies of the same physical systems studied with XPS are presented. Using the RHEED and XPS results, a real-time, indirect measurement of surface exchange reactions was developed.

Anatomical and developmental patterns of interleukin-2 gene expression in the mouse: analysis of IL-2 expressing cells and partial characterization of interactions involved in mediating IL-2 gene expression in vivo

Interleukin-2 (IL-2) is an important mediator in the vertebrate immune system. IL-2 is a potent growth factor that mature T lymphocytes use as a proliferation signal and the production of IL-2 is crucial for the clonal expansion of antigen-specific T cells in the primary immune response. IL-2 driven proliferation is dependent on the interaction of the lymphokine with its cognate multichain receptor. IL-2 expression is induced only upon stimulation and transcriptional activation of the IL-2 gene relies extensively on the coordinate interaction of numerous inducible and constitutive trans-acting factors. Over the past several years, thousands of papers have been published regarding molecular and cellular aspects of IL-2 gene expression and IL-2 function. The vast majority of these reports describe work that has been carried out in vitro. However, considerably less is known about control of IL-2 gene expression and IL-2 function in vivo.

To gain new insight into the regulation of IL-2 gene expression in vivo, anatomical and developmental patterns of IL-2 gene expression in the mouse were established by employing in situ hybridization and immunohistochemical staining methodologies to tissue sections generated from normal mice and mutant animals in which T -cell development was perturbed. Results from these studies revealed several interesting aspects of IL-2 gene expression, such as (1) induction of IL-2 gene expression and protein synthesis in the thymus, the primary site of T-cell development in the body, (2) cell-type specificity of IL-2 gene expression in vivo, (3) participation of IL-2 in the extrathymic expansion of mature T cells in particular tissues, independent of an acute immune response to foreign antigen, (4) involvement of IL-2 in maintaining immunologic balance in the mucosal immune system, and (5) potential function of IL-2 in early events associated with hematopoiesis.

Extensive analysis of IL-2 mRNA accumulation and protein production in the murine thymus at various stages of development established the existence of two classes of intrathymic IL-2 producing cells. One class of intrathymic IL-2 producers was found exclusively in the fetal thymus. Cells belonging to this subset were restricted to the outermost region of the thymus. IL-2 expression in the fetal thymus was highly transient; a dramatic peak ofiL-2 mRNA accumulation was identified at day 14.5 of gestation and maximal IL-2 protein production was observed 12 hours later, after which both IL-2 mRNA and protein levels rapidly decreased. Significantly, the presence of IL-2 expressing cells in the day 14-15 fetal thymus was not contingent on the generation of T-cell receptor (TcR) positive cells. The second class of IL-2 producing cells was also detectable in the fetal thymus (cells found in this class represented a minority subset of IL-2 producers in the fetal thymus) but persist in the thymus during later stages of development and after birth. Intrathymic IL-2 producers in postnatal animals were located in the subcapsular region and cortex, indicating that these cells reside in the same areas where immature T cells are consigned. The frequency of IL-2 expressing cells in the postnatal thymus was extremely low, indicating that induction of IL-2 expression and protein synthesis are indicative of a rare activation event. Unlike the fetal class of intrathymic IL-2 producers, the presence of IL-2 producing cells in the postnatal thymus was dependent on to the generation of TcR+ cells. Subsequent examination of intrathymic IL-2 production in mutant postnatal mice unable to produce either αβ or γδ T cells showed that postnatal IL-2 producers in the thymus belong to both αβ and γδ lineages. Additionally, further studies indicated that IL-2 synthesis by immature αβ -T cells depends on the expression of bonafide TcR αβ-heterodimers. Taken altogether, IL-2 production in the postnatal thymus relies on the generation of αβ or γδ-TcR^+ cells and induction of IL-2 protein synthesis can be linked to an activation event mediated via the TcR.

With regard to tissue specificity of IL-2 gene expression in vivo, analysis of whole body sections obtained from normal neonatal mouse pups by in situ hybridization demonstrated that IL-2 mRNA^+ cells were found in both lymphoid and nonlymphoid tissues with which T cells are associated, such as the thymus (as described above), dermis and gut. Tissues devoid of IL-2 mRNA^+ cells included brain, heart, lung, liver, stomach, spine, spinal cord, kidney, and bladder. Additional analysis of isolated tissues taken from older animals revealed that IL-2 expression was undetectable in bone marrow and in nonactivated spleen and lymph nodes. Thus, it appears that extrathymic IL-2 expressing cells in nonimmunologically challenged animals are relegated to particular epidermal and epithelial tissues in which characterized subsets of T cells reside and thatinduction of IL-2 gene expression associated with these tissues may be a result of T-cell activation therein.

Based on the neonatal in situ hybridization results, a detailed investigation into possible induction of IL-2 expression resulting in IL-2 protein synthesis in the skin and gut revealed that IL-2 expression is induced in the epidermis and intestine and IL-2 protein is available to drive cell proliferation of resident cells and/or participate in immune function in these tissues. Pertaining to IL-2 expression in the skin, maximal IL-2 mRNA accumulation and protein production were observed when resident Vγ_3^+ T-cell populations were expanding. At this age, both IL-2 mRNA^+ cells and IL-2 protein production were intimately associated with hair follicles. Likewise, at this age a significant number of CD3ε^+ cells were also found in association with follicles. The colocalization of IL-2 expression and CD3ε^+ cells suggests that IL-2 expression is induced when T cells are in contact with hair follicles. In contrast, neither IL-2 mRNA nor IL-2 protein were readily detected once T-cell density in the skin reached steady-state proportions. At this point, T cells were no longer found associated with hair follicles but were evenly distributed throughout the epidermis. In addition, IL-2 expression in the skin was contingent upon the presence of mature T cells therein and induction of IL-2 protein synthesis in the skin did not depend on the expression of a specific TcR on resident T cells. These newly disclosed properties of IL-2 expression in the skin indicate that IL-2 may play an additional role in controlling mature T-cell proliferation by participating in the extrathymic expansion of T cells, particularly those associated with the epidermis.

Finally, regarding IL-2 expression and protein synthesis in the gut, IL-2 producing cells were found associated with the lamina propria of neonatal animals and gut-associated IL-2 production persisted throughout life. In older animals, the frequency of IL-2 producing cells in the small intestine was not identical to that in the large intestine and this difference may reflect regional specialization of the mucosal immune system in response to enteric antigen. Similar to other instances of IL-2 gene expression in vivo, a failure to generate mature T cells also led to an abrogation of IL-2 protein production in the gut. The presence of IL-2 producing cells in the neonatal gut suggested that these cells may be generated during fetal development. Examination of the fetal gut to determine the distribution of IL-2 producing cells therein indicated that there was a tenfold increase in the number of gut-associated IL-2 producers at day 20 of gestation compared to that observed four days earlier and there was little difference between the frequency of IL-2 producing cells in prenatal versus neonatal gut. The origin of these fetally-derived IL-2 producing cells is unclear. Prior to the immigration of IL-2 inducible cells to the fetal gut and/or induction of IL-2 expression therein, IL-2 protein was observed in the fetal liver and fetal omentum, as well as the fetal thymus. Considering that induction of IL-2 protein synthesis may be an indication of future functional capability, detection of IL-2 producing cells in the fetal liver and fetal omentum raises the possibility that IL-2 producing cells in the fetal gut may be extrathymic in origin and IL-2 producing cells in these fetal tissues may not belong solely to the T lineage. Overall, these results provide increased understanding of the nature of IL-2 producing cells in the gut and how the absence of IL-2 production therein and in fetal hematopoietic tissues can result in the acute pathology observed in IL-2 deficient animals.

Characterization of diverse megathrust fault behavior related to seismic supercycles, Mentawai Islands, Sumatra

Long paleoseismic histories are necessary for understanding the full range of behavior of faults, as the most destructive events often have recurrence intervals longer than local recorded history. The Sunda megathrust, the interface along which the Australian plate subducts beneath Southeast Asia, provides an ideal natural laboratory for determining a detailed paleoseismic history over many seismic cycles. The outer-arc islands above the seismogenic portion of the megathrust cyclically rise and subside in response to processes on the underlying megathrust, providing uncommonly good illumination of megathrust behavior. Furthermore, the growth histories of coral microatolls, which record tectonic uplift and subsidence via relative sea level, can be used to investigate the detailed coseismic and interseismic deformation patterns. One particularly interesting area is the Mentawai segment of the megathrust, which has been shown to characteristically fail in a series of ruptures over decades, rather than a single end-to-end rupture. This behavior has been termed a seismic “supercycle.” Prior to the current rupture sequence, which began in 2007, the segment previously ruptured during the 14th century, the late 16th to late 17th century, and most recently during historical earthquakes in 1797 and 1833. In this study, we examine each of these previous supercycles in turn.

First, we expand upon previous analysis of the 1797–1833 rupture sequence with a comprehensive review of previously published coral microatoll data and the addition of a significant amount of new data. We present detailed maps of coseismic uplift during the two great earthquakes and of interseismic deformation during the periods 1755–1833 and 1950–1997 and models of the corresponding slip and coupling on the underlying megathrust. We derive magnitudes of Mw 8.7–9.0 for the two historical earthquakes, and determine that the 1797 earthquake fundamentally changed the state of coupling on the fault for decades afterward. We conclude that while major earthquakes generally do not involve rupture of the entire Mentawai segment, they undoubtedly influence the progression of subsequent ruptures, even beyond their own rupture area. This concept is of vital importance for monitoring and forecasting the progression of the modern rupture sequence.

Turning our attention to the 14th century, we present evidence of a shallow slip event in approximately A.D. 1314, which preceded the “conventional” megathrust rupture sequence. We calculate a suite of slip models, slightly deeper and/or larger than the 2010 Pagai Islands earthquake, that are consistent with the large amount of subsidence recorded at our study site. Sea-level records from older coral microatolls suggest that these events occur at least once every millennium, but likely far less frequently than their great downdip neighbors. The revelation that shallow slip events are important contributors to the seismic cycle of the Mentawai segment further complicates our understanding of this subduction megathrust and our assessment of the region’s exposure to seismic and tsunami hazards.

Finally, we present an outline of the complex intervening rupture sequence that took place in the 16th and 17th centuries, which involved at least five distinct uplift events. We conclude that each of the supercycles had unique features, and all of the types of fault behavior we observe are consistent with highly heterogeneous frictional properties of the megathrust beneath the south-central Mentawai Islands. We conclude that the heterogeneous distribution of asperities produces terminations and overlap zones between fault ruptures, resulting in the seismic “supercycle” phenomenon.

Preparation and characterization of an intramolecular electron transfer in a pentaammineruthenium derivative of Candida krisei cytochrome c

A semisynthetic binuclear metalloprotein has been prepared by appending the pentaammineruthenium moiety to histidine 39 of the cytochrome c from the yeast Candida krusei. The site of ruthenium binding was identified by peptide mapping. Spectroscopic and electrochemical properties of the derivative indicate the protein conformation is unperturbed by the modification. A preliminary (minimum) rate constant of 170s^(-1) has been determined for the intramolecular electron transfer from ruthenium(II) to iron(III), which occurs over a distance of at least 13Å (barring major conformational changes). Electrochemical studies indicate that this reaction should proceed with a driving force of ~170mV. The rate constant is an order of magnitude faster than that observed in horse heart cytochrome c for intramolecular electron transfer from pentaammineruthenium(II)(histidine 33) to iron(III) (over a similar distance, and with a similar driving force), suggesting a medium or orientation effect makes the Candida intramolecular electron transfer more favorable.

Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MSCL)

The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.

It has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 °C – 60 °C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.

Extensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.

MscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.

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.

Fabrication, Characterization, And Deformation of 3D Structural Meta-Materials

Current technological advances in fabrication methods have provided pathways to creating architected structural meta-materials similar to those found in natural organisms that are structurally robust and lightweight, such as diatoms. Structural meta-materials are materials with mechanical properties that are determined by material properties at various length scales, which range from the material microstructure (nm) to the macro-scale architecture (μm – mm). It is now possible to exploit material size effect, which emerge at the nanometer length scale, as well as structural effects to tune the material properties and failure mechanisms of small-scale cellular solids, such as nanolattices. This work demonstrates the fabrication and mechanical properties of 3-dimensional hollow nanolattices in both tension and compression. Hollow gold nanolattices loaded in uniaxial compression demonstrate that strength and stiffness vary as a function of geometry and tube wall thickness. Structural effects were explored by increasing the unit cell angle from 30° to 60° while keeping all other parameters constant; material size effects were probed by varying the tube wall thickness, t, from 200nm to 635nm, at a constant relative density and grain size. In-situ uniaxial compression experiments reveal an order-of-magnitude increase in yield stress and modulus in nanolattices with greater lattice angles, and a 150% increase in the yield strength without a concomitant change in modulus in thicker-walled nanolattices for fixed lattice angles. These results imply that independent control of structural and material size effects enables tunability of mechanical properties of 3-dimensional architected meta-materials and highlight the importance of material, geometric, and microstructural effects in small-scale mechanics. This work also explores the flaw tolerance of 3D hollow-tube alumina kagome nanolattices with and without pre-fabricated notches, both in experiment and simulation. Experiments demonstrate that the hollow kagome nanolattices in uniaxial tension always fail at the same load when the ratio of notch length (a) to sample width (w) is no greater than 1/3, with no correlation between failure occurring at or away from the notch. For notches with (a/w) > 1/3, the samples fail at lower peak loads and this is attributed to the increased compliance as fewer unit cells span the un-notched region. Finite element simulations of the kagome tension samples show that the failure is governed by tensile loading for (a/w) < 1/3 but as (a/w) increases, bending begins to play a significant role in the failure. This work explores the flaw sensitivity of hollow alumina kagome nanolattices in tension, using experiments and simulations, and demonstrates that the discrete-continuum duality of architected structural meta-materials gives rise to their flaw insensitivity even when made entirely of intrinsically brittle materials.