Utilizing the diverse functionality of amino acids, a new class of amphiphilic graft copolymers has been synthesized, characterized, and explored for applications in biomaterials and nucleic acid delivery. This thesis research focused on the syntheses of oligopeptide-functionalized polyesters and polyolefins. Polyester functionalization was geared towards applications in biomaterials, tissue engineering, and drug delivery by incorporating sequences that promote cell-adhesion. These polyester-graft-oligopeptide materials were prepared by a 1,3-Huisgen cycloaddition reaction, “click” chemistry, of an azide-terminated oligopeptide (prepared by Fmoc-based solid phase peptide synthesis (SPPS)) and alkyne-containing polyester (synthesized by ring-opening polymerization). Following the syntheses of these materials, they were analyzed by nuclear magnetic resonance (NMR) and organic gel permeation chromatography (GPC). The oligopeptide-functionalized polyolefins were designed for nucleic acid complexation, and therefore the oligopeptide sequences were intended to incorporate positively-charged moieties (e.g., oligolysine) for DNA and short interfering RNA (siRNA) complexation. These graft copolymers, prepared by SPPS followed by ring-opening metathesis polymerization, have highly tunable structures that enable control over charge density and polymer backbone rigidity. Moreover, non-ionic hydrophilic grafts such as polyethylene glycol were integrated into these polyelectrolytes such that the charges along the polymer backbone are spaced accordingly while maintaining the hydrophilicity of the polymer. While numerous applications for such charged, “bio-tailored” materials can be envisioned, this work is geared towards positively-charged polyelectrolytes for their potential application in nucleic acid therapy, specifically the delivery of plasmid DNA and siRNA. These graft copolymers were characterized (1H, 13C NMR, organic and aqueous GPC), studied for their solution properties (static and dynamic light scattering), and investigated as polyplexes with plasmid DNA.

Chloroplast biogenesis relies on the import of thousands of nuclear encoded proteins into the organelle and proper sorting to their sub-organellar compartment. The majority of nucleus-encoded chloroplast proteins are synthesized in the cytoplasm and imported into the organelle via the Toc-Tic translocation systems of the chloroplast envelope. In many cases, these proteins are further targeted to subcompartments of the organelle (e.g. the thylakoid membrane and lumen or inner envelope membrane) by additional targeting systems that function downstream of the import apparatus. The inner envelope membrane (IEM) plays key roles in controlling metabolite transport between the organelle and cytoplasm, and is the major site of lipid and membrane biogenesis within the organelle. In contrast to the protein import and thylakoid targeting systems, our knowledge of the pathways and molecular mechanisms of protein targeting and integration at the IEM are very limited. Previous reports have led to the conclusion that IEM proteins are transferred to the IEM during protein import via a stop-transfer mechanism. Recent studies have shown that at least two components of the Tic machinery (AtTic40 and AtTic110) are completely imported into the stroma and then re-inserted into the IEM in a post-import mechanism. This led me to investigate the mechanisms and pathways involved in the integration of chloroplast IEM proteins in more detail. I selected candidates (AtTic40 for post-import and IEP37 for stop-transfer) that are predicted to have only one membrane-spanning helix and adopt the same IEM topology to facilitate my analysis. My studies confirm the existence of both stop-transfer and post-import mechanisms of IEM protein targeting. Furthermore, I conclude that the IEP37 transmembrane domain (TMD) is a stop-transfer signal and is able of diverting AtTic40 to this pathway in the absence of AtTic40 IEM targeting information. Moreover, the IEP37 TMD also functions as a topology determinant. I also show that the AtTic40 targeting signals are context dependent, with evidence that in the absence of specific information in the appropriate context, the AtTic40 TMD behaves as a stop-transfer signal. This is an indication that the stop-transfer pathway is the default mechanism of protein insertion in the IEM.

Wnt ligands are a family of highly conserved glycoproteins that act as morphogens to regulate development in many organisms. Drosophila Wnt-1 (Wingless) is involved in directing cell fate decisions and pattern formation during differentiation. Wnt signaling are of high interest of many developmental biologists due to their important functions, yet little is known about how these ligands function on a biochemical level. Previously it was found that Porcupine, an ER-membrane-bound acyltransferase is required for Wingless secretion. But it is unclear how a secreted morphogen requires an acyltransferase to function. Studies reported here demonstrated that Wingless undergoes a hydrophobic modification, in which a lipid moiety containing a palmitate group is covalently attached to the polypeptide through an ester linkage. And it partitions with the specialized detergent insoluble lipid raft microdomains in the plasma membrane. Porcupine is required for the modification and the raft targeting of Wingless. Blocking Wingless modification with a specific inhibitor results in the loss of rafts-association as well as loss of protein secretion. Disrupting raft microstructures by cholesterol depletion reagents also impaired Wingless secretion, indicating that the ligand secretion is dependent on its specific association with the plasma membrane. This work provided the first insight on the function of Porcupine and the important biochemical evidence on the role of specialized membrane microdomains in Wnt signaling.

In mammalian eggs, the fertilizing sperm evokes intracellular Ca 2+ ([Ca2+]i) oscillations that are essential for initiation of egg activation and embryonic development. Although the exact mechanism leading to initiation of [Ca2+]i oscillations still remains unclear, accumulating studies suggest that a presently unknown substance, termed sperm factor, is delivered from the fertilizing sperm into the ooplasm and triggers [Ca2+]i oscillations. Recent evidence shows that sperm/sperm factor stimulates persistent production of inositol 1,4,5-trisphosphate (IP3) and that a sperm-specific phosphoinositide-specific phospholipase C (PLC), PLCζ, may be the long sought-after [Ca 2+]i oscillation-inducing molecule in sperm. In the first study, we show that subtle modification of the sequence of events that lead to fertilization, such as that seen in fertilization achieved by intracytoplasmic sperm injection (ICSI), alters the pattern of [Ca2+]i oscillations and embryonic development, and this alternation is possibly due to the non-physiological release of sperm factor (Chapter 3). Thereafter, we demonstrate that activation of a Src family kinase, which is essential for fertilization-associated Ca2+ release in non-mammalian species, is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]i oscillations in mouse (Chapter 4). Finally (Chapter 5), we demonstrate the complete extraction of the [Ca2+] i oscillation-inducing activity from porcine sperm, and show that regardless of the cellular location or solubility, the same molecule/complex appears to be responsible for [Ca2+]i oscillations induced by injection of porcine sperm extracts (pSEs). Moreover, our results suggest that PLCζ may not be the only active molecule in pSEs. We demonstrate that biotin-labeled peptide A7 (B-A7), a Src kinase inhibitory peptide, specifically binds to and precipitates the active component of sperm factor with avidin beads. Although the proteins that we have isolated and identified thus far are, however, unlikely to be involved in activation of the phosphoinositide pathway when introduced into mouse eggs, future studies using B-A7 should lead to identification of the putative sperm factor molecule.

The strength of the interfacial interactions and the length scale over which these interactions occur are two important factors to understand the behavior of polymer blends, diblock copolymers, cell recognition, adhesion and wettability on a surface. This has important implications in pattern recognition applications, biosensors and random recognition processes. A simple means of examining patterns and the influence of patterns on many length scales simultaneously is with gradient surfaces, where the lateral distribution of the chemical nature and functionality of surface interactions can be varied in a systematic manner. Surfaces with gradient heterogeneous topographies were prepared, using blends of homopolymers and diblock copolymers, to vary the lateral size scale of the heterogeneities from the microscopic to nanoscopic correlation of heterogeneity. By tuning the lateral size scale of the heterogeneities, surface patterning can be engineered to have a specific function. Mixtures of homopolymers macroscopically phase separate, whereas diblock copolymers microphase separate on nanoscopic length scales. By gradually varying the relative concentrations of homopolymers and block copolymers, the length scale of the domains can be continuously varied from the nanoscopic to the macroscopic length scale. A method to generate gradient surfaces based in such mixtures is described in Chapter 1. Several examples demonstrating their utility are shown in Chapters 2 and 3. Polystyrene film dewetting and polystyrene/poly(methyl methacrylate) phase separation in thin films are discussed in Chapter 2. In Chapter 3, cell adhesion and migration are shown to be vastly different when the cells are grown on surfaces with nanometer to micrometer features. In Chapter 4, cell migration on patterned surfaces is investigated to tackle the sole effect of topography. Cell migration on polystyrene surfaces with micron sized topographic features is compared to cell migration on flat polystyrene substrate. Actin cytoskeleton, focal adhesion and cell migration speed were characterized to understand cell movement on topographic surfaces. Cell movements on hydrophilic polystyrene posts show strong similarities to cells cultured in 3D environment. This may provide a simple model system to study cell migration in physiological relevant conditions and contribute to our understanding in cell migration to better design surfaces for medical applications.

The research presented here seeks to advance the understanding of the chemotaxis system in Escherichia coli through the use of a novel surface-templating approach. The chemosensory system in E. coli is an example of a two-component pathway and is common in many prokaryotes and is also found in eukaryotes. The system allows the cell to constantly sample the surrounding chemical environment, swimming toward attractants and away from repellants. The mechanism of signal propagation across the membrane to the kinase is not well understood. For many years a ligand-induced shift or rotation within a receptor dimer was thought to be alone responsible for signal propagation across the membrane bilayer. However, evidence for receptor clustering has mounted during the past decade from this lab and others. Inter-dimer interactions, more broadly, receptor clustering, has been observed making it possible for a signal to be propagated laterally as well as vertically through the membrane thus revealing a new avenue for communication and information processing within the cell. Receptor clustering is now believed responsible, in part or wholly, for the remarkable sensitivity, due to large gain, and also the system's ability to respond to a wide variety of environmental stimuli including pH, temperature, and many chemical signals. Reconstitution of this cluster, or 'brain' as referred to in some instances, is paramount in elucidating how this system functions and is the main goal of the research presented here. Reconstitution of membrane proteins has proved difficult, thus we have developed a new method to assemble a 2D array of activating cytoplasmic fragments (CFs) of the aspartate receptor of E. coli onto a lipid membrane. Histidine-tagged CFs of the Tar receptor were assembled on the surface of sonicated and extruded unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup. In the presence of the adaptor protein CheW, the protein kinase CheA bound to and was activated up to 475-fold by vesicle-bound CF. Surface-assembled CF was also found to serve as a substrate of the receptor methyltransferase, CheR. Since neither significant CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it was concluded that surface-templating generates the organization among CF subunits required for biochemical activity. This novel reconstitution method will be generally applicable to other systems where signaling occurs at or near the membrane surface and also for the generation of new anchoring chemistries for the marriage of proteins to surfaces.

Thymocytes undergo negative and positive selection during their development in the thymus. During this selection process, the majority of thymocytes are eliminated by apoptosis. In the first part of this dissertation, I examined the role of oxidative stress in thymocyte apoptosis. My initial observations show that thymocytes require molecular oxygen to undergo apoptosis, and treatment with N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibits thymocyte apoptosis in vivo as well as ex vivo. Various apoptosis-inducing stimuli increase intracellular hydrogen peroxide (H2O2) levels in thymocytes ex vivo, and treatment with NAC reduces the levels of intracellular H2O2 during apoptosis. The degree of reduction of H2O2 by NAC correlates well with the decrease of apoptosis, except in cells treated with γ-irradiation. These results indicate that the level of intracellular H2O 2 influences a cell's vulnerability to undergo apoptosis under many conditions, but not all. I also show that cell death-related mitochondrial events are attenuated by NAC treatment in protected cells. By using various inhibitors of the mitochondrial electron transport chain, I identified the production site for H2O2 under all apoptosic conditions tested as complex III of the mitochondria. The results show that when the inhibitors decrease the production of H2O2 at the mitochondria, the mitochondrial cell death events are also significantly reduced under all conditions. I also show that the production of H2O2 and the mitochondrial cell death events are controlled by proteosomal activities during thymocyte apoptosis. The second part of this dissertation focused on the role of hyperbaric oxygen (HBO) in enhancing apoptosis and/or suppressing cellular proliferation. This study provides evidence that HBO treatment increases intracellular H 2O2, which is partly responsible for enhancing apoptosis in HL-60 cells, a granulocytic cell line. Since HBO is effective in treating chronic wounds, these results suggest HBO may exert its beneficial effect by inducing apoptosis in neutrophils, known to mediate chronic inflammation. I also provide a piece of evidence that exposure to HBO can stop the proliferation of breast cancer cells at various stages of the disease. This could be due to abrogated antioxidative defense mechanisms, which are commonly found in rapidly dividing cells.

During mitosis, the microtubule cytoskeleton is completely rearranged to form a bipolar spindle that functions to congress and segregate a complete set of genetic material into two nascent daughter cells. The kinesin-5 family of molecular motor proteins is required for spindle pole separation in most organisms. By cross-linking and sliding apart antiparallel microtubules extending from opposite poles, Eg5, the human kinesin-5 family member, produces the outward force necessary to establish spindle bipolarity. Eg5 has recently been demonstrated to interact with the spindle assembly factor targeting protein for Xklp2, or TPX2. TPX2 contributes to many aspects of spindle assembly, including activating the mitotic kinase Aurora A, nucleating microtubules around chromosomes, and targeting several proteins to the spindle. In this dissertation, I use in vitro experiments to explore the regulation of TPX2 and Eg5 and the physiological significance of their interaction. By assaying the activity of populations of Eg5 motors, I show that TPX2 inhibits Eg5-driven microtubule gliding and relative microtubule sliding; an interaction between TPX2 and Eg5 contributes to the inhibition of the motor. Using total internal reflection fluorescence (TIRF) microscopy, I show that Eg5 accumulates on microtubules in the presence of TPX2, but less in the presence of TPX2-710, a truncated TPX2 construct lacking the Eg5 binding domain. These results contribute to a model where, in vivo, TPX2 alters the activity of Eg5 on, and also localizes the motor to, spindle microtubules, to achieve spindle formation. Using TIRF microscopy in live cells combined with automated particle tracking, I explore the dynamics of localization and purification (LAP)-tagged Eg5 punctae under two conditions: dynein inhibition and TPX2 knock-down. I show that on astral microtubules, dynein activity is required for the minus-end-directed movement of Eg5 punctae, and this movement is dependent on TPX2. On overlapping microtubules in the spindle midzone, the magnitude of the velocity of Eg5 is similar under all conditions tested, demonstrating that perturbations to Eg5 activity might have an effect on the overall structure of the spindle without affecting the dynamics of Eg5. These results contribute to a model where Eg5 is transported poleward on astral microtubules by dynein, in a TPX2-dependent manner, and that the dynamics of Eg5 punctae in the spindle midzone, but not the spindle structure, are unaffected by dynein inhibition or the absence of TPX2. I use in vitro assays with epifluorescence and TIRF microscopy to explore the binding dynamics of TPX2 on microtubules. TPX2 binds to both GTP-like and GDP-like microtubule lattices, in a concentration-dependent manner, and also exhibits microtubule binding activity within both of its N- and C- termini. The electrostatic interactions that TPX2 makes with microtubules are dampened by the addition of salt to the TPX2 binding assay, but binding does not require the negatively-charged tubulin E-hook. The dwell time of TPX2 when assayed by single molecule TIRF microscopy is 85 seconds on average, and some molecules of TPX2 exhibit bidirectional diffusion along the microtubule lattice. When assayed by single molecule TIRF microscopy, TPX2 and TPX2-710 similarly inhibit fluorescently tagged Eg5 (Eg5-EGFP) motors, suggesting that TPX2 can act as a roadblock, or can sterically block Eg5 motors from translocating along a microtubule protofilament. An interaction between TPX2 and Eg5, however, contributes to enhanced inhibition of Eg5 motors, demonstrating that TPX2 is also a brake, or interacts specifically with Eg5 to tether the motor to the microtubule as a mode of inhibition. Adding an excess of a purified C-terminal construct of TPX2 comprised of the Eg5 binding domain, CT35, partially alleviates the inhibition of TPX2 on Eg5-EGFP motors in TIRF, but does not alleviate the inhibition of TPX2 on the microtubule-gliding activity of populations of dimeric Eg5 motors. A purified, truncated construct of TPX2, comprised of the N-terminal half of the protein sequence (GST-NT), inhibits the microtubule-gliding activity of populations of dimeric Eg5 motors, but not as strongly as full-length TPX2, further indicating that an interaction with Eg5 contributes to the braking effect of TPX2 on Eg5. Preliminary data shows that the dynactin subunit p150 speeds up Eg5-EGFP motor activity, but in the presence of both p150 and TPX2, Eg5-EGFP motor activity is inhibited. These results suggest that Eg5 could be differentially regulated by TPX2 and p150 - TPX2 acting as a brake and p150 acting as an accelerator.

The overall goal of this dissertation was to examine inflammatory and regenerative responses to acute skeletal muscle damage and to define molecular mediators of repair. Study I examined the effects of an oral anti-inflammatory supplement on exercise-induced muscle damage (EIMD) and systemic inflammation in a human model. Quercetin has been shown in animal and in vitro models to downregulate nuclear factor-kappa beta (NF-κB) nuclear transactivation and monocyte chemoattractant protein 1 (MCP-1) secretion, which regulate muscle regeneration and inflammatory signaling between muscle and immune cells after injury. Subjects ingested quercetin (N=15) or placebo (N=15) before and after performing 24 eccentric contractions of the elbow flexors. Subjects experienced moderate strength losses and delayed onset muscle soreness, indicating damage, but no supplementation effect was observed. The null effect of quercetin in the human model (with its complex inflammatory response) encouraged us to explore basic injury-induced inflammation in a controlled in vitro model, to better understand the post-injury roles of NF-κB and MCP-1. Study II used an in vitro injury model (scratch of C 2C12 myotubes) to identify the roles and interplay of NF-κB and MCP-1 in muscle regeneration and inflammation following acute injury. Protein expression changes of NF-κB and MCP-1, and morphological changes in regenerating muscle cultures were monitored for 24-72 hours (h) post-injury (3-6 replicates per experiment). NF-κB activation was significantly downregulated (-30±1.4% to -44±1.1%) at 6-12h post-injury. Pharmacological blockade of NF-κB downregulated satellite cell proliferation by 19±9% after 19h and 72h, evidence for a role of NF-κB signaling in post-injury regeneration. Furthermore, NF-κB activation strongly correlated (R=0.69) with MCP-1 secretion from injured muscle cultures, and blockade of NF-κB reduced MCP-1 secretion at 1-24h (-33±0.1%) and strongly correlated (R=0.74) with NF-κB activation. These data support recent in vivo findings to demonstrate that NF-κB and MCP-1 signaling are critical regulators of inflammatory and regenerative responses following muscle injury. Moreover, this work provides the first kinetic profile of early (<24 hours) molecular responses of NF-κB and MCP-1 to acute muscle injury, and introduces novel evidence that NF-κB regulates MCP-1 protein secretion, indicating an indispensible role of NF-κB signaling in muscle inflammation in vitro.

The purpose of the study is to describe the development of infant melatonin levels form birth to six months, and to examine the effects of seasonal luminosity, and nutrition on the development of melatonin levels and changes in circadian sleep:wake cycles. Additionally the study will investigate the role of infant, maternal and care giving variables on the development of infant sleep. This longitudinal study will be conducted in Tromso, Norway, which, because of its far northern location (70° North), provides extremes in seasonal variations and light/dark cycles. For two months in the Winter the sun never rises above the horizon and for two months in the Summer it never sets below the horizon. A total of 146 infants (ranging in age from birth to 6 months) and 146 mothers participated in the study. Four sampling periods occurred within a two week interval surrounding the Summer and Winter solstices and the Spring and All equinoxes. Results show that age is the primary developmental influence and seasonal light influences actual levels of development at three and six months of age.