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.

Simian virus 40 (SV40) entry through caveolae represents a novel form of endocytosis. SV40 translocation to caveolae was mainly examined by cell fractionation and electron microscopy (EM) in CV-1 African green monkey kidney fibroblast cells. SV40 translocated to the caveolin-enriched low density Triton-insoluble complexes as identified by Western blotting. SV40 partitioning into this fraction was inhibited by nystatin. EM analysis indicated that SV40 translocation to caveolae was prevented by the tyrosine kinase inhibitor genistein (thin sections), but SV40 was not blocked from entering the Triton-insoluble low density fraction (negative staining). Northern analysis suggested that an SV40 signal that upregulates c-myc is transmitted through caveolae. The role of the major histocompatibility complex (MHC) class I proteins was not clear in SV40 translocation to caveolae. This is partly because of the high background level of class I molecules in the low density fraction isolated by sodium carbonate sucrose gradients (LDF-C). Class I proteins were enriched in the LDF-C relative to the whole cell extract or to the plasma membrane isolated by silica coating. The clustering of class I molecules was independent of any induction. Indirect immunofluorescence labeling of class I proteins and caveolin indicated that class I molecules colocalize with caveolae. MHC class I signaling and its physiological relevance were analyzed using a monoclonal antibody (mAb) W6/32. By immunoprecipitation and thin layer chromatography, ras was found to be activated by W6/32. W6/32 did not seem to increase the tyrosine-phosphorylation level in phospholipase C-$\gamma1.$ Engagement of class I molecules did not seem to induce apoptosis, but resulted in a distinct physiological response, the adhesion strengthening in CV-1 cells to Jurkat cells. The adhesion strengthening seemed to be sensitive to the protein kinase C inhibitor calphostin C, but enhanced by the tyrosine kinase inhibitor genistein. Furthermore, the mAb-induced adhesion strengthening was transient, antibody-concentration dependent, but independent of the Fc portion of the mAb. A serum factor was found to inhibit the basal level of adhesion in CV-1 cells to Jurkat cells. These findings represent one of the first investigations of MHC class I signaling in non-lymphoid cell lines.

U14 small nucleolar RNA (snoRNA) is required for processing of 18S ribosomal RNA. It was hypothesized that U14 might base pair with 18S RNA through two highly conserved U14 sequence elements known as domains A and B. Using Saccharomyces cerevisiae as the experimental system, I showed that: (1) the domain A and B elements are functionally interdependent, and (2) single-point mutations in domain A combined with complete substitution of domain B causes lethality while either mutation alone does not. Direct interaction of U14 with 18S RNA was shown by demonstrating that a lethal mutation in U14 domain A can be suppressed with a mutation which restores complementarity in the corresponding region of 18S RNA. Y-domain in yeast U14 was postulated to serve as a recognition element for vital intermolecular or intramolecular interactions. Consistent with this assumption, mutations in several conserved nucleotides of the loop cause growth defects. In contrast, alterations to the stem have little or no effect. Using a lethal mutation in the loop, three different intragenic suppressor mutations were mapped to three positions adjacent to the primary mutation, and are predicted to influence the structure of the loop. An extragenic suppressors (UF1) able to rescue a cold-sensitive mutation in the loop encodes an essential putative ATP-dependent RNA helicase. Loss of UF1 gene expression caused a reduction in 18S rRNA production, without affecting accumulation of 25S rRNA or U14 snoRNA. Pulse-chase analysis showed that depletion of UF1 protein impaired pre-18S rRNA processing. Finally, an effort was made to define minimum pre-rRNA substrates that can be used to produce functional 18S and 25S rRNAs in vivo. The rDNA operon was split either between the 18S RNA and 5.8S/25S coding units, or between the 18S/5.8S RNA and 25S RNA coding units. The test fragments were expressed from GAL7 promoters. The results showed that functional rRNAs could be produced in trans, but only when the operon was divided between the 18S RNA and 5.8S/25S RNA coding sequence.

Senescence in plants is a genetically controlled developmental event that enables the plant to eliminate individual cells, tissues, organs and even the entire plant in order to adapt to change in environmental conditions, reproductive needs, or pathogenic attack. An integral part of the cell death occurring during senescence is the selective degradation of proteins. Proteins may be degraded via the activity of several classes of enzymes, characterized by the different amino acids present at their catalytic sites, known as proteinases. Alternatively, protein degradation in eukaryotic cells can be accomplished through the action of an ATP dependent pathway known as the ubiquitin system, which is capable of selecting specific proteins for degradation by creating ubiquitin-protein conjugates that are then hydrolyzed by the 26S proteasome, a multisubunit proteinase. As a first step in determining whether the ubiquitin pathway was involved in the death of cells during vascular tissue development, experiments were conducted to localize the presence of ubiquitin and ubiquitin-protein conjugates to differentiating vascular tissues. Immunolabeling on tissue prints from cross sections of Phaseolus vulgaris petioles, Gossypium hirsutum hypocotyls and Coleus x hybridus internodes showed accumulation of ubiquitin-protein conjugates in regions of vascular tissues. Immunohistochemical labeling confirmed the presence of elevated levels of ubiquitin-protein conjugates in differentiating and regenerating xylem tissues of Coleus. In addition, labeling of ubiquitin-protein conjugates was observed in parenchymatous tissues and elevated levels of ubiquitin-protein conjugates was also observed in vascular cambia. The senescence of daylily (Hemerocallis hybrid cv Stella d'Oro) petals is accompanied by a rapid decline in protein over a 24 h time period. To determine how this loss of protein is regulated we conducted experiments to characterize and measure the activity of several classes proteinases. Treatment with class specific inhibitors indicated the presence of serine, cysteine, and metalloproteinases. The activity of these proteinases increased after flower opening and this increase in activity could be eliminated by treatment with the protein synthesis inhibitor cycloheximide. Ion leakage was delayed by treatment with inhibitors of the 26S proteasome, suggesting a link between protein hydrolysis and membrane permeability. Immunoblots labeling ubiquitin-protein conjugates and ubiquitin system enzymes revealed that overall profiles of ubiquitinated proteins do not change during senescence but that several enzymes of the ubiquitin system decline after flower opening. Northern analysis showed slight accumulation polyubiquitin message during petal senescence. We propose that senescence in daylily petals is controlled in part by the appearance of at least three classes of proteinases which may work in concert with the ubiquitin system.

Apoptosis is a type of programmed cell death that is essential for the development and maintenance of tissue homeostasis in multicellular organisms. Because apoptosis is important in many biological situations, laboratories are working to delineate the signaling pathways responsible for this process. This thesis work attempts to identify new components of the signaling pathways by examining a role for mitochondria and proteasomes in T cell apoptosis. Interest in mitochondria was initiated when a library screen performed in the laboratory revealed that portions of the mitochondrial genome, called apt-1 and apt-3, were downregulated in thymocytes during negative selection. In this thesis work, two aspects of mitochondrial function were examined in the T cell hybridoma, DO11.10: the integrity of the mitochondrial genome as assessed by levels of apt-l and apt-3 and the integrity of the mitochondrial membrane as assessed by membrane potential. Both the steady-state levels of apt-l and apt-3 RNA and mitochondrial transmembrane potential declined rapidly in apoptotic cells. Recent studies from other laboratories indicate that these changes are the result of the active participation of mitochondria in apoptosis. Apoptosis relies heavily on proteolysis, and this dependence has encouraged investigators to analyze the significance of many proteases and proteolytic pathways. The second part of this thesis work focused on describing a role for the ubiquitin-proteasome pathway in apoptosis. Proteasome inhibitors were used to determine whether the proteasome is required in thymocyte apoptosis. Treatment of thymocytes with acetyl-leu-leu-methioninal (LLM), acetyl-leu-leu-norleucinal (LLnL), carbobenzoxyl-leu-leu-leucinal (MG132), or lactacystin inhibited death induced by dexamethasone, ionizing radiation, or phorbol 12-myristate 13-acetate (PMA). These results suggest that proteasome activity is necessary for the progression of many apoptotic pathways in thymocytes. Inhibitor studies also indicate that the proteasome functions upstream of many apoptotic events, including: PARP cleavage, caspase-3 activation, and mitochondrial transmembrane potential depolarization. Although a precise role for the proteasome in apoptosis cannot be assigned without an identification of its proteolytic targets, the work described in this thesis provides the first definitive link between proteasomes and apoptosis in mammalian systems.

The antioxidant activities of compounds endogenous to bovine rod outer segments (ROS) were investigated by measuring the loss of polyunsaturated fatty acids (PUFA's) from membranes exposed to the water-soluble oxidant $2,2\sp\prime$-azobis(2-amidinopropane) dihydrochloride (AAPH). Osmotically intact ROS, ROS membranes, and unilamellar liposomes prepared from ROS phospholipids (PL) were compared. Intact ROS were most resistant to oxidative loss of PUFA's, followed by ROS membranes and then PL liposomes. The development of a model membrane system allowed the investigation of putative antioxidants singly and in combination. Lipid-soluble compounds (vitamin E, free fatty acids, retinol, retinaldehyde) were incorporated into PL liposomes. Water-soluble compounds (reduced glutathione, taurine) were dissolved in incubation buffer. It was found that normal physiological concentrations of alpha-tocopherol (vitamin E), free fatty acids (16:0, 18:0, 18:1, 22:6), retinol, and glutathione significantly decreased oxidative loss of PUFA's. When the major free fatty acids were added to PL liposomes at the same concentrations found when ROS phospholipase A is stimulated, the oxidative loss of PUFA's was reduced by 31%. The antioxidant effect of free fatty acids suggests that endogenous phospholipase A's may act to protect membranes by releasing fatty acids from phospholipids in proportions and concentrations that afford protection to membrane lipids. The antioxidant activities of the two major retinoid compounds involved in the visual cycle in ROS, all-trans retinaldehyde and all-trans retinol, were compared. The addition of retinol partially protected PUFA's in ROS PL liposomes, whereas retinaldehyde promoted lipid peroxidation. When isolated ROS were stimulated to produce endogenous retinol, PUFA loss was inhibited by up to 17%. These findings suggest that there is an antioxidant function for the enzymatic reduction of retinaldehyde to retinol during photoreception. Water-soluble antioxidants, taurine and reduced glutathione (GSH), were investigated individually and in combination with retinol in ROS PL liposomes. GSH significantly protected PUFA's in ROS PL liposomes. Taurine alone showed little antioxidant activity, but in combination with retinol protected lipids by up to 47.9% (an increase in antioxidant protection of 24.8% over retinol alone). These data support previous findings that taurine protects ROS lipids during exposure to cyclic light conditions.