Expression of the cell cycle dependent FO10S human H4 histone gene is regulated at both the transcriptional and post-transcriptional levels. We have investigated the 5' promoter elements mediating the transcriptional aspects of its regulation. A detailed in vivo and in vitro transcriptional analysis of promoter deletion mutants from this gene has identified three positive regulatory elements and two potentially negative regulatory elements within the first 1000 base pairs upstream of the transcription initiation site. In addition, the minimal promoter located within the first 70 base pairs is required for accurate transcription initiation and contains one of two in vivo identified protein-DNA interactions, site II. Binding of the nuclear factor HiNF-D to this region was correlated with the turn-on of histone gene transcription following stimulation of quiescent normal diploid fibroblasts to re-enter the proliferative phase. The most proximal positive regulatory element contains the other in vivo identified protein-DNA interaction, site I. Results from a series of in vitroprotein-DNA interaction studies revealed the binding of two nuclear factors to this element. One protein, HiNF-C, is indistinguishable from the transcription factor Sp1 while the other, HiNF-E, is a novel, potentially histone-specific member of the ATF transcription factor family. Binding of HiNF-C was required to stabilize the interaction of HiNF-E and together this region stimulated transcription 5 fold. The near-distal transcription activator region lies between -418 and -213 base pairs and forms a single protein- DNA complex, H4UA-1. The interaction domain for H4UA-1 contains recognition sequences for both the thyroid hormone receptor and the nuclear factor CTF/NF-1. The far-distal activator region (-730 and -589 base pairs) was the strongest positive regulatory element identified in the H4 promoter. This region increased transcription 10 fold and contains three protein-DNA interactions. One of the factors, H4UA-2, is an ATF transcription factor closely related to the HiNF-E interaction in the proximal positive element. These studies have defined the functional human H4 histone promoter to be a complex, modular structure extending at least 1000 base pairs.

The questions outlined in this thesis dissertation were proposed in order to provide insight regarding the mechanism by which the Drosophila Son of sevenless (dSOS) protein activates Ras. Ras proteins are GTP-binding proteins which bind guanine nucleotides very tightly and cycle between the inactive GDP-bound state and the active GTP-bound state. To address the mechanism by which the dSOS proteins activates Ras, a structure-function analysis of the dSOS protein was performed using truncation and deletion mutants of dSOS. In vivo Ras activation experiments using transiently transfected cells revealed that the NH2-terminal domain of dSOS is required in order for the catalytic domain of dSOS to exhibit exchange activity in cultured mammalian cells. The COOH-terminal GRB2 (Growth Factor Receptor Binding Protein) binding domain on the otherhand was insufficient to confer Ras exchange activity to the dSOS catalytic domain. Further analysis of the NH2-terminal domain of the dSOS protein demonstrated that the function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin (PH) and DBL (Diffuse B-cell Lymphoma) homology sequences. Fractionation studies of cells transiently transfected with various dSOS mutant proteins demonstrated that the NH2-terminus of dSOS is also necessary for membrane association. These findings suggested that the model proposing that the recruitment of SOS via the adaptor protein GRB2 to the membrane is the main mechanism by which SOS activates Ras is unlikely to be the only mechanism by which SOS can activate Ras. From our data, a model can be proposed which postulates that SOS can activate Ras as a consequence of at least two steps. One step involves the SOS/GRB2 interaction and the second step involves the NH2-terminal domain of SOS associating with unidentified cellular elements.

For many paramyxoviruses, including Newcastle disease virus (NDV), syncytia formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (23, 73). Because a potential interaction in paramyxovirus infected cells has never been clearly demonstrated, such an interaction was explored in Chapter 2 using coimmunoprecipitation and crosslinking. Both HN and F proteins could be precipitated with heterologous antisera after a five minute radioactive pulse as well as after a two hour chase in non-radioactive media, but at low levels. Chemical crosslinking increased detection of complexes containing HN and F proteins at the cell surface. After crosslinking, intermediate as well as high molecular weight species containing both proteins were precipitated with monospecific antisera. Precipitation of proteins with anti-HN after crosslinking resulted in the detection of complexes which electrophoresed in the stacker region of the gel, from 160-300 kD, at 150 kD and at 74 kD. Precipitates obtained with anti-F after crosslinking contained species which migrated in the stacker region of the gel, between 160-300 kD, at 120 kD and at 66 kD. The 3-4 discrete complexes ranging in size from 160-300 kD contained both HN and F proteins when precipitated with either HN or F antisera. That crosslinking of complexes containing both HN and F proteins was not simply a function of overexpression of viral glycoproteins at the cell surface was addressed by demonstrating crosslinking at early time points post infection, when levels of viral surface glycoproteins are low. Use of cells infected with an avirulent strain of NDV showed that chemically crosslinked HN and F proteins were precipitated independent of cleavage of F0. Furthermore, under conditions that maximized HN protein binding to its receptor, there was no change in the percentages of HN and F0 proteins precipitated with heterologous antisera, but a decrease in F1protein precipitated was observed upon attachment. These data argue that the HN and F proteins interact in the RER. Upon attachment of the HN protein to its receptor, the HN protein undergoes a conformational change which causes a subsequent change in the associated F protein, releasing the hydrophobic fusion peptide into the target membrane and initiating fusion. Chapter 3 explores the stalk region of the NDV HN protein, which has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (7, 51, 57). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled-coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha-helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic "a" position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins.

Although much progress has been made in understanding the events that lead to successful cell division, many details of this process remain a mystery. This dissertation presents findings which help to explain events that occur in the latest stages of cytokinesis, with an emphasis on the role of centrosome proteins. The first chapter introduces the novel centrosome protein centriolin. We show that this protein is localized specifically to the subdistal appendages of the maternal centriole in interphase, and it localizes to the midbody during cytokinesis. Disruption of this protein results in a unique cytokinesis defect in which cleavage furrow formation and ingression appear normal, but the cells remain connected by a thin intracellular bridge for extended periods of time. These results lead us to the conclusion that centriolin has an important function in cytokinesis. The second chapter describes our attempt to identify centriolin interacting partners. A yeast two hybrid screen was performed, and the results of this screen revealed an interaction between centriolin and proteins involved in vesicle target specificity and fusion. Further studies of these proteins revealed a novel localization to the midbody in cycling cells and a novel function in the final stages of cytokinesis, similar to centriolin. The third chapter discusses my attempts to clone and characterize a novel GTPase Activating Protein (GAP), which was also discovered in the screen for centriolin interacting proteins.

Chemical warfare agents continue to pose a global threat despite the efforts of the international community to prohibit their use in warfare. For this reason, improvement in the detection of these compounds remains of forensic interest. Protein adducts formed by the covalent modification of an electrophilic xenobiotic and a nucleophilic amino acid may provide a biomarker of exposure that is stable and specific to compounds of interest (such as chemical warfare agents), and have the capability to extend the window of detection further than the parent compound or circulating metabolites. This research investigated the formation of protein adducts of the nitrogen mustard chemical warfare agents mechlorethamine (HN-2) and tris(2-chloroethyl)amine (HN-3) to lysine and histidine residues found on the blood proteins hemoglobin and human serum albumin. Identified adducts were assessed for reproducibility and stability both in model peptide and whole protein assays. Specificity of these identified adducts was assessed using in vitro assays to metabolize common therapeutic drugs containing nitrogen mustard moieties. Results of the model peptide assays demonstrated that HN-2 and HN-3 were able to form stable adducts with lysine and histidine residues under physiological conditions. Results for whole protein assays identified three histidine adducts on hemoglobin, and three adducts (two lysine residues and one histidine residue) on human serum albumin that were previously unknown. These protein adducts were determined to be reproducible and stable at physiological conditions over a three-week analysis period. Results from the in vitro metabolic assays revealed that adducts formed by HN-2 and HN-3 are specific to these agents, as metabolized therapeutic drugs (chlorambucil, cyclophosphamide, and melphalan) did not form the same adducts on lysine or histidine residues as the previously identified adducts formed by HN-2 and HN-3. Results obtained from the model peptide and full protein work were enhanced by comparing experimental data to theoretical calculations for adduct formation, providing further confirmatory data. This project was successful in identifying and characterizing biomarkers of exposure to HN-2 and HN-3 that are specific and stable and which have the potential to be used for the forensic determination of exposure to these dangerous agents.

Affecting 1 in 68 children, Autism Spectrum Disorder (ASD) is one of the most prevalent cognitive disorders in the global population. Symptoms of ASD, although typically not life-threatening, have a large impact on the social wellbeing of diagnosed individuals. Inflammation in the brain, or neuroinflammation, has previously been shown to increase the severity of the behavioral deficits associated with ASD. The exact etiology of the neuroinflammation observed in ASD remains unclear, especially in regards to protein expression that initiates the inflammatory pathway. This experiment examines two specific markers of neuroinflammation, glial fibrillary acidic protein (GFAP) and myelin-associated glycoprotein (MAG) in a previously characterized mouse model of ASD. GFAP is astrocyte-specific, cytoplasmic, and has been shown to be upregulated in trauma or disease pathologies in the brain. MAG is found in the membrane of oligodendrocytes and is a major regulator of development and regeneration of nervous tissue. Control C57bl/6j mice and ASD-representative BTBR T+tf/J (BTBR) mice were sacrificed twenty-one days after birth. Immunoblotting was performed on cingulate cortical tissue using anti-GFAP and anti-MAG primary antibodies to quantify levels of GFAP and MAG protein expression between the control and ASD models. These findings provide further evidence that changes in GFAP and MAG expression may alter the neuroinflammatory pathways observed in ASD-representative mice.

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicles are targeted to sites of exocytosis on the plasma membrane in part by a conserved multi-subunit protein complex termed the exocyst. In addition to tethering vesicles to the plasma membrane, the exocyst complex and components therein may also add a layer of regulation by directly controlling assembly of the SNARE complex, which is required for membrane fusion, as well as other regulatory factors such as Sec1p. In the past, we have shown that Sec6p interacts with Sec9p in vivo and that that interaction retards binary SNARE complex formation in a SNARE assembly assay. Though many interactions have been mapped using in vitro methods, confirming them in vivoand placing them into the context of a complete model that accounts for all observed interactions (and lack of interactions) has proven difficult. In order to address these problems, I have studied the interactions between Sec6p and other factors involved in exocytosis at the plasma membrane via in vivo methods. My hypothesis was that Sec6p interaction with Sec9p and subsequent inhibition of SNARE complex assembly in vitro was an intermediate state and Sec6p was part of a set of cofactors that accelerated SNARE complex assembly in vivo. To test this hypothesis I showed that the interaction between the plasma membrane t-SNARE Sec9p and the yeast exocyst subunit Sec6p can be observed in vivoand designed point mutations to disrupt that interaction. Interestingly, I also showed that Sec6p:Sec9p interaction involves the free pool of Sec6p rather than the exocyst bound fraction of Sec6p. Point mutations in the N-terminal domain of Sec6p result in temperature sensitive growth and secretion defects, without loss of Sec6p-Sec9p interaction. However, at the non-permissive temperature, the exocyst subunits Sec5p, Sec10p and Sec15p are mislocalized and are absent from the exocyst complex. The resulting subcomplex, containing Sec3p, Sec8p, Exo70p and Exo84p, remains stably assembled and localized at sites of polarized secretion. This subcomplex is likely due to disruption of interaction between Sec6p and Sec5p, and may be similar to that observed at restrictive temperatures in the sec6-54temperature sensitive mutant. Additionally, one of the sec6 temperature sensitive mutants displays a loss of binding to the yeast regulatory protein Sec1p. In vitro binding studies indicate a direct interaction between Sec1p and the free pool of the wild-type Sec6p protein, suggesting close interplay between Sec6p and Sec1p in the regulation of SNARE complexes. A coherent model which incorporates all these interactions has continued to be elusive. However, the results I have found do suggest several hypotheses which should prove testable in the future.

Axon pathfinding is an important process required for the establishment of proper neuronal connections during development. An increasing number of secreted and membrane-anchored molecules have been identified as axon guidance cues, which can act as positive or negative factors to increase or decrease the growth of axons and influence the direction of axonal growth. These axon guidance factors present in the extracellular environment interact with receptors present on the growth cone, a structure located at the tip of the axon which functions as the motor unit for the axon. Upon binding to their receptors on the growth cone, the guidance factors then elicit an intracellular signaling cascade within the axon that ultimately influences the direction of axon growth, often through a direct, non-transcriptional mechanism. In this dissertation, we show that Sonic hedgehog (Shh) acts as an axon guidance factor for chick retinal ganglion cell (RGC) axons in a concentration-dependent manner. At a low concentration, Shh functions as a positive factor that induces axon growth and attractive turning while, at a high concentration, Shh functions as a negative factor that induces axon retraction and repulsive axon turning. We further characterized the effects of Shh on macropinocytosis, a fluid-phase type of endocytosis, in the axons. A high concentration of Shh significantly increased macropinocytosis in the axons. Macropinocytosis resulted in the generation of large, dextran-positive, clathrinindependent vesicles in the axonal growth cones, prior to growth cone collapse, axon retraction and repulsive axon turning. These vesicles were found to require dynamic F-actin, nonmuscle myosin II and dynamin for their formation but were formed independently of PI3 kinase signaling. Interestingly, a low concentration of Shh had an opposite effect on macropinocytosis. A low concentration of Shh and soluble laminin decreased macropinocytosis and additionally increased the turnover of these vesicles within the axons, suggesting positive axon guidance factors can additionally regulate downstream processing or maturation of these vesicles. The effect of Shh on regulating the motility of macropinosomes within the axons was investigated. A low concentration of Shh appeared to increase the motility of these vesicles along axonal microtubules in a cAMPdependent manner. However, a high concentration of Shh did not appear to affect the motility of the macropinosomes, suggesting that it likely plays a more predominant role in the formation of these vesicles within the growth cone. When we began this work, a large body of research existed describing the effects of guidance factors on regulating the cytoskeleton during axon motility. However, the role of membrane trafficking events during axon growth and guidance were very poorly characterized. Since we began this project, an increasing number of reports have shown that endo- and exocytosis are important for axon growth and, here, we show that macropinocytosis induced by negative axon guidance factors plays a critical role in growth cone collapse, axon retraction and repulsive axon turning. Positive axon guidance factors also affect macropinocytosis within the axons and additionally regulate their maturation, suggesting that membrane trafficking events mediated by axon guidance factors are important for regulating axon growth and pathfinding.

The overall objective of this study was to understand the mechanisms that control antigen processing and binding of peptides to major histocompatibility complex (MHC) molecules. Towards this goal I investigated (a) the structural features of T cell-presented peptides with a focus on the role of recurrent hydrophobic residues in catalysis of helix formation by these peptides and (b) the biochemical events that determine the fates of the invariant chain molecule (Ii) in its various post-translational processing pathways. In the structural studies, I tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an α-helix can lead to helical nucleation on a hydrophobic surface.For a series of HPLC-purified peptides, including some T cell-presented peptides varying considerably in primary sequence, percentage helicity in the presence of lipid vesicles correlated with strip-of-helix hydrophobicity index (SOHHI), as shown by circular dichroism (CD) analysis. A prototypic helix peptide PH-1.0 (LYQELQKLTQTLK) was designed with a strong axial hydrophobic strip of 4 leucine residues. PH-1.0 formed about 38% helical structure in 10 mM phosphate buffer at pH 7.0 with di-O-hexadecyl phosphatidylcholine (DHPC) lipid vesicles, but no helical structure was detected when the peptide was in phosphate buffer alone. The helix-forming tendencies of 9 analogs of PH-1.0 with one or two amino acid variations from the parent peptide were examined in the presence of lipid vesicles and the results showed that (a) decreasing the strip-of-helix hydrophobicity by substituting even one of the four leucine residues in the axial hydrophobic strip with a less hydrophobic threonine residue reduced the helix-forming tendency of a peptide in the presence of lipid vesicles; (b) the placement of recurrent hydrophobic residues within the axial hydrophobic strip appeared to be critical for a peptide to be induced to form an α-helix by a hydrophobic surface; (c) there was an orientation preference for these peptides to interact with lipid vesicles and to form helical structure; (d) extra hydrophobic residues in other positions appeared to compete with the hydrophobic residues within the axial hydrophobic strip for interaction with the lipid vesicles and therefore to decrease the helix-forming tendency of peptides. For the biochemical studies of the function of Ii, a 17-residue peptide, Ii-3 (Ii 148-164), was synthesized. The CD analysis of Ii-3 showed mainly an α-helical conformation when the peptide was examined in the presence of lipid vesicles. [125I]-labeled Ii-3, after coupling at the N-terminus with a photoactivatable, heterobifunctional crosslinker N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), was able to bind to both α and β chains of class II MHC molecules, indicating that this region of Ii might cover the desetope of class II MHC molecules from the time of their synthesis until their charging with foreign peptides at an endosomal compartment. The biosynthesis of a chondroitin sulfate proteoglycan-form of Ii (CS-Ii) was examined in a class II MHC-negative cell line P3HR-1. [35S]sulfate-labeled microsomal membrane proteins of P3HR-1 were immunoprecipitated with anti-Ii monoclonal antibody and the results of SDS-PAGE analysis demonstrated that P3HR-1 could process Ii to CS-li in the absence of class II MHC molecules and the chondroitin sulfate identity of this molecule was confirmed by chondroitinase-ABC treatment. We conclude that there might be a class II MHC-independent pathway to process Ii to a chondroitin sulfate proteoglycan form as compared to the pathway in which Ii was associated with class II MHC and later cleaved by proteases residing in the endosomal compartment. In an effort to demonstrate in vitro that the class II MHC-associated Ii was eventually dissociated from class II MHC molecules by a proteolytic cleavage process, it was found that cathepsin B could completely remove Ii without damage to class II α and β chains. In order to identify those cleaved Ii fragments, three polyclonal anti-Ii peptide sera were produced by immunizing rabbits with keyhole limpet hemocyanin (KLH)-conjugated Ii peptides. Anti-Ii (146-169) was shown to be able to precipitate a p18 molecule only in cells expressing Ii. Anti-Ii (148-164 )and anti-Ii(78-92) were specific for their respective antigenic peptides as tested by enzyme-linked immunosorbent assay (ELISA).

Cell migration involves complex mechanical interactions between cells or between cells and the underlying substrate. Using a newly developed technique, "traction force microscopy", I have been able to visualize the dynamic characteristics of mechanical forces exerted by migrating fibroblasts such as magnitude, direction, and shear. For NIH 3T3 fibroblasts, I found that the lamellipodium provides nearly all of the force necessary for cell migration. A high shear zone separates the lamellipodium from the remainder of the cell body, suggesting that they are mechanically distinct entities. The timing of the tractions at the leading edge, as well as the spatial distribution, bears no apparent relationship to concurrent local protrusive activities, yet changes in traction force patterns often precede changes in migration direction. In H-ras transformed cells I found isolated regions of weak, transient traction forces in pseudopods all along the cell that appeared to act against one another. The resulting shear pattern suggested that there were multiple disorganized mechanical domains. These results support a frontal towing model for cell migration where the dynamic traction forces at the leading edge served to actively pull the cell body forward. In H-ras transformed cells, the weak poorly coordinated traction forces coupled with weak cell substrate-adhesions were likely responsible for the abnormal motile behavior of these cells. To probe the mechanical interactions beneath various regions of migrating fibroblasts, a cell substrate inhibitor (GRGDTP peptide) was locally applied while imaging stress distribution on the substrate utilizing traction force microscopy. I found that both spontaneous and GRGDTP induced detachment of the trailing edge resulted in extensive cell shortening with no change in overall traction force magnitude or cell migration. Conversely, leading edge disruption resulted in a dramatic global loss of traction forces pnor to any significant cell shortening. These results suggested that fibroblasts transmit their contractile forces to the substrate through two distinct types of adhesions. Leading edge adhesions were unique in their ability to transmit active propulsive forces whereas trailing end adhesions created passive resistance during cell migration and readily redistributed their loads upon detachment. I have also investigated how fibroblasts regulate traction forces based on mechanical input. My results showed that stretching forces applied through the flexible substrate induced increases in both intracellular calcium concentration and traction forces in fibroblasts. Treatment with gadolinium, a well known stretch-activated ion channel inhibitor, was found to inhibit both traction forces and cell migration without inhibiting cellular spread morphology or protrusive activities. Gadolinium treatment also caused a pronounced decrease in vinculin and phosphotyrosine concentrations from focal adhesions. Local application of gadolinium to the trailing region had no detectable effect on overall traction forces or cell migration, whereas local application to the leading edge caused a global inhibition of traction forces and an inhibition of migration. These observations suggest that stretch activated entry of calcium ions in the frontal region serves to regulate the organization of focal adhesions and the output of mechanical forces. Together my experiments elucidate how fibroblasts exert mechanical forces to propel their movements, and how fibroblasts utilize mechanical input to regulate their movements.