<p> Osteosarcoma (OS) is the most common type of solid bone cancer, mainly arising in children and young adults, and remains the second leading cause of cancer-related death in this age group. Chemotherapy resistance underlying latent recurrence and metastasis represent major contributors to poor outcome for many cancer patients especially those with OS. Tumor hypoxia is an essential element intrinsic to most solid tumor microenvironments and is associated with resistance to therapy and a malignant phenotype, while metastatic dissemination is dependent on a cells ability to resist anoikis, i.e., programmed cell death in the absence of attachment to an extracellular matrix. We sought to better characterize hypoxia and anoikis resistance in human OS using established and novel patient-derived OS cells and OS animal models with the long-term goal of identifying and validating targetable signaling pathways. We show that hypoxia-inducible factors (HIFs), canonical proteins associated with the hypoxic response, are present and can be induced in human OS cells. We demonstrate that the Wnt/&beta;-catenin signaling pathway, a key pathway in OS pathogenesis, is down-regulated in response to hypoxia in OS cells, and that this appears to result from both HIF-dependent and HIF-independent mechanisms. Hypoxia promotes resistance of human OS cells to standard chemotherapy, which is mitigated by treatment with Wnt/&beta;-catenin signaling inhibitors. Using an anchorage-independent growth model, we show that anoikis-resistant OS subpopulations have altered growth rates, increased resistance to standard chemotherapies, and display distinct changes in gene expression and DNA methylation. Finally, we validate the use of two FDA-approved epigenetic therapies predicted by expression profiling in both inhibiting anchorage-independent growth and sensitizing anoikis-resistant OS cells to chemotherapy. In summary, despite the heterogeneity of human OS, our work suggests that unique and effectively targetable signaling pathways underlie the phenotypic consequences in response to hypoxia and anoikis resistance.</p><p>

<p> Microfluidic devices are widely used for cell-based analysis. There are always needs to develop simpler, more effective and/or less costly devices than the existing ones for this application. A simple microfluidic device has been fabricated and tested for studying chemotaxis of macrophages in this study. The device was made of polydimethylsiloxane bound to a cell culture dish. It consisted of a millimeter-sized cavum and two arrays of straight channels of 5 um in width and 6um height and about two millimeters in length. The channels connected the cavum, in which a chemoattractant was loaded, with the surrounding environment, in which the macrophages were cultured. The device was first tested with a known chemoattractant-fetal bovine serum and the chemoattractive property of myelin basic protein (MBP) was then studied using the device. The macrophages were found to migrate towards to the MBP-loaded cavum in larger quantity and greater distance than those in the control samples. The results prove the usefulness of the microfluidic device for chemotaxis assay and indicate that MBP is a chemoattractant for the macrophages.</p><p>

<p> Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42. They regulate many cellular functions, including cell polarity, cytoskeletal dynamics, and transcription. Pak1 and Pak2 are redundantly essential for embryonic skeletal myoblast fusion in <i> Drosophila</i>, with Pak2 playing the more important role. Both are expressed in mammalian skeletal muscle, but little is known as to their function in myogenesis. We find that Pak1 and Pak2 are expressed in mammalian myoblasts and are activated specifically during differentiation. Individual genetic deletions of <i>Pak1</i> and <i>Pak2</i> in mice show no overt defects in muscle development or regeneration. However, young adult mice with muscle-specific deletion of <i>Pak1</i> and <i>Pak2 </i> together (dKO mice) present with reduced muscle mass and a higher proportion of myofibers with smaller cross-sectional area compared to controls. This phenotype is exacerbated after repair to acute injury. Primary myoblasts from dKO animals show delayed differentiation, with lower expression of myogenic markers and inefficient myotube formation. Additionally, with age, dKO mice develop a chronic myopathy. Histological analyses of resting muscle show the presence of central nuclei in the majority of fibers, as well as significant fibrosis, inflammation, necrosis, and hypertrophy with fiber splitting. Ultrastructural analysis revealed grossly elongated and branched intermyofibrillar mitochondria, known as megaconial mitochondria, along with occasional accumulation of subsarcolemmal mitochondria. Moreover, dKO mice show impaired mitochondrial function, with significantly reduced Complex I and II activity. These characteristics are absent in control animals. We conclude that the role of Pak1 and Pak2 in embryonic myoblast fusion, first identified in the fly, is not conserved in mammals. Rather, our data demonstrate that Pak1 and Pak2 function redundantly in regulating myoblast differentiation, thereby impacting overall postnatal muscle size. Furthermore, their major function appears to be in muscle homeostasis. Few protein kinases have been implicated in muscle disease. Group I Paks have wide roles in cell regulation, and the generation of dKO mice provides a genetic system to gain new mechanistic insights into muscle maintenance, as well as to discover the substrates of Paks that regulate this process.</p><p>

U14 is an evolutionary conserved small nucleolar RNA required for 18S ribosomal RNA production in the yeast Saccharomyces cerevisiae. This study attempts to identify cellular components that interact with U14 RNA with the aim of gaining insight into U14 function and identifying strategies for characterizing this activity. The objectives include: (1) development of a hypothetical secondary folding model for U14 RNA; (2) biochemical characterization of the U14 small nuclear ribonucleoprotein particle (snRNP); (3) identification of U14 binding proteins through biochemical and genetic approaches, and; (4) a genetic test to determine if U14 interacts with 18S RNA through an essential, complementary segment in U14 RNA, i.e. domain 18S-A. Two U14 consensus secondary structures were established by phylogenetic folding analysis. These structures are supported by a variety of genetic observations, but are only partially consistent with biochemical probing data developed by others. Additional research is required to determine the actual folding properties of U14 in vivo. Biochemical characterization showed that natural U14 RNA does not contain a trimethylguanosine (TMG) cap, while U14 synthesized from the GAL1 promoter does. Natural U14 occurs in two classes of RNP complexes: (i) a 10S RNP presumed to be a free snRNP and, (ii) a polydisperse, larger complex containing fibrillarin. The direct association of fibrillarin with in vitro synthesized U14 was not detected using a variety of assays. Analysis of the higher order structures sedimenting at approximately 90S revealed the presence of over one dozen snRNAs, two-thirds of which appear to be associated with fibrillarin. Several extragenic suppressor mutations were collected that relieve a lethal mutation in the box C sequence. One of these was identified as a putative sugar transport protein. It seems unlikely that this protein is directly related to U14 function per se, but may play a role in the regulation of U14 activity, possibly by influencing proteins that interact with U14 RNA or DNA. Mutations in a domain A-related sequence of 18S rDNA resulted in under-accumulation of 18S RNA, demonstrating the importance of this region for 18S RNA stability. This effect was not influenced by complementary changes in domain A of U14 RNA. The significance of these results are discussed in the context of U14 function and ribosome biogenesis.

The factors controlling desensitization to ATP stimulation were investigated in PC12 cells. Reducing the concentration of ATP to produce half maximal response reduced the degree to which cells desensitize to ATP. Increasing external Me concentration, which produced a comparable decrease in the secretory response of the cells, had no effect on the degree of desensitization. Neither did decreasing external Ca2+ concentration, which produced a similar decrease in secretory response. Desensitizing cells in 0.5 mM Ca2+ did not result in a corresponding decrease in response when cells were subsequently tested in 2.2 mM Ca2+. PC12 cells desensitized in 2.2 mM Ca2+ were found to show the same degree of desensitization when tested in 0.5 mM Ca2+. A similar pattern was found when desensitization to 30 and 300 uM ATP was examined. The role of the ATP receptor subtypes, P2x and P2y, was studied using the ATP agonists 2-MeS ATP and UTP, respectively. 60 uM 2-MeS ATP was found to cause desensitization to the same degree as 30 uM ATP. As with ATP, the initial response to 2-MeS ATP was found to be sensitive to changes in external Mg2+. Unlike ATP, the desensitization to 2-MeS ATP was sensitive to changes in external Mg2+. When cells were co-stimulated with 2-MeS ATP and UTP, the sensitivity of 2-MeS ATP desensitization to Mg2+ remained. UTP in the background solution, however, increased desensitization to ATP and 2-MeS ATP. The effect of voltage-operated Ca2+ channel (VOCC) blockers on the response and desensitization to ATP and 2-MeS ATP was examined. Cd2+ produced a significant increase in the secretory response to both stimulants. Both nicardipine and Cd2+ increased the rate of desensitization to ATP. Only nicardipine was able to increase the rate of desensitization to 2-MeS ATP. These results were integrated to provide insights into the relationship between ATP receptors and VOCCs.

Polychlorinated biphenyls are one type of organic pollutant. Although they are no longer produced due to their resistance to degradation they have persisted in the environment and have become globally distributed. They have been found within the tissues of nearly every organism that has been tested for their presence. Because they are capable of interacting with biological systems their effect on environmental and human health have become a topic of interest and research. It is known that PCBs alter the function and levels of thyroid hormone. This is significant because of the importance of properly regulated thyroid hormone levels during development. To gain a better understanding of the specific effects of PCBs on thyroid hormone function and to better assess the risks associated with PCB exposure I set out to identify mechanisms by which PCBs alter thyroid hormone-directed brain development. I used a number of approaches to identify impacts of PCBs on thyroid hormone-directed brain development. I first looked at gene expression and identified new targets of thyroid hormone, the cofactors N-CoR and SRC-1. I next looked at the effects of PCB exposure on these genes. I found that PCBs did not affect the expression of these genes. I next went on to look at cell and death and proliferation in the cerebellum and found that PCBs did not alter these processes. When I tried an in vitro approach to look at the impact of PCB exposure on receptor-independent effects of thyroid hormone on actin polymerization I found that my cultures did not respond to thyroid hormone as reported. In the last experiment I used a differential display screen to identify targets of PCB exposure and identified number of genes whose expression is putatively affected by PCB exposure. These genes included Stathmin, BESH, and Zic-1 all of which are associated with the cytoskeleton. It has also been shown that the effects of thyroid hormone can alter neuronal migration. These data suggest that PCBs may be altering neuronal migration and that the genes identified in this screen are downstream consequences of the thyroid hormone receptor -independent effects of thyroid hormone.

The epidermal growth factor receptor (EGFR) plays important roles in the control of many fundamental cellular processes including cell cycle, cell migration, cell metabolism and survival, cell proliferation and differentiation, as well as regulation of oocyte maturation and embryonic development. In the first part of this work I studied the EGFR expression and activation in cumulus cells (CCs). CCs are special cells immediately surrounding the oocyte. It has been shown that CCs in the isolated cumulus cell/oocyte complexes (COCs) exhibit both a slow rise in intracellular calcium concentration ([Ca 2+]i) and plasma membrane permeabilization in response to epidermal growth factor (EGF) stimulation. But cultured individual bovine CCs rarely showed a [Ca2+]i increase. The lack of response was confirmed to be due to a decrease of expression of endogenous EGFRs after dissociation. After CCs were reconstituted EGFR expression they showed robust, prolonged, EGF-stimulated [Ca2+] i elevations characteristic of CC responses in intact COCs followed by CC permeabilization and death. These responses were also confirmed being mediated by the IP3 signaling pathway. This EGFR activated Ca 2+ response in CCs followed by cell death may play an important role in the regulation of oocyte maturation. In the second part of this work, I identified an EGFR intramolecular regulation mechanism through study of the tyrosine phosphorylation in the EGFR regulatory domain (RD). EGFR signaling is partly controlled by tyrosine phosphorylation on the RD. There are 5 major tyrosine phosphorylation residues (992, 1068, 1086, 1148 and 1173) whose phosphorylation functions as the main platform for recruitment of downstream components. In order to understand the effect of intramolecular interactions among EGFR RD tyrosine residues, we constructed a series of single site mutant RDs. Each one replaces one major tyrosine phosphorylation residue with phenylalanine. After in vitro phosphorylation, the phosphorylation degree of each major tyrosine residue was quantitatively compared between mutant RDs and wild type RD using LC-ESI Ion Trap mass spectrometry. Our results indicate that Y1068 increases the phosphorylation of Y1148 and Y1173, Y1086 inhibits the phosphorylation of Y1068, Y1148 inhibits the phosphorylation of Y992 and Y1086, and Y1173 inhibits the phosphorylation of Y1068. Thus the EGFR exhibits extensively intramolecular interaction among its major tyrosine phosphorylation sites. Such intramolecular interaction may increase the complexity of EGFR signal transduction as well as modulate its efficacy.

The flow of information through neuronal circuits relies on the ability of neurons to form synaptic connections with specific temporal and spatial properties. These properties are not static but have the ability to change, allowing synaptic connections to be strengthened or weakened. It is this plastic nature of synapses that is central to higher order processes such as learning and memory. However, major gaps remain in our understanding of this process. Throughout my dissertation work I have examined mechanisms of a form of structural synaptic plasticity by analyzing the roles of a variety of proteins that we have found serve to regulate the formation and maintenance of glutamatergic synapses at the Drosophila NMJ. These proteins include APPL, the Drosophila homolog of Alzheimer's disease-associated β-Amyloid Precursor Protein (APP), the tumor suppressor protein Scribble (Scrib), the secreted signaling molecule Wingless (Wg), and the cell adhesion molecule Fasciclin II (FasII). In this work, in collaboration with members of the labs of Dr. Vivian Budnik, Dr. Kalpana White, and Dr. Susan Cumberledge, I have demonstrated that Wingless (Wg) provides a secreted signal that is required to initiate the formation of pre- and postsynaptic structures. Further, I have also demonstrated that once synapse formation is initiated, presumably by Wg signaling, APPL regulates synaptic bouton proliferation. This process also involves signaling by FasII, a protein required for synapse maintenance, and growth. Moreover, I have also demonstrated that Scrib is a scaffolding protein that plays a key role at these synapses in influencing neurotransmitter release.

The ER quality control machinery maintains the fidelity of the protein maturation process by sorting aberrant proteins for ER-associated protein degradation (ERAD), a process requiring retranslocation from the ER lumen to the cytosol and degradation by the proteasome. To understand the role of N-linked glycans in ERAD, degradation of wild-type (Tyr) and mutant (Tyr(C85S)) tyrosinase was examined. Here, we demonstrated that both wild-type and mutant tyrosinase were substrates of the 26S proteasome. Tyr(C85S), however, was less stable, and the cell line harboring the C85S mutation exhibited an up-regulated unfolded protein response as measured by XBP-1 mRNA splicing. Inhibiting mannose trimming or accumulating Tyr(C85S) in a monoglucosylated form led to stabilization, supporting a role for lectin chaperones in ER retention and mannose trimming in proteasomal degradation. In contrast, preventing glucose trimming caused rapid disappearance of protein. Upon closer examination employing procedures which monitored the appearance of degradation product (small peptides) rather than the disappearance of full-length protein, ablating lectin chaperone binding induced the formation of aggregates. Colocalization of tyrosinase with BiP and PDI, but not calnexin, implicated the latter two in aggregate dissolution. The fact that aggregates were disassembled and cleared from the ER at a rate similar- to non-aggregated species and degraded by the proteasome suggests a model of glycoprotein degradation in which non-lectin molecular chaperones function in the quality control of glycoproteins, at least in part, in the absence of lectin chaperones.

Variability in the discharge of human motor neurons was investigated in the context of aging and skill acquisition. In the aging experiment, young and older adults performed constant-force and complex sinusoidal isometric force-matching tasks while motor unit action potentials were recorded from the first dorsal interosseous (FDI) muscle. In three manuscript chapters, the characteristics of motor unit inter-spike interval (ISI) distributions were compared, patterns in the time series of ISIs were investigated with two experimental measures of complexity, and the extent of motor unit synchronization and common drive was compared between young and older adults. The sinusoidal force-matching conditions required substantial motor unit rate coding compared to the constant-force task. In both tasks, older adults utilized a greater proportion of motor units that were discharging at lower rates and with greater discharge variability. Positive skewness of ISI distributions from older adults indicated that motor unit discharge rates varied more freely towards lower rates and de-recruitment. That skewness was greater in older adults indicates that disruption of consistent discharge through de-recruitment and subsequent recruitment may be one reason why force fluctuations are greater and force-matching performance was poor in older adults. The complexity measures indicated that the fluctuations in motor unit discharge during constant-force contractions were random. Detrended fluctuation analysis, but not approximate entropy, was able to distinguish between the constant-force and sinusoidal force-matching tasks. It was demonstrated with simulated data that these techniques are sensitive to the manner in which motor unit data are pre-conditioned. Indeed, different methods of creating firing rate time series resulted in opposite conclusions regarding complexity. These complexity measures should be further evaluated with discharge data from patient populations in which patterns might be expected due to altered neural function. Motor unit synchronization and common drive were similar in young and older adults and greater synchronization was associated with greater discharge variability. (Abstract shortened by UMI.)