<p> <i>Chromobacterium violaceum</i> (CV) produces a violet color pigment known as Violacein. It has been reported that violacein has anticancer activity. This compound is produced by CV a gram-negative facultatively anaerobic bacterium found in soil and water environmental samples. The purpose of this study was to determine the effect of purified violacein on select cancer cell lines. Violacein used in this study was purified from CV strain (14N23), a strain isolated from environmental samples collected in the Tennessee Copper Basin. The previous reports used a crude extract preparation of violacein; thus, it was of interest to determine the effect of the pure compound on cancer cell growth was similar to that of the crude extracts. The compound purified following the method of Mehta, et al. was exposed to cancer cells and cell death assessed using the Alamar Blue procedure. It was found that violacein had no effect on A549, BT549, and PC3 cancer cell growth; however, there was a significant effect on Colo-320 cancer cells. It was concluded that further studies are required to assess the effect of violacein on enzymes and proteins involved in the cancer cell apoptotic pathways. Such studies will explain why cancer cell death was observed in certain cancer cells and not others.</p>

<p> Angiogenesis, the formation of new blood vessels, requires the reorganization of microtubules (MT), which are polar cytoskeletal structures, consisting of a free&#129;-plus end and a minus&#129;-end that is free or anchored. CAMSAP2 and CAMSAP3 have been shown to bind and stabilize MT minus&#129;-ends; yet, this activity&#129;'s contribution to MT organization and directional migration is unknown. To investigate this contribution, we performed live&#129;-cell imaging of polarized HUVECs expressing CAMSAP2 or CAMSAP3. Our results show that CAMSAP2 and CAMSAP3 localized to the trailing edge of cells. Pharmacologic disassembly of MTs resulted in CAMSAP reorganization to the leading edge. MCAK expression is not sufficient for CAMSAP reorganization, but may recruit CAMSAP to the MT minus&#129;-end. MT growth dynamics analysis revealed that CAMSAP2 and CAMSAP3 promoted dynamic MT growth. These results suggest that CAMSAP2 and CAMSAP3 protect MTs against MCAK&#129;]mediated disassembly and also function to nucleate new, dynamic MTs at the leading edge.</p>

<p> Growth is one of the fundamental characteristics of biological systems. The study of eye growth regulation presents an interesting window that allows for the investigation of the role of the visual environment on internal processes. We now know that there is an intricate circuitry within the eye, independent of higher brain processes, that controls the growth of the eye but more needs to be elucidated about these local regulatory circuits. An improved understanding of this circuitry is critical to developing new therapies for abnormalities in eye growth regulation such as myopia, which is impacting more and more individuals around the world each day and in its more severe from, is linked to potentially blinding ocular complications. </p><p> The role of retinal glucagon, a neuropeptide, in the regulation of eye growth and refractive error has attracted the interest of researchers over the past 15 years yet there remain many unresolved questions. The research described in this dissertation aimed to elucidate the respective roles in eye growth regulation of specific subpopulations of retinal glucagonergic amacrine cells, which have been the subject of much speculation as the source of inhibitory growth signals, i.e. stop signals, yet not thoroughly investigated. </p><p> The approach taken to investigate this problem is to ablate glucagonergic amacrine cells in vivo using different neurotoxins, and to examine how this affects the sign-dependent circuitry of eye growth regulation. In addition, with the advent of advancements in high resolution imaging and electrophysiology, we were able to characterize the effects of these neurotoxins on the region-specific and time-sensitive changes in the structure and function of the living retina. </p><p> That the inhibitory response induced by imposed myopic defocus remains intact, in spite of total ablation of glucagon cells (Chapter 5) or elimination of the peripheral glucagon cells (Chapter 3) and other unintended adverse retina effects, compared to findings from previous studies involving QUIS (Chapter 2) of this thesis, is a novel finding. These results point to the same conclusion that glucagon cells themselves are not responsible for the decoding of the sign of optical defocus, but appear to have a role in fine-tuning of compensatory growth responses. The results of our experiments also suggest that the choroid may serve as an intermediate relay, and the altered anterior chamber development raise the further possibility that retina-derived growth modulatory factors also regulate the anterior segment, perhaps reaching this more remote site by diffusion forward through the vitreous chamber or via the uvea.</p>

<p> A living cell is a complex soft matter system far from equilibrium. While its components have definite mechanical properties such as stiffness and viscosity, cells consume energy to generate force and exhibit adaptation by modulating their mechanical properties through regulatory pathways. In this dissertation, we explore cell mechanics by stretching single fibroblast cells and simultaneously measuring their traction stresses. Upon stretch, there is a sudden, drastic increase in traction stresses, often followed by a relaxation over a time scale of about 1 minute. Upon release of stretch, traction stresses initially drop and often recover on a similar time scale of about 1 minute. We show that a minimal active linear viscoelastic model captures essential features of cell response to stretch. This model is most successful in describing the response of cells within the first 30 seconds of stretch. While perturbations of myosin and vinculin change quiescent traction stresses, they surprisingly have no significant impact on the stiffness or viscoelastic timescale of the cells. On longer time scales, cells may show an adaptive response to stretch that contradicts the minimal mechanical model. The probability of an adaptive response is significantly reduced by myosin de-activation and vinculin knockout. Therefore, we find that while vinculin and myosin are not important in determining passive mechanical properties of cells, such as stiffness and viscosity, they play a significant role in the adaptive mechanisms of cell response to stretch. To perform this work, we have built a novel micro stretching device compatible with live cell microscopy and developed a computational tool to analyze data from large traction stresses. Therefore, this dissertation's contribution is two-fold: (1) a novel experimental approach to explore the mechanics of living cells, and (2) a new model and framework for understanding the mechanical response of cells to stretch.</p>

<p> The membrane-bounded lysosome is a dynamic organelle responsible for macromolecule degradation, receptor down regulation, stress survival and pH balance. Intact lysosomal function is critical for proper cell function, as defects in lysosomal trafficking have been associated with neurodegenerative diseases such as Alzheimer&rsquo;s disease. <i>Saccharomyces cerevisiae </i>, budding yeast, is an ideal model organism for studying lysosomal protein trafficking because its vacuole is functionally analogous to the mammalian lysosome and both share conserved trafficking machinery components. </p><p> A previous yeast genomic deletion strain screen in our lab uncovered fourteen deletion strains with h&barbelow;ypersensitivity to <u>hy </u>gromycin B (<i>hhy</i> mutants), all of which exhibited defective vacuolar trafficking, morphology and/or function. Additionally, recent microscopic findings in our lab support that the late-endosome dependent pathway is the affected pathway and that <i>hhy</i>s have compromised T&barbelow;arget of R&barbelow;apamycin C&barbelow;omplex 1&barbelow; signaling. TORC1 regulates cell growth and cell proliferation and its hyperactivity is associated with cancer and metabolic diseases. As with lysosomes, the vacuole also serves as the platform for TORC1 signaling. </p><p> To further assess if the late-endosome dependent pathway is affected by hygromycin B treatment in <i>hhy</i> mutants, vacuolar delivery of two vacuolar markers was assessed. Alkaline phosphatase (ALP) delivery was assessed as a late-endosome independent pathway marker and carboxypeptidase Y (CPY) delivery was assessed as a late-endosome dependent pathway marker. To date, we have observed that hygromycin B treatment disrupts CPY trafficking via the late-endosome dependent pathway while ALP trafficking via the late-endosome independent pathway is unaffected by hygromycin B treatment. </p><p> Rapamycin is the direct inhibitor of TORC1. Rapamycin and its analogs (rapalogs) are currently administered in the treatment of TORC1 hyperactivity. However, therapeutic inhibition of TORC1 signaling by rapamycin is associated with severe toxicities. Since our recent results implicate compromised vacuolar trafficking of Tor1 kinase in <i>hhy</i> mutants, we tested vesicular trafficking inhibiting drugs in combination with rapamycin in order to explore a cumulative effect on TORC1 inhibition assessed by growth of wild type cells. Additionally, we explored the effects of combining two vesicular trafficking drugs on wild type cell growth. </p><p> We established a cumulative effect on wild type growth upon using low concentrations of rapamycin in combination with vesicular trafficking inhibitory drugs. Thus, rapamycin and vesicular trafficking inhibitory drugs have potential for drug combination therapy against TORC1 hyperactivity at lower drug concentrations. Drug combination treatment may be a new and effective way to regulate TORC1 function at sub-toxic levels of rapamycin.</p>

<p> HIV-infected cells can reach the central nervous system (CNS) and release HIV virions, which productively infect macrophages and microglia. Infected cells release both viral and host factors that can induce inflammation in bystander immune cells, to initiate neuropathogenesis beyond the initial site of the infected cell. While antiretroviral therapy can effectively control CNS viral loads, HIV-1 RNA and Tat protein can persist in cerebrospinal fluid of treated patients. Ongoing neurotoxic insults can result in HIV associated neurocognitive disorders (HAND). While the incidence of the severe form of HAND has declined with increasing treatment, milder forms of HAND have increased in prevalence. Delineating the viral determinants of cytotoxicity that can lead to HAND is critical to developing effective neuroprotective agents.</p><p> HIV-1 Tat protein contributes to HIV neuropathogenesis in multiple ways, including direct neurotoxicity and recruitment of HIV infected monocytes to CNS. Tat can promote transcriptional transactivation of cellular proinflammatory cytokines in the infected cells. Tat's ability to be secreted and subsequently taken up by bystander cells can lead to an induction of inflammatory cytokines in the uninfected cells as well. Tat's cellular uptake is mediated by an Arginine-rich region termed the cell penetration peptide (CPP). We now describe the functional consequences of a polymorphism in the Tat basic domain (residues 48-57) that diminishes extracellular Tat's uptake by cells. Earlier work, using lade B HIV-1 Tat CPP containing sixArginines, showed that each of the sixArginines are essential for maximal Tat CPP uptake. However, we observed significant variability in the conservation of R57 residue within group M clades &ndash; while R57 is well conserved (>75%) in many, other clades contain a non-Arginine residue at this position.</p><p> We hypothesized that the absence of R57 reduces Tat uptake. Cells incubated with fluorescently-labeled peptides corresponding to residues 48-57 in Tat, with R57, S57 or G57 residues revealed a 3-4 fold higher level of CPP-R57 uptake compared to the others. In order to further study the biological implication of this variation, we chose to compare clade B (most studied clade) with clade C (globally pre-dominant clade). To test the effects of R57S substitution on full-length Tat protein, we established a transcellular Tat transactivation system, where HeLa cells are transfected with expression constructs for Tat-B (R57), Tat-C (S57) proteins or their R57S or S57R mutants respectively. Media are collected from the transfected cells, relative levels of Tat determined by ELISA and equal amounts of Tat applied to TZM-bl reporter cells. We demonstrated that R57S mutation in Tat-B diminished its transcellular reporter signal response, while the S57R mutation in Tat-C enhanced the transcellular response. Furthermore, we investigated the role of R57S substitution on the establishment of an inflammatory environment by exposing monocytic and microglial cells lines to Tat-containing media and quantifying the inflammatory cytokines gene induction. We found significantly higher levels (~2-3 fold) of expression of several cytokine genes, in cells exposed to the R57-containing Tat variants compared with the S57-variants.</p><p> Based on our results, we conclude that Tat R57 is an important viral determinant for efficient cellular uptake of Tat. It contributes to the intercellular, systemic spread of inflammation, and may contribute to the inflammatory phenotype in the CNS of HIV-infected individuals. Intriguingly, a small data set of Tat sequences from CNS samples in HIV-1 C patients indicated an enrichment for R57 variants, compared with the consensus peripheral sequences (77% vs 13%, respectively), suggesting that the rare CNS HIV disease that is observed in some clade C patients may be associated with a R57-containing Tat. Our studies have identified a novel signature of HIV-1 Tat that enhances the induction of inflammatory signal, and may play an important role in neurovirulence. </p>

<p> In order to adapt to changing conditions and life stages, cells must remodel the composition of the proteome by specifically removing obsolete or damaged proteins. Failure to remove proteins at the appropriate time can cause a variety of cellular dysfunctions that can lead to cell death and conditions such as cancer, Huntington's, Parkinson's and Alzheimer's disease. Protein quality control is especially important during aging and stress, as the accumulating load of damaged, misfolded, and aggregated proteins can overwhelm the cell's ability to maintain the proteome. The ubiquitin-proteasome system is responsible for the specific degradation of many proteins within eukaryotic cells. The 26S proteasome degrades proteins that have been &ldquo;tagged&rdquo; with a chain of ubiquitin molecules. Ubiquitin is a highly-conserved 8.5 kDa protein that is conjugated post-translationally to a target protein after passing through a complex series of enzymes. Using the nematode <i> C. elegans</i> as a model, we sought to 1) Characterize the roles of specific ubiquitin-conjugation enzymes (UBCs) in the response to aggregation of a polyglutamine-containing protein in body wall muscle cells and 2) Examine the subcellular localization of proteasomes in these cells during normal aging and stress. Q82::GFP, a fusion protein expressed from a transgene encoding a polyglutamine tract fused to Green Fluorescent Protein (GFP), was found to aggregate rapidly (~58 minutes) in a manner that is not directly dependent on ubiquitin but dependent on the concentration of Q82::GFP protein, which was altered in response to RNA interference of several E2 ubiquitin-conjugating enzymes. In a separate transgenic worm strain, a fluorescently-labeled proteasome subunit, RPT-1, was observed in live animals to localize to the nucleus and cytoplasm during the adult stage. Within the nucleus, RPT-1::GFP localizes diffusely and to foci, and was excluded from heterochromatin. Within the muscle contractile apparatus, RPT-1::GFP localizes to I-bands, regions of thin actin filaments, and is excluded from dense bodies. After a prolonged heat stress, RPT-1::GFP in adult worms relocalizes to dense bodies. After starvation, these foci are associated with high proteolytic activity. These studies demonstrate some of the spatial dynamics of the ubiquitin-proteasome system in response to development, aging, and stress.</p>

<p>Abstract</p><p>During embryonic development many structures crucial for breathing and eating arise from the pharyngeal and anterior foregut epithelium (FGE), which contains the oral ectoderm and the foregut endoderm. Proper differentiation and signaling within and from this epithelial tissue is necessary for the development of the mandible, the esophagus, and the trachea. Many birth defects occur in these structures that greatly disrupt the ability of affected infants to breathe and eat. This dissertation investigates the importance of the pharyngeal and anterior FGE in mandible and foregut development. </p><p>The most rostral portion of the pharyngeal epithelium contributes to the development of the mandible. At embryonic day 10.5 the mandible is a bud structure, composed of neural crest-derived mesenchyme and core mesoderm surrounded by pharyngeal epithelium. The mesenchyme needs to receive Hedgehog signaling for mandible development, but the epithelial tissue that signals to the mesenchyme has not been identified in mammals. Data presented in Chapter 2 show that Sonic Hedgehog is necessary at two distinct stages of mandible development by using a tissue specific genetic ablation to remove Sonic Hedgehog from the pharyngeal endoderm. First, we show that Sonic Hedgehog promotes cell survival prior to cartilage differentiation through immunostaining for Caspase-9, an apoptosis marker. Second, a rescue of early cell death with the p53 inhibitor pifithrin-&#945; shows that Sonic Hedgehog is necessary for cartilage condensation and differentiation later in development. Without cartilage differentiation the mandible is unable to elongate properly and hypoplasia occurs.</p><p>Caudal to the pharyngeal epithelium is the anterior FGE, which develops into the larynx, esophagus and trachea. The anterior FGE is a single endodermal tube at E9.5 and by E11.5 compartmentalizes into two distinct tubes: the esophagus and trachea. While the signaling pathways involved in proper compartmentalization of the foregut are well studied, nothing is known about the cellular behaviors that drive this complex event. One important event during foregut compartmentalization is the establishment of dorso-ventral patterning, which is necessary for separation to occur. To elucidate the importance of dorso-ventral patterning, we take advantage of two genetic mouse models with disrupted patterning, an activation of and a removal of &#946;-catenin in the ventral foregut endoderm. Data presented in Chapter 3 show that &#946;-catenin is important for epithelial pseudostratification and the establishment of a region of double-positive cells at the dorso-ventral midline through close examination of epithelial morphogenesis at E10.5 prior to compartmentalization. This data has established two mouse models for studying changes in epithelial morphology during foregut compartmentalization. In total, this body of work details how signals originating in the pharyngeal and anterior foregut epithelium regulate both mesenchymal and epithelial behaviors during mandible and foregut development.</p> / Dissertation

<p>Bacterial tubulin homolog FtsZ assembles straight protofilaments (pfs) that form the scaffold of the cytokinetic Z ring. These pfs can adopt a curved conformation forming a miniring or spiral tube 24 nm in diameter. Tubulin pfs also have a curved conformation, forming 42 nm tubulin rings. We have previously provided evidence that FtsZ generates a constriction force by switching from straight pfs to the curved conformation, generating a bending force on the membrane. In the simplest model the membrane tether, which exits from the C terminus of the globular FtsZ, would have to be on the outside of the curved pf. However, it is well established that tubulin rings have the C terminus on the inside of the ring. Could FtsZ and tubulin rings have the opposite curvature? In the present study we explored the direction of curvature of FtsZ rings by fusing large protein tags to the N or C terminus of the FtsZ globular domain. FtsZ with a protein tag on the N terminus did not assemble tubes. This was expected if the N terminus is on the inside, because the protein tags are too big to fit in the interior of the tube. FtsZ with C-terminal tags assembled normal tubes, consistent with the C terminus on the outside. The FN extension was not visible in negative stain, but thin section EM gave definitive evidence that the C-terminal tag was on the outside of the tubes. This has interesting implications for the evolution of tubulin. It seems likely that tubulin began with the curvature of FtsZ, which would have resulted in pfs curving toward the interior of a disassembling MT. Evolution not only eliminated this undesirable curvature, but managed to reverse direction to produce the outward curving rings, which is useful for pulling chromosomes.</p> / Dissertation

Adrenomedullin (AM) and pro-adrenomedullin N-terminal 20-peptide (PAMP) are peptides recently identified from a rat pheochromocytoma. Both of these peptides are cleavage products of pre-pro-AM. Specific receptors for AM have been characterised in several species, including rat and human. The aim of this study was to investigate the role of PAMP and AM in the adrenal cortex. Using an intact rat capsule preparation PAMP was shown to cause a dosedependent increase in aldosterone secretion, which was accompanied by a dosedependent increase in cAMP release. The effects of PAMP were inhibited by HA1004, an inhibitor of protein kinase A. These results suggest that PAMP stimulates aldosterone secretion from the zona glomerulosa via cAMP. Ligandbinding studies were then used to demonstrate the presence of specific PAMP receptors. Two classes of receptor were shown in the rat zona glomerulosa (Kdi 1.9 nmol/l, Bmai, 53 fmol/mg protein; Kd2 10 nmol/l, Bmac,2 225 fmol/mg protein). At the latter receptor PAMP was displaced by AM. None of the other competitors tested displaced PAMP. Using the H295R cell line, both PAMP and AM were shown to increase aldosterones ecretion in a dose-dependenmt anner. In both casesa corresponding dose-dependent increase in cAMP was observed. Both PAMP and AM also effected a dose dependent increase in cortisol secretion. mRNA analysis showed that the gene encoding pre-pro-AM was expressed in these cells. Immunocytochemistry confirmed that these cells were producing both PAMP and AM. Immunocytochemistry and mRNA analysis also revealed that both of the candidate receptors for AM, L1 and CRLR, are expressed in this cell line. Taken together these findings demonstrate that both AM and PAMP are produced by adrenocortical cells and likely to have a role in regulating adrenal steroidogenesis. Furthermore, these studies suggest the presence of a specific PAMP receptor in the rat adrenal gland.