The dynamic processes that occur at specific times and locations in cells and/or whole organisms during cellular division, migration, morphogenesis and development are critical. When these molecular events are not properly regulated, disease states can develop. Tools that can allow us to better understand the specific events that, when misregulated, result in disease development can also allow us to determine better ways to combat such misregulation. Specifically, tools that could allow us to better visualize cellular processes or those that allow us to control cellular functioning in a spatiotemporal manner could present great insight into the detailed inner workings of cells and/or whole organisms. Where chemistry and biology intersect presents a powerful starting point for the development of such tools. The first half of this thesis addresses tools to allow the better visualization of cellular events, in particular the intriguing process of bioluminescence and the work that has been done to better understand and optimize its utilization, particularly in living organisms. The novel work presented here details a parallel approach to improve our ability to observe cellular functioning specifically by improving bioluminescence imaging through the generation and characterization of mutant luciferase proteins that can better utilize novel small molecule luciferin substrates. The second half of this thesis discusses methods that have been developed to better control cellular events through the control of protein activity, specifically a family of proteins called the Rho GTPases. This family’s activation at specific times and locations is essential to proper cellular function and exemplifies the need for spatiotemporal control. Described are methods to control the activation states of the Rho GTPases to probe their cellular roles in a temporal and spatial manner using photosensitive small molecules. Taken together, the findings described herein demonstrate the application of chemistry to allow for the better observation and control of cellular processes, toward the ultimate goal of improving our understanding of the regulatory processes involved in the control of key factors leading to disease states.

Different cell types in multi-cellular organisms heritably maintain different gene expression patterns despite carrying the same genome; a phenomenon termed epigenetics. It is widely believed that the packaging state of the genome, known as chromatin structure, carries epigenetic information. How chromatin states are inherited and how chromatin structure changes during development, moreover how different epigenomes, such as chromatin and DNA modifications communicate with each other during these processes are important questions. Accordingly, understanding the mechanisms that govern pluripotency and differentiation requires details of chromatin dynamics. The major goal of my doctoral thesis was to understand the genome wide view of chromatin dynamics in embryonic stem cells. My studies centered on two aspects of chromatin dynamics in mouse embryonic stem cells—localization and function of two antagonistic chromatin regulators and genome-wide histone variant dynamics. In the first part, we examined the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We found that two such regulators, Mbd3 and Brg1, control a large number of genes in ES cells via antagonistic effects on promoter nucleosome occupancy. Moreover, we found that both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC), a newly identified DNA modification. Mbd3, which was named by homology to known cytosine methyl binding domains, yet does not bind methylcytosine in vitro, co-localized in ES cells with 5hmC. Furthermore, Mbd3 localization was lost in knockdown cells lacking the major 5mC hydroxylase, Tet1. Our results suggest, contrary to current dogma, that 5hmC is more than just an intermediate in cytosine demethylation pathways, that it may regulate genes via the Mbd3/NuRD complex. Finally, we showed that both Mbd3 and Brg1 are themselves required for normal levels of 5hmC in vivo, identifying a feedback loop between 5hmC and Mbd3. Together, our results identified a possible effector for 5hmC, thereby suggesting a functional role for this DNA modification. Moreover, Brg1 and Mbd3 can now be added to the growing list of regulators with opposite effects on ES cell gene expression, suggesting that pairs of antagonistic chromatin binding proteins may be a common phenomenon in ES cell transcription regulation (Yildirim et al., Cell 2011). The second part of my dissertation concerns the dynamics of several histone variants. Seminal studies in the Henikoff lab showed that certain histone variants are replaced throughout the cell cycle, in contrast to the predominant replication-coupled mode of histone assembly. Work in yeast and flies showed that rapid histone turnover occurs at epigenetically-regulated genomic regions, such as chromatin boundary elements or Polycomb/Trithorax binding sites. Notably, promoter regions of actively transcribed genes exhibit rapid turnover, suggesting that histone turnover may have an important role in gene regulation, as higher histone turnover rate would provide higher probability of DNA element exposure and faster erasure of chromatin marks of the replaced histones. In order to extend such studies to a model for pluripotency and differentiation, we developed a system for measuring histone replacement in mouse ES cells. To be able to carry out turnover experiments in ES cells, we generated stable ES cell lines that can be induced to express epitope-tagged histone variants. Our results confirmed that histone turnover patterns are conserved from yeast to mammals and that turnover profiles are histone variant specific. Murine H3.3 turnover is similar to H3.3 turnover in flies, with peaks at the promoters of highly transcribed genes. MacroH2A2, a variant generally linked to gene repression, had a more complex turnover profile. Surprisingly, we found rapid exchange of macroH2A2 occurring around transcription start sites of a number of highly expressed genes. At poorly expressed genes, on the other hand, macroH2A2 localizes upstream or downstream of transcription start sites and is incorporated slowly, either via slow turnover or via replication-coupled incorporation. Finally, we have used those inducible ES cell lines to generate mice, which will enable future studies on tissue-specific histone replacement in vivo. In summary, my thesis work not only significantly extends our understanding of chromatin regulation in general but also provides a more detailed landscape of chromatin structure and regulation in ES cells. Extending these analyses to differentiating cells and in vivo tissue specific dynamics should provide us with a better understanding not only of cell type specific chromatin organization but also improve our ability to program and re-program genomic landscapes in vitro.

The generation of complex behaviors often requires the coordinated activity of diverse sets of neural circuits in the brain. Activation of neuronal circuits drives behavior. Inappropriate signaling can contribute to cognitive disorders such as epilepsy, Parkinson’s, and addiction (Nordberg et al., 1992; Quik and McIntosh, 2006; Steinlein et al., 2012). The molecular mechanisms by which the activity of neural circuits is coordinated remain unclear. What are the molecules that regulate the timing of neural circuit activation and how is signaling between various neural circuits achieved? While much work has attempted to address these points, answers to these questions have been difficult to ascertain, in part owing to the diversity of molecules involved and the complex connectivity patterns of neural circuits in the mammalian brain. My thesis work addresses these questions in the context of the nervous system of an invertebrate model organism, the nematode Caenorhabditis elegans. The locomotory circuit contains two subsets of motor neurons, excitatory and inhibitory, and the body wall muscle. Dyadic synapses from excitatory neurons coordinate the simultaneous activation of inhibitory neurons and body wall muscle. Here I identify a distinct class of ionotropic acetylcholine receptors (ACR-12R) that are expressed in GABA neurons and contain the subunit ACR-12. ACR-12R localize to synapses of GABA neurons and facilitate consistent body bend amplitude across consecutive body bends. ACR-12Rs regulate GABA neuron activity under conditions of elevated ACh release. This is in contrast to the diffuse and modulatory role of ACR-12 containing receptors expressed in cholinergic motor neurons (ACR-2R) (Barbagallo et al., 2010; Jospin et al., 2009). Additionally, I show transgenic animals expressing ACR-12 with a mutation in the second transmembrane domain [ACR-12(V/S)] results in spontaneous contractions. Unexpectedly, I found expression of ACR-12 (V/S) results in the preferential toxicity of GABA neurons. Interestingly loss of presynaptic GABA neurons did not have any obvious effects on inhibitory NMJ receptor localization. Together, my thesis work demonstrates the diverse roles of nicotinic acetylcholine receptors (nAChRs) in the regulation of neuronal activity that underlies nematode movement. The findings presented here are broadly applicable to the mechanisms of cholinergic signaling in vertebrate models.

For 30 years, the tumor suppressor p53 has been a subject of intense research in nearly every discipline of scientific inquiry. While numerous surprising roles for p53 in health and disease are uncovered each year, the central role of its activation in preventing neoplastic transformation has been and will remain at the forefront of p53 research as investigators work to address an unexpectedly complex question—precisely how does p53 integrate upstream stress signals to coordinate activation of its target genes in response to stress? One manner in which to address this question is at the level of transcription initiation—after upstream signals converge on p53 and produce a number of pools of post-transcriptionally modified p53, how exactly are specific target promoters activated in such a sensitive, context-specific manner? The work presented herein aims to address the role of histone acetylation at the p21 promoter—a critical mediator of G1/S arrest—by the P/CAF acetyltransferase in response to a variety of p53-activating stresses. We show that depletion of P/CAF strongly inhibits p21 expression in response to a variety of stresses, despite normal stabilization of p53 and recruitment to target promoters. This defect in p21 expression correlates closely with abrogation of stress-induced cell-cycle arrest. Strikingly, a p53 allele lacking putative P/CAF acetylation sites was still able to direct p21 expression, which was still dependent upon P/CAF. We show further that histone acetylation at H3K14 at the p21 promoter following stress is dependent upon P/CAF. Rescue of p21 expression with wild-type P/CAF or a ∆HAT point mutant indicates that P/CAF requires an intact HAT domain, suggesting that histone acetylation at H3K14 is catalyzed by P/CAF HAT activity, not the molecular bridging of a heterologous HAT by P/CAF. Furthermore, RNA polymerase II (RNAP II) was present at the p21 proximal promoter under all basal and stress conditions, but elongation of RNAP II after stress required the presence of P/CAF. These data indicate that H3K14 acetylation by P/CAF closely correlates with the activation status of the p21 promoter, and may be necessary for activation of a larger subset of p53-responsive promoters. In addition to its critical role in p21 expression, we noted that p53 stabilization and cell-cycle arrest in response to p14ARF, but not other p53-stabilizing stresses, were also dependent on P/CAF. Cell-cycle arrest induced by p16INK4A was intact after P/CAF ablation, indicating a role for P/CAF in cell-cycle arrest specific to p14ARF-p53 signaling. Basal MDM2 levels were unaffected by P/CAF knockdown, as were p53- MDM2 and ARF-MDM2 complexes. A preliminary analysis of MDM2 localization was inconclusive, due to vastly different quantities of MDM2 in different conditions making analysis of subcellular localization difficult; however, the role of P/CAF in the relocalization of MDM2 to the nucleolus by p14ARF could potentially explain the defect in p53 stabilization, and should be explored further. These observations, underscored by recent reports that P/CAF undergoes loss of heterozygosity in several tumor types, suggest that P/CAF plays a critical role in p53-mediated cell-cycle arrest through multiple, independent mechanisms. Further study should clarify whether P/CAF is lost in tumors maintaining wild-type p53, and whether its reintroduction into these tumors confers any potential therapeutic benefit.

The hydrophobic effect and hydrogen bonding interactions have long been considered to be the dominant forces in protein folding. However, the contribution of hydrogen bonds to stabilizing proteins has been difficult to clarify. As the intramolecular hydrogen bonds are formed in place of hydrogen bonds with solvent during folding, measures of stability fail to give a significant change in free energy. Previous studies on hydrogen bonding interactions have shown that they are only marginally important. Three long-range side chain-main chain hydrogen bonds have been found in the alpha subunit of tryptophan synthase (αTS), a (βα)8TIM barrel protein. These long-range noncovalent interactions connect either the N-terminus of one β-strand with the C-terminus of the succeeding and anti-parallel α-helix (F19-D46 and I97-D124) or the N-terminus of an α-helix with the C-terminus of the succeeding β-strand (A103-D130). By analogy, these interactions are designated as βα- or αβ-hairpin clamps. Surprisingly, the removal of any one of these clamp interactions, by replacement of the aspartic acid with alanine, results in significantly decreased thermodynamic stability for the native state and a substantial loss of secondary structure. When compared to several other side chain-side chain and short-range side chain-main chain interactions in αTS, these hairpin clamps clearly play a unique role in the structure and stability of αTS. The generality of these observations for βα-hairpin clamps in TIM barrel proteins was tested by experimental analysis of the clamps in a pair of homologous indole-3-glycerol phosphate synthase (IGPS) TIM barrels of low sequence identity. The results suggest that only the subset of conserved βα-hairpin clamps with hydrogen bond length less than 2.80 Å make substantive contributions to stability and/or structure. Those clamps with longer hydrogen bonds make modest contributions to stability and structure, similar to other types of side chain-main chain or side chain-side chain hydrogen bonds. The role of these clamps in defining the structures of the super-family of TIM barrel proteins was examined by a survey of 71 TIM barrel proteins from the structural database. Conserved features of βα-hairpin clamps are consistent with a 4-fold symmetry, with a predominance of main chain amide hydrogen bond donors near the N-terminus of the odd-number β-strands and side chain hydrogen bond acceptors in the loops between the subsequent α-helices and even-numbered β-strands. In this configuration, the clamps provide an N-terminal cap to odd-number β- strands in the β-barrel. Taken together, these findings suggest that βα-hairpin clamps are a vestigial signature of the fundamental βαβ building block for the (βα)8 motif and an integral part of the basic TIM barrel architecture. The relative paucity of βα-hairpin clamps remaining in TIM barrel structures and their variable contributions to stability imply that other determinants for structure and stability of the barrel have evolved to render a subset of the clamp interactions redundant. Distinct sequence preferences for the partners in the βα-hairpin clamps and the neighboring segments may be useful in enhancing algorithms for structure prediction and for engineering stability in TIM barrel proteins.

The dissemination of tumor cells to distant organs i.e. metastasis is an exceedingly complex process leading to 90% of all cancer deaths. Despite being so clinically important, little is known about this process that requires tumor cells to leave the primary tumor site, intravasate and transport through the blood stream, extravasate and colonize at secondary sites leading to distant metastases. Survivin, a member of the IAP (Inhibitor of Apoptosis) family with known functions in apoptosis and mitosis, is highly expressed in aggressive tumors and is associated with poor prognosis and adverse clinical outcome. But the mechanistic role of survivin in metastatic dissemination has not been investigated. In this study, we demonstrate an important and novel role of survivin in activating a broad gene expression program in tumor cells. Of particular importance is the upregulation of a distinct class of cell adhesion molecules, particularly fibronectin. This IAP mediated gene regulation requires synergistic intermolecular cooperation between survivin and its related cofactor molecule, XIAP that results in activation of NF-κB dependent fibronectin gene expression. The binding of fibronectin with its cognate cell surface receptors initiates outside–in signaling leading to the autocrine and paracrine activation of cell motility kinases, FAK and Src, in turn leading to enhanced tumor invasion and metastasis. The importance of survivin and XIAP in the process of metastasis has also been demonstrated in vivousing intrasplenic injections in mouse models. Overall this study is the first to place survivin upstream of transcriptional activation of gene expression particularly fibronectin. In addition, it also demonstrates the importance of survivin-XIAP complex in mediating NF-κB activation which in turn switches on the expression of various target genes involved in tumor metastasis. Hence this study dissects the upstream and downstream requirements of survivin- XIAP complex mediated tumor dissemination and metastasis. Significance of this Study The hallmark of end-stage cancer is metastasis, an incurable condition almost invariably associated with death from disease. Despite a better understanding of the metastatic process, and the identification of key gene expression requirements of this pathway, the development of anti-metastatic therapies has lagged behind, with no viable options being currently offered in the clinical setting. Our findings that Inhibitor of Apoptosis (IAP) proteins functions as metastasis-promoting genes independently of cell survival, but through activation of cell motility could have important ramifications for the broader application of IAP antagonists currently in early clinical trials, as novel anti-metastatic therapies.

Calmodulin is a prevalent calcium sensing protein found in all cells. Three genes exist for calmodulin and all three of these genes encode for the exact same protein sequence. Recently mutations in the amino acid sequence of calmodulin have been identified in living human patients. Thus far, patients harboring these mutations in the calmodulin sequence have only displayed an altered cardiac related phenotype. Calcineurin is involved in many key physiological processes and its activity is regulated by calcium and calmodulin. In order to assess whether or not calcineurin contributes to calmodulinopathy (a pathological state arising from dysfunctional calmodulin), a comprehensive search of relevant literature has been performed. Herein, the physiological roles of calcineurin and consequences of dysfunction have been reviewed for literature focused on the heart.

There is an emerging threat of Neisseria gonorrhoeae strains that are resistant to all antibiotics. Because of this, the purpose of this research is to isolate, analyze, and partially characterize a new inhibitor(s) of N. gonorrhoeae. Since there is an unknown molecule secreted by Candida albicans that inhibits N. gonorrhoeae, this molecule can be partially characterized using 1H NMR Spectroscopy to assist in the development of a new antibiotic compound. It was hypothesized that quorum-sensing molecules, trans, trans- farnesol, tyrosol, phenylethyl alcohol, and tryptophol, could be possible candidates for the inhibitor. Because of this, 1H NMR spectra for these quorum-sensing molecules were obtained to serve as controls. Column chromatography and fractionation was used to isolate the inhibitor in large scale from C. albicans grown in salts-based media. Attempts to isolate the inhibitor in large scale, however, was unsuccessful since no inhibition of N. gonorrhoeae was observed. Because of this, analysis of growth media was conducted to test the media effect on producing the inhibitor. C. albicans was grown in liquid chocolate, liquid white chocolate, salts-based, and YPD media in aerobic and candle jar environments. Analysis of growth media in different environments suggests that liquid chocolate and salts-based media retain the inhibitory activity. 1H NMR spectra were obtained for the isolated molecule in liquid chocolate and salts-based media in both aerobic and candle jar environments. Analysis of this 1H NMR suggested that the inhibitor could be isolated from either the aerobic or candle jar environment for both liquid chocolate and salt-based media because a clear peak between 3.5 and 4.0 ppm was observed in all spectra. Comparison of 1H NMR spectra from quorum-sensing molecules with spectra from the isolated molecule suggests that the inhibitor is not a quorum-sensing molecule. The peaks represented by the inhibitor cannot be fully characterized and thus, either correspond to a single molecule or a complex molecular structure. It can be concluded that the inhibitor secreted by C. albicans to inhibit N. gonorrhoeae is a new unknown compound.

Due to the blood-brain barrier, delivery of many drugs to the brain has required intracranial surgery which is prone to complication. Here we show that Dopamine Neuron Stimulating Peptide 11 (DNSP-11), following non-invasive intranasal administration, protects dopaminergic neurons from a lesion model of Parkinson’s disease in the rat. A significant and dose-dependent increase in an index of dopamine turnover (the ratio of DOPAC to dopamine) was observed in the striatum of normal young adult Fischer 344 rats by whole-tissue neurochemistry compared to vehicle administered controls. Among animals challenged with a moderate, unilateral 6-hydroxy-dopamine (6-OHDA) lesion of the substantia nigra, those treated repeatedly with intranasally administered DNSP-11 exhibited greater numbers of tyrosine hydroxylase (TH) positive dopaminergic neuronal cell bodies in the substantia nigra and greater TH+ fiber density in the striatum when compared to animals treated intranasally with vehicle only or a scrambled version of the DNSP-11 sequence. Lesioned animals that received intranasal DNSP-11 treatment did not exhibit abnormal, apomorphine-induced rotation behavior, contrasted with animals that received only vehicle or scrambled peptide that did exhibit significantly greater rotation behavior. In addition, the endogenous expression of DNSP-11 from the pro-region of GDNF was investigated by immunohistochemistry with a custom, polyclonal antibody. Signal from the DNSP-11 antibody was found to be differentially localized from the mature GDNF protein both spatially and temporally. While DNSP-11-like immunoreactivity extensively colocalizes with GDNF immunoreactivity at post-natal day 10, the day of maximal GDNF expression, DNSP-11-like signal was found to be present in the 3 month old rat brain with signal in the substantia nigra, ventral thalamic nucleus, dentate gyrus of the hippocampus, with the strongest signal observed in the locus ceruleus where GDNF is not expressed. Results from immunoprecipitation of brain homogenate were not consistent with the synthetic, amidated 11 amino-acid rat DNSP-11 sequence. However, binding patterns in the literature of NPY, the only homologous sequence present in the CNS, do not recapitulate the immunoreactive patterns observed for the DNSP-11 signal. This study provides evidence for a potential easy-to-administer intranasal therapeutic using the DNSP-11 peptide for protection from a 6-OHDA lesion rat model of Parkinson’s disease.

Several lipopeptidyl nucleoside antibiotics that inhibit bacterial translocase I (MraY) involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. A-90289 and muraminomicin are the two representative antibiotics that belong to this family. Bioinformatic analysis of the biosynthetic A-90289 gene clusters revealed that five enzymes are likely involved in the assembly and attachment of the aminoribosyl unit. These enzymes of A-90289 are functionally assigned by in vitro characterization. The results reveal a unique ribosylation pathway that highlighted by uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor. Muraminomicin, which has a structure similar to A-90289, holds the distinction in that both ribose units are 2-deoxy sugars. The biosynthetic gene cluster of muraminomicin has been identified, cloned and sequenced, and bioinformatic analysis revealed a minimum of 24 open reading frames putatively involved in the biosynthesis, resistance, and regulation of muraminomicin. Similar to the A-90289 pathway, fives enzymes are still likely involved in the assembly of the 2,5-dideoxy-5-aminoribose saccharide unit, and two are now functionally assigned and characterized: Mra20, a 5′-amino-2′,5′-dideoxyuridine phosphorylase and Mra23, a UTP:5-amino-2,5-dideoxy-α-D-ribose-1-phosphate uridylyltransferase. The cumulative results are consistent with the incorporation of the ribosyl appendage of muraminomicin via the archetypical sugar biosynthetic pathway that parallels A-90289 biosynthesis