<p> Beta-arrestins are adaptor proteins that can scaffold a number of signaling proteins to promote localized activity within the cell. Downstream of some GPCRs, &beta;-arrestins can promote activation of the actin filament severing protein, cofilin, through two mechanisms: one involving inhibition of LIM Kinase (LIMK) which negatively regulates cofilin activity through phosphorylation on serine 3. The mechanism by which &beta;-arrestin-1 regulates LIMK activity has not been elucidated; however, it has been shown to be important for cell migration downstream of protease-activate-receptor-2 (PAR-2), dendritic spine formation and opioid receptor function. Here my work demonstrate that &beta;-arrestin-1 directly binds both cofilin and LIMK, and inhibits LIMK activity directly and investigate the mechanism by which inhibition of kinase activity occurs. Using serial truncations and site-directed mutagenesis, I identify crucial residues for cofilin and LIMK interaction within amino acids 1-99 of &beta;-arrestin-1 and show that charged residues at 50 and 51 are crucial for binding to LIMK and R51 is required for LIMK inhibition, PAR2 stimulated cofilin dephosphorylation and cell migration. Additionally, our work reveals that amino acids 1-99 aminos of &beta;-arrestin-1 bind both cofilin and LIMK with a higher apparent affinity than the full length and blocks PAR2-stimulated cofilin dephosphorylaton in HEK293 cells, suggesting it functions as a selective dominant negative &beta;-arrestin-1, inhibiting specifically the cofilin pathway. Thus, residues in the N-terminus of &beta;-arrestin-1 are involved in LIMK inhibition and cofilin activation and this, in turn, is important for cell migration downstream of PAR-2.</p>

<p> TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not &Delta;Np63, p53 or p73, induces the expression of CCDC3 mRNA level by directly binding to the p63 consensus DNA binding sequence within the CCDC3 enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but inversely with &Delta;Np63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance, and fatty liver (steatosis) formation in transgenic CCDC3 mice on the high-fat diet by markedly reducing hepatic PPAR&gamma; expression and consequently leading to a drastic decrease of the PPAR&gamma; target gene, CIDEA, and other genes involved in de novo lipogenesis and of lipid droplets formation in their livers. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on high-fat diet. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.</p>

<p> The research presented in this dissertation deals with the phenomena of bacterial efflux pump inhibition by natural products and plant extracts. Bacterial efflux pumps are active transport proteins, primarily deriving their energy source from the proton motive force, which functions to export toxic compounds outside the cell. This is a natural defense mechanism that bacteria utilize to protect themselves from toxic environments. The over-production of efflux pumps is one mechanism by which bacteria can evolve resistance to clinical antibiotics, as well as other antimicrobials. Thus, the study of efflux pump inhibitors is important because it holds the potential to reverse some forms of antibiotic resistance. In light of this importance, a series of studies were designed to improve the ability to study this phenomenon, to investigate the distribution of efflux pump inhibition in land plants, and to improve our ability to identify an important class of efflux pump inhibitors, the flavonoids. </p><p> The first aim of this research was to develop an improved method for experimentally quantifying efflux pump inhibitory activity of small molecules. Preexisting methods made this difficult due to several limitations including: the collection of indirect results, time consuming materials handling techniques, and/or matrix interference problems pertaining to the quenching of fluorescent signal. An improved method relying on mass spectrometry measurements was developed that addressed the aforementioned limitations. The importance of this improved method lies in its ability to produce data sets useful in calculating IC₅₀ values for a wider range of samples than was previously possible. </p><p> The second aim was to evaluate the presence of efflux pump inhibitor production across the land plant lineage. This is important to botanical science and the understanding of plant-microbe interactions and plant evolutionary biology. The most ancient lineages of land plants have not been previously evaluated for efflux inhibitory activity. Additionally, land plants play an important role in many traditional medicinal systems and in modern complementary and alternative medicine. Thus, understanding the distribution of efflux pump inhibitor production in this group increases our understanding of these common forms of medical treatment. In order to gain these data, a set of 14 plant species spanning the major lineages within the land plant group (bryophytes, pterophytes and lycophytes, gymnosperms, and angiosperms) were extracted and assayed to determine efflux pump inhibitory activity of the extracts. Positive results (indicating the presence of an efflux pump inhibitor) were observed for many (but not all) of the plant species tested. The observation of activity in extracts prepared from the most ancient plants tested (bryophytes--the liverworts and mosses) lends credence to the hypothesis that the production of efflux pump inhibitors is of great antiquity in land plants. </p><p> The last component of this work was the evaluation of methods for the analysis of flavonoids via mass spectrometry. This is of importance to this study due to the commonality of flavonoids in the literature pertaining to efflux pump inhibitors, and the consistency activity of the flavonoids evaluated in Chapter II. The goal of this work was to compare two methods for the tentative identification of signals in complex data produced via high-resolution mass spectrometry that could be labeled as "possible flavonoids." The methods evaluated were firstly the use of mass spectrometry fragmentation spectra to identify key diagnostic fragments of the flavonoid ring structure and secondly the use of mass defect filtering directly applied to high resolution data to select a short list of signals for further processing. The former method was not fruitful due to a combination of the frequent poor fragmentation and the dependency on standards for all samples. The latter proved more useful, successfully producing a list of potential flavonoids to be carried forward to other methods such as database searching and molecular formula calculation. This method was also successfully applied to a complex extract of <i>Hydrastis canadensis </i>, identifying three flavonoids known from previous work to be present. Further, the mass defect method is an intrinsic property of molecules, and therefore does not change with experimental conditions. For all of these reasons mass defect was selected as the more useful of the two methods evaluated for the identification of "possible flavonoid" signals in crude extracts.</p>

Thesis (PH.D.) -- Syracuse University, 2007. / "Publication number AAT 3281716"

Thesis (Ph.D.)--Drexel University, 2010. / Includes abstract. Includes bibliographical references (leaves 166-182).

<p> The molecular gatekeepers of nearly all gene expression in living cells are the proteins that function in the process of transcription. Transcription occurs when a cell must respond to a signal. These signals can be in the form of metabolic responses, signals for growth or differentiation, signals to defend against stress or pathogenic invasion, to name a few. The fundamentals of transcription have been extensively studied in bacterial systems and model organisms, but technical limitations have hindered their studies in mammalian and human systems. Recent developments in mass spectrometric methodologies, next-generation sequencing and techniques to study difficult-to-detect post-translational protein modifications are extensively reviewed here to highlight an important regulatory network through which gene expression is regulated. In addition, I present two vignettes: the first, a study of the regulatory mechanisms of monomethylation of the HIV-1 Tat protein in regulating HIV-1 gene expression and latency; the second, a study investigating the role of acetylation in regulating RNA Polymerase II protein modifications and gene expression in mammalian systems. Together, these studies combine new mass spectrometric techniques, modification-specific antibodies, protein purification methods, and next generation sequencing to better understand the role of these modifications in regulating the transcriptional response in mammalian systems. These findings can be applied to better understand mechanisms that regulate HIV-1 viral latency, along with fundamentally shifting the field of mammalian transcription by pinpointing unique modes of regulation only found in higher eukaryotes relevant to HIV-1 infection and cancer.</p><p>

<p> Hyper-O-GlcNAcylation of proteins is a subsequent artifact of metabolic disorder and is indicative of many cancers, including cancers of the female reproductive tract. While the incidence of most cancer types has been declining in the U.S., endometrial and cervical cancer remain among the most common cancers diagnosed in women. Diabetic women have a 2-3 fold increased risk of developing endometrial cancer, and tend to have more aggressive cases of cervical cancer, however, the molecular aspects of these risks are not fully understood. This study investigated the alteration of cellular O-GlcNAcylation of proteins as the potential mechanistic connection between diabetes and tumorigenicity in cancers of the female reproductive tract. The cervical cancer cell line (SiHa) and the endometrial cancer cell line (Ishikawa) were utilized to study the effect of dysregulation of O-GlcNAcylation on the proliferation, migration, invasion, and related molecular mechanisms. In cervical cancer, O GlcNAcylation was found to be an important regulator of tumorigenicity. Overall, inhibition of O-GlcNAcylation (via the inhibitor, OSMI-1) in SiHa cells impaired cell proliferation (p&lt;0.01) and invasion (p&lt;0.01) yet did not affect cell cycle progression. These effects occurred concomitantly with an alteration of cellular morphology, principally the disruption/decline of K8/18 and &beta;-actin filament expression. The results suggest O-GlcNAcylation regulates several aspects of tumorigenesis in cervical cancer cells, and cytoskeletal proteins are among the targets. Similarly, in endometrial cancer cells, hyper-O-GlcNAcylation (via 1&mu;M Thiamet-G/ThmG or 25mM Glucose) enhanced the expression of EMT-associated genes (WNT5B and FOXC2), and the E-Cadherin suppressor, Snail. Reorganization of actin filaments into stress filaments, consistent with EMT, was also noted in ThmG-treated cells. Interestingly, Hypo-O GlcNAcylation (via 50 &mu;M OSMI-1) also upregulated WNT5B, inferring that any disruption to O-GlcNAc cycling impacts EMT. However, Hypo-O-GlcNAcylation reduced cellular proliferation/migration and the expression of the pro-EMT genes (AHNAK, TGFB2, FGFBP1, CALD1, TFPI2). Finally, Ishikawa cells were used to investigate the effect hyper-O GlcNAcylation on the efficacy of progesterone (P4) in therapy for endometrial cancer proliferation and invasion. Ishikawa cells were exposed to ThmG, to induce hyper-O GlcNAcylation, and 100nM P4. P4 alone, significantly decreased cell proliferation, however, the addition of ThmG, and subsequent hyper-O-GlcNAcylation, negated this affect, returning the cells to control level proliferation (p&lt;0.05). A similar pattern was noted in Matrigel invasion assays, where Hyper-O-GlcNAcylation augmented invasion compared to P4 treatment alone, both with and without progesterone treatment (p&lt;0.05). Progesterone treatment has been shown to induce the expression of p21 and p27, reducing cell growth. In this study, P4 maintained p21 expression and increased p27 expression, however, ThmG decreased p27 expression and the expression of endogenous progesterone receptor B (PR B) despite P4 treatment. These results suggest that hyper-O-GlcNAcylation, common in obese and diabetic patients, may promote tumorigenicity in female cancers and could impair the efficacy of progesterone treatment. O GlcNAcylation has the potential to serve as a biomarker for early diagnosis and could predict treatment success. O-GlcNAc cycling enzyme inhibitors could prove to be useful tools for providers when treating cancer patients with metabolic disorders.</p><p>

<p> Biophysical and biochemical imbalance of mechanisms relevant to muscle function, can result in morphological changes to the tissue. While the purpose of activities involving exercise is to modify the shape and size of skeletal muscle, and the length of these muscles allows wide ranges of stiffness and stretch to be applied, cardiac tissue is not meant to change much. However, stressful extrinsic factors (poor diet, chemotherapy, etc) or intrinsic factors like inherited mutations in muscle functioning genes can result in a myopathy or a disease of the muscle. In fact, another biological process that requires much compliance of many molecules is embryogenesis. Although the timeline of an embryonic structure is limited, compared to an adult heart and muscle composition, continuous and coordinated movement is essential, but cumulative, prolonged disruptions can be harmful. At the core of muscle biology is the myosin molecule which is a motor protein that hydrolyzes ATP, binds to actin, and the spatial dynamics of its function (contraction-relaxation) alter the length of muscle. Myosin cyclically follows specific steps and undertakes well-defined structural conformations during these events, but mutations can alter the time and stability of any of these aspects. In this thesis I did a comprehensive analysis of the ATPase cycle parameters for both embryonic and cardiac myosin and studied the effects of specific associated or linked mutations have on function. The multiple mutations were in the interest of cataloging common features and defects to identify mechanistic patterns. In a collaborative effort I also used these wet-lab measurements to simulate the cycle using a working kinetic model for the myosin ATPase cycle. We have found distinct differences between three different myopathies that will be discussed in the following chapters.</p><p>

<p> The discovery of viroids in 1971 opened the door to a whole new field of RNA biochemistry. Viroids subsequently became the first of many facets of RNA biochemistry: the first single stranded covalently closed RNA discovered in nature, the first subviral pathogen discovered, and the first pathogen of a eukaryotic system to have its genome sequenced. Viroids are the smallest known agents of infectious disease and they represent the borders of life. They replicate autonomously within their host and since they do not code for their own proteins, they act as scavengers of the host transcriptional machinery. By doing so, viroids find ways of trafficking, localizing, and replicating within their host based on the sequence and structure of the RNA alone. Once in their hosts, viroids are incredibly resilient and can cause economic damage on several commercial crops. Apart from controlling viroids for economic reasons, the more enticing feature of viroid study is the use of viroids as model systems to study essential underlying questions about the evolution of RNA pathogens, and to use viroids as models to study non-coding RNAs. The field of non-coding RNA research has surged within the past decade and viroids are becoming important vehicles to bring insight into this field of study. The study of viroids has been extensive through the years, but several questions remain: What structural conformations do viroids employ to recruit host enzymes, and what are the enzymes that cleave and ligate viroids into mature progeny. To answer some of these questions, we have looked at processing of the potato spindle tuber viroid (PSTVd) RNA in the budding yeast <i> Saccharomyces cerevisiae</i>. We found that one specific construct will process into a mature viroid circle in yeast and we also found that processing in this system is distinct from other plant and non-plant based host systems. This processing is a delicate interplay of ligation and degradation by host machinery. Yeast is a great system to study viroid processing as yeast allows for use of the entire toolbox of temperature-sensitive and knockout protein mutants. By employing yeast, focus can be driven towards the mechanisms of host protein recruitment, viroid processing requirements, and degradation mechanisms from the host. We have ascertained insight into PSTVd processing using yeast. We have found methods to transform and process PSTVd, investigated enzymes that effect processing, and started to establish an <i>in vitro </i> yeast system. Through these studies, we have also developed a method to enrich viroid RNAs from total RNA extractives. This has been vital to assays specific around viroid transcription and cleavage. Overall, this research is further testament that viroids are minimalist scavengers of a very diverse array of cellular transcriptional machinery. They can process in higher eukaryotes (plants) and simple eukaryotes (yeast). They are shown to affect each host in distinct manners using fundamental RNA biology that all organisms share. </p><p>

<p> Dietary fat provides essential nutrients, contributes to energy intake and regulates blood lipid levels. These functions are important to health; however, when dysregulated they contribute to dyslipidemia and increase risk for development of obesity, diabetes and cardiovascular disease. </p><p> Dietary fat absorption is efficiently mediated by the small intestine. The digested products of dietary fat in the gut lumen are taken up by enterocytes, the absorptive cells of the intestine, where they are re-synthesized to triacylglycerol (TAG). The resulting TAG is packaged onto chylomicrons (CMs) for secretion into the circulation, contributing to postprandial blood lipid levels. When levels of dietary fat are high, TAG is also packaged into cytoplasmic lipid droplets (CLDs) for temporary storage within enterocytes. The CLDs increase and then decrease overtime in response to fat consumption, indicating that the stored TAG is mobilized for secretion or to the other fates at later time points. The intestinal metabolism of CMs and CLDs together regulate the rate and the amount of TAG secreted into the circulation. The objective of this dissertation work is to explore the mechanisms through which the TAG is partitioned or mobilized to certain metabolic fates in enterocytes. </p><p> First, we investigated the effect of endurance exercise on genes of intestinal lipid metabolism using Otsuka Long-Evans Tokushima Fatty (OLETF), an obese and diabetic rat model. We found that exercise training in these animals resulted in parallel upregulations of genes involved in TAG anabolic and catabolic processes and promoted mitochondrial biosynthesis in enterocytes compared to sedentary rats. We proposed that these changes lead to a more efficient fatty acid oxidation in the intestine and a consequent reduction of intestinal TAG secretion in this model. Overall, this work highlights that endurance exercise training programs intestinal lipid metabolism, contributing to the beneficial effect of endurance exercise on improving obesity and metabolic disease. </p><p> Nest, we investigated the differential roles of acyl CoA: diacylglycerol acyltransferase 1 (Dgat1) and Dgat2, in regulating dietary fat absorption. Mice with intestine-specific overexpression of Dgat1 (<i>Dgat1</i>^Int) or Dgat2 (<i>Dgat2</i>^Int), or lack of Dgat1 (<i>Dgat1</i>^{&ndash;/&ndash;}) were previously reported to have different intestinal phenotypes in response to fat consumption and altered susceptibilities to obesity and hepatic steatosis; the underlying mechanism(s) is unknown. By conducting an ultrastructural analysis on enterocytes from these Dgat mouse models in response to fat consumption, we found that Dgat1 and Dgat2 altered intracellular TAG distribution for CM and CLD synthesis. Based on the observations in the study, Dgat1 is proposed to preferentially synthesize TAG for the subcellular pool that promotes CM expansion in the ER lumen and thus limits TAG storage in CLDs. In addition, Dgat2 is proposed to preferentially synthesize TAG for the subcellular pool that determines the number of CMs generated in the ER lumen and for storage in CLDs. In this study, we provide the mechanism of how intestinal Dgat1 and Dgat2 exert regulatory effects on postprandial blood lipid levels and whole-body physiology. Overall, this work demonstrates non-redundant cellular roles of Dgat1 and Dgat2 in dietary fat absorption. </p><p> Lastly, we investigated the regulation of lipophagy, where CLDs are targeted by autophagy and catabolized in acidic lysosomes, in enterocytes of <i> Dgat1</i>^{&ndash;/&ndash;} mice. We found an increased number of autophagic vesicle (AV) and abnormal TAG accumulation within AVs in enterocytes of <i>Dgat1</i>^{&ndash;/&ndash;} compared to WT mice, suggesting an impaired AV turnover and an inefficient lipophagy by Dgat1 deficiency. In addition, we identified that this impaired lipophagy process was due to a lysosome dysfunction, as indicated by the decreased mRNA levels of genes involved in lysosome acidification and higher lysosome pH in enterocytes of <i>Dgat1</i>^{&ndash;/&ndash;} compared to WT mice. Furthermore, we found alterations in cellular lipid composition and levels of reactive oxygen species (ROS) in enterocytes of <i>Dgat1 </i>^{&ndash;/&ndash;} compared to WT mice. These changes may possibly contribute to the lysosome dysfunction seen in <i>Dgat1 </i>^{&ndash;/&ndash;} mice. Based on the results in the study, we propose that the lysosome dysfunction limits lipid supply from the storage pool for secretion, resulting in a greater intestinal TAG storage and a reduced rate of intestinal TAG secretion seen in <i>Dgat1</i>^{&ndash;/&ndash;} mice. Together, this study highlights that lysosome function plays a critical role in lipophagy and that lipophagy may serve as a potential target for treating postprandial hyperlipidemia and its related diseases. </p><p> The findings presented in this dissertation expand the current knowledge of regulation of dietary fat absorption. The proposed models generated from these studies provide novel therapeutic strategies for managing postprandial blood lipid levels and preventing obesity and its related diseases.</p><p>