Plants produce a vast array of secondary metabolites. A group of phenolic compounds, the flavonoids, are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have been found to possess anti-inflammatory, anti-cancer, and antioxidant properties. Flavonoid’s ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases (GT). Citrus paradisi contains a glucosyltransferase that is specific for adding glucose to the 3-O position of flavonols (Cp-F3-O-GT). To further understand the reactions it catalyzes, Cp-F3-O-GT structure was modeled against an anthocyanidin/flavonol 3-O-GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then generated using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline. Biochemical characterization of the mutant D344P protein was performed in order to determine whether the mutation has an effect on the substrate specificity of Cp3-O-GT. An initial quick-screening assay using radioactive UDP-glucose as a sugar donor suggested there may have been an expansion of substrate acceptance. The time course assays did not support observation. Additionally, results show that D344P protein has decreased activity with flavonols as compared to the wild-type Cp3-O-GT. with no expansion of substrate specificity. Homology models supported experimental results.

No description available.

No description available.

Nosocomial lung infections are a growing concern in the United States, with more than 300,000 cases reported annually. More than 30 % of which are caused by the Gram-negative bacteria, Pseudomonas aeruginosa and Klebsiella pneumoniae. Similarly, Gram-negative bacteria establish chronic infections in individuals with cystic fibrosis (CF) that are difficult or impossible to eradicate.P. aeruginosa has historically been one of the most prevalent pathogens of adults with CF. However, as antipseudomonal therapy has improved, more antibiotic resistant species have taken hold, including Stenotrophomonas maltophilia, which now colonizes more than 10 % of individuals with CF. Regardless of the cause or source, Gram-negative respiratory infections are becoming increasingly difficult to treat due to the rising incidence of multiple drug resistance among these organisms. To aid in the development of new therapeutics, a greater understanding of how these organisms transition from the environment to the host lung is needed. Here we utilized a combination of transcriptomics and molecular genetics to examine how P. aeruginosa, K. pneumoniae, and S. maltophilia, recognize and exploit the host lung milieu during the initiation of infection. One of the first components of the host lung environment that aspirated bacteria are exposed to is pulmonary surfactant (PS). This phospholipid-rich substance coats the distal airways of the lung and is thought to contain molecular cues that facilitate lung colonization by pathogenic bacteria. Here, we characterized the transcriptional response of K. pneumoniae to purified PS to examine how this organism interreacts with the host lung during colonization. This work revealed numerous virulence and colonization-related genes that are expressed by K. pneumoniae under these conditions. We also tested the contribution of other surfactant-induced transcripts to K. pneumoniae pathogenesis using engineered gene deletion strains and a mouse model of pneumonia. This work revealed the polyamine efflux pump, MdtJI, and glycine betaine transporter, ProU are required for K. pneumoniae virulence. Phosphatidylcholine is the primary constituent of PS. P. aeruginosa is capable of completely metabolizing the phosphocholine head group of this lipid, and readily does so when exposed to PS. We previously observed that the most highly expressed genes in P. aeruginosa in response to PS were those involved in the catabolism of a downstream choline metabolite, sarcosine. Although our group had previously characterized the choline catabolic pathway of P. aeruginosa, the transcriptional regulation of sarcosine catabolism was not known. We utilized a genetic screen to identify the regulator controlling the expression of the sarcosine catabolic genes in P. aeruginosa. This regulator, which we named SouR (Sarcosine oxidase utilization Regulator) is the first sarcosine-responsive regulator to be characterized. The thick, viscous mucus (sputum) that accumulates within the CF lung serves as the primary nutrient source for microbes colonizing the CF lung. Here, we characterized the transcriptional responses of three S. maltophilia strains during growth in synthetic CF sputum media (SCFM2) to gain insight into how this organism interreacts with the host lung. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates with the SCFM2 transcriptome of the acute infection model strain, K279A. This revealed CF isolate-specific signatures in gene expression that reflect adaptation to the CF lung.

No description available.

My research is designed to test the hypothesis that a single nucleotide polymorphism (SNPs) in the non-coding region of the GCH1 gene suppresses GCH1 mRNA translation and generates a novel peptide, and that both of these phenomena contribute to downregulation of the dopamine synthesis pathways. The GCH1 gene codes for the protein guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1), a key regulator of dopamine synthesis. I focused on a SNP resulting from substitution of a cytosine (C) with thymine (T) at location +142 (position from the transcription start site) in the 5’ untranslated region (UTR) of the GCH1 gene (+142C>T SNP), because it is associated with a heritable movement disorder and subtype of dystonia called DYT5. Thus, my research is designed to investigate molecular mechanisms associated with the pathogenesis of DYT5. To test my hypothesis, I used an in vitro model in which human embryonic kidney (HEK) 293T cells were transfected with cDNA for wild type GCH1, GCH1 containing the +142C>T SNP sequence, or control sequences. Firefly luciferase (Fluc) reporter assays showed that the +142C>T SNP introduces an upstream open reading frame (uORF), which suppresses translation of GCH1. The reduced GCH1 translation fits well with the reduction in GCH1 protein and dopamine reported in DYT5. The uORF introduced by the +142C>T SNP is predicted to synthesize a 73 amino acid novel peptide, which I have named DRDp73. As a first step toward understanding the role of DRDp73 cellular function, I performed a bioinformatics analysis. This analysis predicted existence of a nuclear localization signal, a large “disordered” region and a nucleic acid binding alpha-helical domain in DRDp73 revealing interesting clues to DRDp73’s potential biological function. To test predictions provided by the bioinformatics analysis, I characterized the expression, subcellular localization and the impact of DRDp73 on cell viability. I used a custom-made DRDp73 antibody, and a commercially available GCH1 antibody to detect both of the proteins. I detected DRDp73 and GCH1 in HEK293T cells overexpressing the +142T GCH1 (GCH1 in DYT5) plasmid. However, GCH1 expression in these cells was significantly lower than that in HEK293T cells transfected with wildtype GCH1 plasmid. Since HEK293T cells divide rapidly, I predicted that DRDp73 is degraded and cleared during cellular division. Therefore, in additional studies, I inhibited the proteasome and autophagy pathways, key regulators of protein degradation. Under these circumstances, DRDp73 was localized to the nucleus, consistent with the predicted nuclear localization signal revealed by bioinformatics, and GCH1 was localized to the cytoplasm. In addition, viability of the HEK293T cells expressing DRDp73 was compromised. These findings confirmed that the +142C>T SNP is associated with generation of DRDp73, downregulation of GCH1 expression, and provided evidence that DRDp73 accumulation may adversely affect cell viability. The HEK293T cells do not synthesize dopamine, the neurotransmitter involved in motor function. Moreover, since the HEK293T cells divide rapidly, analysis of the long term effects of protein accumulation becomes difficult. Therefore, I used human origin SK-N-BE(2)-M17 (a twice sub-cloned cell line from the SK-N-BE (2) neuroblastoma cell line) and SH-SY5Y cells (third sub-clone of SK-N-SH neuroblastoma cell line). Both cell lines differentiate into non-dividing cells, express GCH1 and neuronal markers upon differentiation, and are excellent models of dopaminergic cells, such as the midbrain dopaminergic neurons affected in DYT5 dystonia. My data from SK-N-BE(2)-M17 and SH-SY5Y cells show that endogenous GCH1 protein is localized to the nucleus, cytoplasm and processes. In both types of cells transfected with the +142T GCH1 plasmid, DRDp73 also localizes to the nucleus, cytoplasm and processes. GCH1 expression is reduced in the nucleus in both neuroblastoma cells expressing DRDp73, and is found predominantly in the cytoplasm and processes. The dopamine biosynthetic pathway is compromised in DRDp73 expressing SK-N-BE(2)-M17 cells. Thus, the +142C>T SNP downregulated GCH1 expression and compromised dopamine biosynthesis in the neuronal-like cells. In the final set of studies, I analyzed GCH1 expression in lymphoblastoid cells from DYT5 patients. GCH1 expression was reduced in the patient-derived cells compared to that in cells from non-DYT5 individuals. In summary, my work identified dual consequences of DYT5-associated +142C>T SNP in the non-coding region of the GCH1 gene: Suppression of GCH1 translation, and production of the novel DRDp73 peptide. DRDp73 is a novel biomarker for DYT5, and the DRDp73 antibody is a potential novel diagnostic tool. My results expand current understanding of the SNP-uORF encoded proteins, and advances the field of SNP-uORF associated diseases by offering novel mechanistic and functional insights into SNP-uORF encoded peptides. My work also identifies future studies, for example using DYT5 patient derived cells, primary neuronal cultures and animal models that could significantly advance this emerging biomedical field. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the Doctor of Philosophy. / Summer Semester 2017. / July 19, 2017. / dopamine, dystonia, gene, regulation, SNP, uORF / Includes bibliographical references. / Pradeep G. Bhide, Professor Directing Dissertation; Debra Ann Fadool, University Representative; Richard S. Nowakowski, Committee Member; Heather Flynn, Committee Member; Akash Gunjan, Committee Member.

The modulation of biological interactions with artificial surfaces is a vital aspect of biomaterials research. Protein adsorption is established as an early biological response to implanted materials that influences biocompatibility, hence an understanding of how to direct specific protein and cellular responses is critical for the development of future biomaterials. The effects of protein adsorption and subsequent cellular interactions on a variety of surfaces are investigated. Acrylic-based hydrogels are used as a model system in which to investigate both tear and serum protein adsorption from simple and complex solutions. The effect of surface topography, created by colloidal silica, on serum protein adsorption and conformation as well as cell adhesion is also investigated. Tantalum (Ta) and oxidised polystyrene (PSox) are investigated for their ability to support cell adhesion when precoated with various serum proteins. Protein interactions are examined using a combination of quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), dual polarisation interferometry (DPI) and enzyme-linked immunosorbent assay (ELISA) while cellular interactions are analysed using QCM-D, microscopy and adhesion assays. The QCM-D technique was evaluated for its ability to provide new insight into cell-surface interactions. Most tear and serum proteins were found to adsorb onto the acrylic hydrogels, however, lysozyme was found to absorb into the hydrogel matrix and decrease the hydration, which may lead to an adverse biological response. Fibronectin adsorbed onto nanotextured colloidal silica surfaces was found to be conformationally changed compared to flat controls which is likely to correlate with the reduced endothelial cell adhesion observed on these textured surfaces. Ta and PSox precoated with either serum or fibronectin were shown to support cell adhesion and spreading, while surfaces precoated with albumin were not. QCM-D responses varied between underlying surfaces, protein precoating, ECM deposition, cytoskeletal activity and length of exposure indicating that alterations in cell-material responses are reflected in QCM-D measurements. QCM-D parameters were found to correlate with adhered cell numbers, cell contact area and cytoskeletal activity. The results highlight that characterisation of interfacial interactions with a wide range of analytical techniques is necessary to gain insight into cell-protein-material interactions which can then be utilised in the development of new generations of biomaterials with improved properties designed for specific applications.

Treatments for the hepatitis C virus (HCV) are currently only partially effective. Research into antivirals directed at HCV viral proteins are commonly based and tested on a single genotype, namely genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach for the isolation of antiviral agents. SELEX allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNAdependent RNA polymerase (RdRp) [non-structural protein B (NS5B)] protein of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 and resultant aptamers cloned from rounds 2, 4, 8 and 10. Sequences of aptamers were aligned to elucidate common motifs and a proportion of the aptamers from rounds 8 and 10 (29/48) were screened for binding ability using the Biacore. The five ???best binding??? aptamers were investigated for inhibition of 3a polymerase activity in an in vitro polymerase assay. Two aptamers, r10/43 and r10/47, were chosen for further studies based on their ability to inhibit polymerase activity. The inhibition constants (Ki) of r10/43 and r10/47 were estimated to be 1.4 + 2.4 nM and 6.0 + 2.3 nM respectively. The affinity (Kd) of these aptamers for the 3a polymerase was estimated to be 1.3 + 0.3 nM (r10/43) and 23.5 + 6.7 nM (r10/47). The estimated inhibition and dissociation constants of these two aptamers are among the best for inhibitory aptamers of the HCV enzymes (polymerase and protease). Inhibition of HCV 3a polymerase appeared to be specific for r10/47, whilst r10/43 also had some inhibitory effect on norovirus and ??6 polymerase activity. This study is the first description of an inhibitor to the HCV subtype 3a polymerase that investigates genotypic specificity of targeted antivirals.

<p>The number of protein-coding genes are likely not sufficient to account for the complexity of higher organisms. It is plausible that the proteome is responsible for the complexity of an organism.</p><p>An important mechanism that increases the protein variability is post-transcriptional modifications that alter the pre-mRNA sequence from that encoded in the genome. In this thesis work I have been focusing on a post-transcriptional process where adenosine (A) is deaminated to inosine (I), A-to-I RNA editing. Inosine is read as a guanosine (G) by the translation machinery, editing within coding regions can therefore give rise to more than one protein isoform from a single gene. A-to-I RNA editing is catalyzed by members of the ADAR enzyme family. ADARs have been found in all metazoans tested and two active ADAR proteins, ADAR1 and ADAR2, have been found in mammals. However, recoding by A-to-I editing is a rarely found event in mammals.</p><p>To detect novel substrates for A-to-I editing we developed an experimental approach to pull down ADAR2 substrates using immunoprecipitations. The captured RNAs were identified by microarray analysis. In this thesis two novel substrates for A-to-I editing are presented that were found using our IP-array approach, in combination with bioinformatic techniques.</p><p>The transcript coding for the GABA_Areceptor subunit α3 (Gabra-3) was found to be selectively edited by both ADAR1 and ADAR2. Editing of Gabra-3 recodes an isoleucine to a methionine and it was found to have a negative effect on the Gabra-3 assembly into the receptor. Moreover, the mouse specific CTN-RNA that codes for the CAT2 Transcribed Nuclear-RNA was shown to be hyper-edited by ADAR2.</p><p>In conclusion, this thesis work has resulted in an experimental method that extracts ADAR substrates. Two novel editing substrates were discovered. Our data adds additional evidence to the fact that RNA editing is of principal significance for a functional brain.</p>

<p>Progression through the G1, S, G2 and M phases of the cell cycle is controlled by cyclin-dependent kinases (Cdks) and cyclins. These proteins form active Cdk:cyclin complexes that phosphorylate specific substrates. The Cdk:cyclin complexes of the G1/S transition regulate the progression of cells into the S phase by phosphorylating the retinoblastoma protein (Rb). This prevents Rb from sequestering E2F, a transcription factor that induces expression of genes required for DNA synthesis. This process is in part regulated by a family of Cdk inhibitors (CKIs) called the Ink4 family (<u>In</u>hibitors of Cd<u>k4</u>). The Ink4 family of CKIs consists of four members; p16^Ink4a, p15^Ink4b, p18^Ink4c and p19^Ink4d, and they bind specifically to Cdk4 and Cdk6, thereby negatively regulating their kinase activities and cell cycle progression. Because of its cell cycle inhibitory role, p16^Ink4a is frequently mutated or deleted in human cancer, whereas the other <i>Ink4</i> genes are only occasionally altered in cancer. The overall aim of this thesis was to study the roles of p18^Ink4c and p19^Ink4d using <i>in vivo</i> models of cancer and embryonic development. In paper I, we analyzed the tumor spectrum in mice lacking <i>p53</i>, <i>Ink4c</i> and <i>Ink4d</i>. p53 is a tumor suppressor and one of the most frequently mutated genes in human cancer. Mice carrying mutated <i>p53</i> alleles are highly tumor-prone but develop predominantly lymphomas. However, the combined loss of <i>p53</i> and <i>Ink4c</i> (but not <i>Ink4d</i>) caused a shift in the tumor spectrum to increased incidences of hemangiomas and hemangiosarcomas, as well as appearance of medulloblastomas, a tumor of the cerebellum. These data, revealed in the absence of p53, suggest a cell-type specific tumor suppressing role for p18^Ink4c. In paper II, loss of <i>Ink4c</i> was evaluated in another tumor-prone mouse model; the Eµ-<i>Myc</i> mouse. This is a transgenic mouse overexpressing c-Myc in B cells causing clonal B cell lymphomas. Surprisingly, precancerous B cells and lymphomas from Eµ-<i>Myc</i> mice exhibited elevated levels of p18^Ink4c mRNA and protein despite high rates of proliferation. Moreover, loss of <i>Ink4c</i> in this model did not affect the rate of cell proliferation or the onset of tumor development. We conclude from these studies that <i>Ink4c</i> is not an important tumor suppressor of Myc-induced lymphomas. To gain insight into the role of <i>Ink4</i> genes in early vertebrate development, the African clawed frog, <i>Xenopus laevis</i>, was analyzed for the presence of <i>Ink4</i> homologs. Paper III describes the cloning and characterization of a gene homologous to <i>Ink4d</i>, <i>Xl-Ink4d</i>. This CKI is expressed throughout frog embryo development, making <i>Xl</i>-Ink4d the only CKI present during the cleavage stages of <i>X. laevis</i>. Antisense morpholino oligonucleotides directed against <i>Xl-Ink4d</i> were used to knock down the protein level of <i>Xl</i>-Ink4d during development. This resulted in defects in head tissues and reduced expression of <i>Twist</i>, a gene important for neural crest cell migration. We therefore propose that <i>Xl</i>-Ink4d is important for proper neural crest differentiation in the frog.</p>