The Rbfox proteins are a family of splicing regulators in post-mitotic neurons, predicted to be required for control of hundreds of alternative exons in neuronal development. However, their contribution to the cellular processes in developing and adult nervous system remains unclear and few candidate target exons were experimentally confirmed due to functional redundancy of the three Rbfox proteins. In this thesis, I combined CRISPR/Cas9 genome engineering with in vitro differentiation of embryonic stem cells into spinal motor neurons to unravel the Rbfox regulatory network and to study the functional importance of Rbfox-dependent splicing regulation for neuronal maturation. Global analysis revealed that neurons lacking Rbfox proteins exhibit developmentally immature splicing profile but little change in the gene expression profile. Integrative modeling based on splicing changes in Rbfox triple knockout (Rbfox tKO) neurons and HITS-CLIP Rbfox binding mapping identified 547 cassette exons directly regulated by Rbfox proteins in maturing neurons. Strikingly, many transcripts encoding structural and functional components of axon initial segment (AIS), nodes of Ranver (NoR) and synapses undergo Rbfox-dependent regulation. I focused on the AIS whose assembly, which occurs during the early stages of neuronal maturation, is poorly understood. I found that the AIS of Rbfox tKO neurons is perturbed and contains disorganized ankyrin G, as revealed by super-resolution microscopy. This is in part due to an aberrant splicing of ankyrin G, resulting in destabilization of its interaction with βII- and βIV-spectrin. Thus, Rbfox factors play a crucial role in regulating a neurodevelopmental splicing program underlying structural and functional maturation of post-mitotic neurons. These data highlight the importance of alternative splicing in neurodevelopment and provide a novel link between alternative splicing regulation and AIS establishment.

<p>It is widely known that orexin A and B peptides as well as their receptors are expressed in the hypothalamus and distributed throughout the central nervous system, but there have been few studies regarding its presences in other parts of the body. There is now evidence that orexin (ORX) and its receptors (ORXR1/2) are present in the avian liver; however, their regulation under different environmental conditions is still unknown. In the current study, we sought to determine the effects of heat and oxidative stress using hydrogen peroxide (H2O2) and 4-hydroxynonenal (4-HNE) on the hepatic expression of ORX and ORXR1/2 in the avian species. Overall, heat stress significantly down regulated the expression of ORX, and ORXR1/2 mRNA and pro1tein in quail liver and LMH cells. LMH cells treated with H2O2 had decreased ORX protein and increased ORX mRNA levels (P < 0.05). There was a biphasic effect of 4-HNE on the expression of ORX and ORXR1/2 in LMH cells. There was a significant upregulation at low doses (10 and 20 ?M) and significant down-regulation at a high dose (30?M) of 4-HNE. In light of the current data, the hepatic expression of orexin could serve as a molecular signature in the heat and oxidative stress response.

abstract: The RASopathies are a collection of developmental diseases caused by germline mutations in components of the RAS/MAPK signaling pathway and is one of the world’s most common set of genetic diseases. A majority of these mutations result in an upregulation of RAS/MAPK signaling and cause a variety of both physical and neurological symptoms. Neurodevelopmental symptoms of the RASopathies include cognitive and motor delays, learning and intellectual disabilities, and various behavioral problems. Recent noninvasive imaging studies have detected widespread abnormalities within white matter tracts in the brains of RASopathy patients. These abnormalities are believed to be indicative of underlying connectivity deficits and a possible source of the behavioral and cognitive deficits. To evaluate these long-range connectivity and behavioral issues in a cell-autonomous manner, MEK1 loss- and gain-of-function (LoF and GoF) mutations were induced solely in the cortical glutamatergic neurons using a Nex:Cre mouse model. Layer autonomous effects of the cortex were also tested in the GoF mouse using a layer 5 specific Rbp4:Cre mouse. Immunohistochemical analysis showed that activated ERK1/2 (P-ERK1/2) was expressed in high levels in the axonal compartments and reduced levels in the soma when compared to control mice. Axonal tract tracing using a lipophilic dye and an adeno-associated viral (AAV) tract tracing vector, identified significant corticospinal tract (CST) elongation deficits in the LoF and GoF Nex:Cre mouse and in the GoF Rbp4:Cre mouse. AAV tract tracing was further used to identify significant deficits in axonal innervation of the contralateral cortex, the dorsal striatum, and the hind brain of the Nex:Cre GoF mouse and the contralateral cortex and dorsal striatum of the Rbp4:Cre mouse. Behavioral testing of the Nex:Cre GoF mouse indicated deficits in motor learning acquisition while the Rbp4:Cre GoF mouse showed no failure to acquire motor skills as tested. Analysis of the expression levels of the immediate early gene ARC in Nex:Cre and Rbp4:Cre mice showed a specific reduction in a cell- and layer-autonomous manner. These findings suggest that hyperactivation of the RAS/MAPK pathway in cortical glutamatergic neurons, induces changes to the expression patterns of P-ERK1/2, disrupts axonal elongation and innervation patterns, and disrupts motor learning abilities. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2018

No description available.

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.