<p> The major barrier to curing HIV-1 infection is latency. HIV-1 latent cells are those in which the viral genome has been integrated into the host cell genome but the virus does not produce the primary infectious agents, viral RNA and proteins. Latency can occur when the virus directly infects long-lived memory CD4+ T cells or infects active CD4+ T cells that have the potential to become memory T cells. The virus persists inside those cells as long as they are alive. This dormancy provides a reservoir of HIV-1 virus in memory T cells, which can cause infection relapse whenever antiretroviral therapy (ART) is discontinued. The situation is further complicated by the fact that multiple reservoirs of HIV-1 virus can be established at early stages of infection. </p><p> The major reservoir lies in the CD4+ T cells present in blood, lymph nodes and the spleen. Unfortunately, ART fails to target hidden HIV-1 virus that persists in resting T-cells. Furthermore, life-long ART use increases the chances that mutant virus will develop which will be resistant to continued therapy. Therefore, various studies have explored mechanisms to eradicate the latent HIV-1 reservoir. One proposed strategy to target this reservoir is known as &ldquo;shock and kill&rdquo;. The proposed shock and kill strategy initially &ldquo;shocks&rdquo; the HIV-1 virus out of latency with latency reversing agents (LRAs). The reactivated virus can then be controlled by ART and cytotoxic CD8+ T cells (CTLs), which kill the infected cells. Despite the great findings regarding reactivating HIV-1 latency <i>in vitro</i> and <i>ex vivo</i>, tested LRAs proved unsuccessful in reactivating HIV-1 virus in clinical trials. </p><p> Different factors can contribute to establishment of HIV-1 latency and different reservoirs in different immune cells and tissues are established early after HIV-1 infection. Therefore, synergy between multiple LRAs should be sought and studied for successful reactivation of the latent viral pool. </p><p> Runt Related Transcription Factor 1 (RUNX1) is a key transcription factor that is important during T cell development and has been shown to recruit different transcription factors in a context dependent manner. It has been shown to be involved in repressing various genes and it also interacts with chromatin modifiers that can alter the landscape of the chromatin and modify its compaction. Our lab has shown that there is a putative RUNX1 binding site on HIV-1 long terminal repeats (LTR) and the transfection of RUNX1 can suppress HIV-1 transcription. In addition, our lab has shown that the benzodiazepine, RO5-3335, which pharmacologically inhibits RUNX1, synergizes with vorinostat (SAHA), an HDAC inhibitor to reactivate latent HIV-1. </p><p> Using DNA cloning, an HIV-1 virus with a mutated RUNX1 binding site was constructed. Then, replication, infectivity and fitness of the mutated virus were examined and compared to a control virus using ELISA, RT, PCR, and TA cloning techniques. We have found that this mutated virus replicates faster and has more fitness and infectivity than the control virus with an intact RUNX1 binding site. Our results show that inhibition of RUNX1 binding to HIV-1 3&rsquo; long terminal repeat (LTR) positively affects viral replication and infectivity. This suggests that RUNX1 host transcription factor suppresses HIV-1 replication through its transcriptional repressor function and it possibly contributes to establishment of latency. </p><p> We screened clinically prescribed benzodiazepines (BDZs) to identify reactivators for latent HIV-1 virus. Using flow cytometry, we have found most of these BDZs synergized with SAHA in reactivation of latent HIV-1. Unlike the other BDZs tested, alprazolam was able to reactivate HIV-1 even when not in combination with SAHA. The effect of alprazolam on RUNX1 responsive genes was further investigated using qPCR. Alprazolam was found to affect RUNX1 responsive genes similarly to RO5-3335, a known RUNX1 inhibitor. The effect of alprazolam on IFN&gamma; and TNF&alpha; that are produced from cytotoxic T cells (CTLs) was also examined. Alprazolam enhanced CTL function that was shown in the literature to be attenuated by SAHA. Thus, alprazolam successfully reverses HIV-1 latency and decreases the side effects of SAHA on CTL function when used in combination. </p><p>

Chu Wai-ling. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 106-114). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.v / Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.8 / Chapter 2.1 --- Introduction to phylogenetic biology --- p.8 / Chapter 2.1.1 --- Definition of phylogenetics --- p.8 / Chapter 2.1.2 --- Why employ molecular genetic markers in phylogenetics? --- p.8 / Chapter 2.2 --- DNA analysis and the contributions to phylogenetics --- p.10 / Chapter 2.2.1 --- Historical development of DNA analysis in phylogenetics --- p.10 / Chapter 2.2.2 --- Nuclear ribosomal DNA (rDNA) --- p.12 / Chapter 2.2.3 --- Animal mitochondrial DNA (mt DNA) --- p.14 / Chapter 2.3 --- Molecular phylogeny of crustaceans --- p.16 / Chapter 2.3.1 --- Phylogenetic studies of crustaceans using nuclear ribosomal DNA --- p.16 / Chapter 2.3.2 --- Phylogenetic studies of crustaceans using mitochondrial DNA --- p.17 / Chapter 2.4 --- Taxonomy of the genus Heterocarpus --- p.22 / Chapter Chapter 3 --- Materials and Methods --- p.36 / Chapter 3.1 --- Collection and storage of specimens --- p.36 / Chapter 3.2 --- DNA extraction --- p.36 / Chapter 3.3 --- Amplification of mitochondrial genes --- p.38 / Chapter 3.3.1 --- PCR profile --- p.39 / Chapter 3.3.1.1 --- 16SrRNA gene --- p.39 / Chapter 3.3.1.2 --- COI gene --- p.42 / Chapter 3.3.1.2.1 --- Amplification of COI gene segments using primers LCD1490/HCO2198 --- p.42 / Chapter 3.3.1.2.2 --- Amplification of COI gene segments using primers COIf/COIa and COIp3/COIa --- p.43 / Chapter 3.4 --- DNA sequencing --- p.44 / Chapter 3.4.1 --- Purification of extension products --- p.45 / Chapter 3.4.2 --- Electrophoresis and data collection --- p.46 / Chapter 3.5 --- Data analysis --- p.47 / Chapter Chapter 4 --- Results --- p.50 / Chapter 4.1 --- PCR products of 16S rRNA and COI genes --- p.50 / Chapter 4.2 --- Genetic variability in Heterocarpus based on partial DNA sequence of 16S rRNA gene --- p.52 / Chapter 4.3 --- Genetic variability in Heterocarpus based on COI gene --- p.61 / Chapter 4.3.1 --- Genetic variability in Heterocarpus based on partial DNA sequence of COI gene --- p.61 / Chapter 4.3.2 --- Genetic variability in Heterocarpus based on amino acid sequence of COI --- p.69 / Chapter 4.4 --- Phylogenetic analysis --- p.75 / Chapter 4.4.1 --- Phylogenetic analysis based on 16S rDNA sequence --- p.75 / Chapter 4.4.2 --- Phylogenetic analysis based on DNA sequence of COI gene --- p.80 / Chapter 4.4.3 --- Phylogenetic analysis based on amino acid sequence of COI --- p.84 / Chapter 4.5 --- Kishino-Hasegawa and Shimodaira-Hasegawa tests --- p.86 / Chapter Chapter 5 --- Discussion --- p.90 / Chapter 5.1 --- Examination on the validity of the four Heterocarpus complexes --- p.90 / Chapter 5.2 --- Phylogenetic relationship of Heterocarpus species within each complex --- p.91 / Chapter 5.2.1 --- Phylogenetic relationship of Heterocarpus species within H.gibbosus complex --- p.92 / Chapter 5.2.2 --- Phylogenetic relationship of Heterocarpus species within H.woodmasoni complex --- p.94 / Chapter 5.2.3 --- Phylogenetic relationship of Heterocarpus species within H. ensifer and H. sibogae complexes --- p.96 / Chapter 5.3 --- Phylogenetic relationship among Heterocarpus complexes --- p.98 / Chapter 5.4 --- "Comparisons of phylogenetic resolving power of 16S rRNA, COI and 28S rRNA genes" --- p.100 / Chapter Chapter 6 --- Conclusions --- p.104 / Literature Cited --- p.106

Cheng Tat Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 106-114). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of content --- p.iii / Abbreviations --- p.iv / List of figures --- p.v / List of tables --- p.vi / Chapter Chapter One --- Introduction / Chapter 1.1 --- Ribozyme --- p.1 / Chapter 1.1.1 --- RNA world hypothesis --- p.2 / Chapter 1.1.2 --- Hammerhead ribozyme --- p.3 / Chapter 1.1.3 --- Applications of hammerhead ribozymes --- p.4 / Chapter 1.1.4 --- Allosteric ribozyme --- p.4 / Chapter 1.1.5 --- Ribozyme screening system --- p.5 / Chapter 1.2 --- Other RNA as gene silencing agents --- p.7 / Chapter 1.2.1 --- RNAi --- p.7 / Chapter 1.2.2 --- Antisense RNA --- p.10 / Chapter 1.3 --- Project Overview --- p.11 / Chapter 1.3.1 --- Construction of anti-GFP ribozymes and their in vitro and in vivo studies --- p.11 / Chapter 1.3.2 --- Construction of anti-Elfin ribozyme and its application on gene silencing study --- p.11 / Chapter Chapter Two --- Materials and Methods / Chapter 2.1 --- Cloning techniques --- p.13 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) --- p.13 / Chapter 2.1.2 --- Restriction digestion of DNA --- p.13 / Chapter 2.1.3 --- Ligation of DNA fragments --- p.14 / Chapter 2.1.4 --- Preparation of competent cells --- p.15 / Chapter 2.1.5 --- Transformation of competent cells --- p.16 / Chapter 2.1.6 --- Gel extraction --- p.16 / Chapter 2.1.7 --- Plasmid preparation --- p.17 / Chapter 2.1.7.1 --- Mini scale plasmid preparation --- p.17 / Chapter 2.1.7.2 --- Medium scale plasmid preparation --- p.19 / Chapter 2.1.8 --- DNA agarose gel electrophoresis --- p.20 / Chapter 2.1.9 --- Buffer and reagents --- p.21 / Chapter 2.2 --- In vitro cleavage of target RNA by ribozymes --- p.22 / Chapter 2.2.1 --- In vitro transcription of target RNA and ribozymes --- p.22 / Chapter 2.2.2 --- Purification of transcription products --- p.22 / Chapter 2.2.3 --- Ribozymatic cleavage reaction --- p.24 / Chapter 2.2.4 --- Preparation of RNA size marker templates --- p.24 / Chapter 2.2.5 --- Urea-acrylamide gel electrophoresis --- p.25 / Chapter 2.2.6 --- Autoradiography --- p.26 / Chapter 2.2.7 --- Buffer and reagents --- p.26 / Chapter 2.3 --- Detection of cellular RNA expression RT-PCR detection --- p.28 / Chapter 2.3.1 --- RNA extraction --- p.28 / Chapter 2.3.2 --- DNase I digestion --- p.29 / Chapter 2.3.3 --- Reverse transcription --- p.29 / Chapter 2.4 --- Mammalian cell culture techniques --- p.31 / Chapter 2.4.1 --- Transfection into mammalian cells --- p.31 / Chapter 2.4.2 --- Counting the number of cells --- p.32 / Chapter 2.4.2 --- Buffer and reagents --- p.32 / Chapter Chapter Three --- Construction of anti-GFP ribozymes and their in vitro and in vivo studies / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.1.1 --- Objectives --- p.34 / Chapter 3.1.2 --- Why anti-GFP ribozymes? --- p.34 / Chapter 3.2 --- Construction of anti-GFP ribozymes that are active in vitro --- p.36 / Chapter 3.2.1 --- Design of the anti-GFP ribozymes --- p.36 / Chapter 3.2.2 --- Construction of DNA templates for in vitro transcription --- p.40 / Chapter 3.2.3 --- GFP RNA was successfully cleaved by ribozymes --- p.45 / Chapter 3.3 --- In vivo activities of anti-GFP ribozymes --- p.48 / Chapter 3.3.1 --- Design of systems that detected anti-GFP ribozyme activities in --- p.48 / Chapter 3.4 --- The first trial - system α --- p.50 / Chapter 3.4.1 --- Cloning of ribozymes into pACYC184 --- p.51 / Chapter 3.4.2 --- IPTG interfered with GFP expression --- p.54 / Chapter 3.5 --- The second trial - system β --- p.57 / Chapter 3.5.1 --- Insertion of new multiple cloning sites into pET3d --- p.58 / Chapter 3.5.2 --- Cloning of GFP into pET-neu --- p.60 / Chapter 3.5.3 --- GFP expression was interfered by the additional T7 promoter --- p.62 / Chapter 3.6 --- The third trial - system δ --- p.65 / Chapter 3.6.1 --- Insertion of a new cloning site into pET-neu --- p.66 / Chapter 3.6.2 --- Cloning of GFP and ribozymes into pET-fn --- p.68 / Chapter 3.6.3 --- Ribozymes sequence upstream of GFP interfered with GFP expression / Chapter 3.7 --- The fourth trial - system co --- p.73 / Chapter 3.7.1 --- Insertion of new cloning sites into pET-neu --- p.74 / Chapter 3.7.2 --- Cloning of GFP and ribozymes into pET-nr --- p.76 / Chapter 3.7.3 --- Anti-GFP ribozymes did not turn off green fluorescence of GFP --- p.79 / Chapter 3.7.4 --- No in vivo cleavage of GFP was detected --- p.81 / Chapter 3.8 --- Summary --- p.83 / Chapter Chapter Four --- Construction of an anti-Elfin ribozyme and its application on gene silencing study / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.1.1 --- Objectives --- p.84 / Chapter 4.1.2 --- Elfin --- p.84 / Chapter 4.1.3 --- Experimental plan --- p.85 / Chapter 4.2 --- In vitro cleavage of Elfin RNA by ribozyme --- p.86 / Chapter 4.2.1 --- Design of anti-Elfin ribozyme --- p.86 / Chapter 4.2.2 --- Preparation of DNA template for in vitro transcription --- p.88 / Chapter 4.2.3 --- Successful in vitro cleavage of Elfin RNA by ribozyme --- p.88 / Chapter 4.3 --- In vivo gene silencing studies of RNA tools --- p.90 / Chapter 4.3.1 --- Design of antisense RNA --- p.90 / Chapter 4.3.2 --- Design of siRNA --- p.92 / Chapter 4.3.3 --- Cloning of RNA tools into pSilencer --- p.94 / Chapter 4.3.4 --- Cloning of a neomycin resistance gene into pSilencer-R --- p.94 / Chapter 4.3.5 --- Elfin RNA was not down-regulated --- p.97 / Chapter 4.4 --- Summary --- p.100 / Chapter Chapter 5 --- Discussions --- p.101 / Reference / Appendix

Lo Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 108-115). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.vi / Abstract (in Chinese) --- p.viii / Table of content --- p.x / List of tables --- p.xv / List of figures --- p.xvi / List of abbreviations --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The secretory pathway --- p.2 / Chapter 1.1.1 --- Endoplasmic reticulum --- p.2 / Chapter 1.1.2 --- Golgi complex --- p.3 / Chapter 1.1.3 --- Vacuoles --- p.3 / Chapter 1.1.4 --- Prevacuolar compartment --- p.4 / Chapter 1.2 --- The secretory pathway in plant cells --- p.5 / Chapter 1.2.1 --- The secretory pathway in yeast and mammalian cells --- p.7 / Chapter 1.2.2 --- The lytic pathway in plant cells --- p.8 / Chapter 1.2.3 --- The protein storage vacuole pathway in plant cells --- p.10 / Chapter 1.3 --- Dynamic studies between organelles --- p.12 / Chapter 1.4 --- Objectives of this thesis research --- p.13 / Chapter Chapter 2 --- Development of Transgenic Cell Lines Expressing PVC and Golgi Markers --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- Putative PVC marker --- p.16 / Chapter 2.1.2 --- Golgi marker --- p.17 / Chapter 2.1.3 --- Dynamic studies --- p.18 / Chapter 2.1.4 --- Cell culture study --- p.18 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Plant material --- p.21 / Chapter 2.2.2 --- Construction of fusion reporters --- p.22 / Chapter 2.2.2.1 --- Cloning materials --- p.22 / Chapter 2.2.2.2 --- Vector preparation --- p.22 / Chapter 2.2.2.3 --- Cloning of pGFP-BP-80K and pGFP-BP-80H --- p.24 / Chapter 2.2.2.4 --- Cloning of pGFP-α-TIPH --- p.28 / Chapter 2.2.3 --- Transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.3.1 --- Agrobacterium transformation --- p.30 / Chapter 2.2.3.2 --- BY-2 cell transformation --- p.30 / Chapter 2.2.4 --- Screening of transgenic BY-2 cells --- p.31 / Chapter 2.2.4.1 --- Killing curve study --- p.31 / Chapter 2.2.4.2 --- Antibiotic selection --- p.32 / Chapter 2.2.4.3 --- Fluorescence microscopy screening (For single-construct cell lines) --- p.33 / Chapter 2.2.4.4 --- Confocal laser scanning microscopy (CLSM) screening (For double-construct cell lines) --- p.33 / Chapter 2.2.5 --- Detection of fluorescent protein expression --- p.35 / Chapter 2.2.5.1 --- Confocal imaging --- p.35 / Chapter 2.2.5.2 --- Protein extraction and subcellular fractionation --- p.36 / Chapter 2.2.5.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.36 / Chapter 2.2.5.4 --- Western blot analysis --- p.37 / Chapter 2.2.5.5 --- Cell culture study --- p.37 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Hygromycin concentration at 50 mg/L was optimal for selection --- p.39 / Chapter 2.3.2 --- Lower transformation efficiency for double-construct cell lines --- p.40 / Chapter 2.3.3 --- Screening of transgenic cell lines --- p.41 / Chapter 2.3.4 --- Both pGFP-BP-80K and pGFP- a -TIPH expressed as punctate signals in single-construct cell lines --- p.45 / Chapter 2.3.5 --- Weak punctate or diffuse signals were detected from PVC markers in double-construct cell lines --- p.47 / Chapter 2.3.6 --- GFP reporters were successfully transformed into BY-2 cells --- p.51 / Chapter 2.3.7 --- Profiles of fluorescent signals in transgenic cells during cell culture --- p.53 / Chapter 2.4 --- Discussion --- p.59 / Chapter 2.4.1 --- Abnormal cell growth might be due to high selection pressure --- p.59 / Chapter 2.4.2 --- Double-construct cell lines developed were not yet suitable for further study --- p.60 / Chapter 2.4.3 --- Single-construct cell lines expressing putative PVC markers were developed --- p.62 / Chapter 2.4.4 --- 2- to 3-day-old cells were more suitable for subsequent studies --- p.63 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Expressing Reporters for Distinct Prevacuolar Compartments --- p.66 / Chapter 3.1 --- Introduction --- p.67 / Chapter 3.1.1 --- Wortmannin --- p.69 / Chapter 3.1.2 --- Brefeldin A --- p.70 / Chapter 3.1.3 --- FM4-64 --- p.71 / Chapter 3.2 --- Materials and Methods --- p.73 / Chapter 3.2.1 --- Plant material --- p.73 / Chapter 3.2.2 --- Confocal immunofluorescence studies --- p.73 / Chapter 3.2.3 --- Drug treatment studies --- p.74 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.74 / Chapter 3.2.3.2 --- BFA treatment --- p.75 / Chapter 3.2.4 --- FM4-64 uptake study --- p.76 / Chapter 3.3 --- Results --- p.78 / Chapter 3.3.1 --- Organelles marked by GFP- a -TIP CT reporters did not localize at Golgi compartment --- p.78 / Chapter 3.3.2 --- Wortmannin induced GFP- a -TIP marked organelles to vacuolated --- p.81 / Chapter 3.3.3 --- GFP- a -TIP CT reporters partially colocalized with VSRin wortmannin-treated cells --- p.83 / Chapter 3.3.4 --- BFA induced GFP- a -TIP marked organelles to form BFA- induced compartments --- p.88 / Chapter 3.3.5 --- GFP-α -TIP CT reporter colocalized with internalized FM4-64 --- p.91 / Chapter 3.4 --- Discussion --- p.94 / Chapter 3.4.1 --- GFP- α -TIP CT reporter was a putative PVC marker --- p.94 / Chapter 3.4.2 --- GFP- a -TIP marked organelles behaved differently from lytic PVCs --- p.95 / Chapter 3.4.3 --- GFP- a -TIP marked organelles were not lytic PVCs --- p.96 / Chapter 3.4.4 --- FM4-64 uptake study reveals a new PVC marker --- p.98 / Chapter Chapter 4 --- Summary and Future Prospects --- p.100 / Chapter 4.1 --- Summary --- p.101 / Chapter 4.1.1 --- Hypothesis --- p.101 / Chapter 4.1.2 --- Development of transgenic cell lines --- p.102 / Chapter 4.1.3 --- Characterization of organelles marked by GFP- a -TIP CT reporter --- p.103 / Chapter 4.2 --- Future prospects --- p.106 / Reference --- p.108

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.

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.

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.