by Chan Ying Chi, Jessica. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 113-127). / Abstract also in Chinese. / Acknowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iv / Contents --- p.vi / Abbreviations --- p.xi / Single Letter Amino Acid Code --- p.xvi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Glycogen Synthase (EC 2.4.1.11) --- p.1 / Chapter 1.2 --- Glycogen Synthase Kinase-3 --- p.4 / Chapter 1.3 --- Structure of Glycogen Synthase Kinase-3 --- p.5 / Chapter 1.4 --- Functions of Glycogen Synthase Kinase-3 --- p.8 / Chapter 1.4.1 --- Substrate Recognition --- p.8 / Chapter 1.4.2 --- Glycogen Synthase Kinase-3 Homologs --- p.10 / Chapter 1.4.2.1 --- Drosophila --- p.10 / Chapter 1.4.2.2 --- Xenopus --- p.11 / Chapter 1.4.2.3 --- Dictyostelium and Others --- p.12 / Chapter 1.4.3 --- Regulation of Glycogen Synthase-3 in Mammalian Systems --- p.13 / Chapter 1.4.4 --- The role of Glycogen Synthase Kinase-3in Mammalian Brain --- p.16 / Chapter 1.4.4.1 --- Glycogen Synthase Kinase-3β --- p.18 / Chapter 1.4.4.2 --- Glycogen Synthase Kinase-3α --- p.21 / Chapter 1.4.5 --- Glycogen Synthase Kinase-3α in Certain Tumor Cells --- p.23 / Chapter 1.5 --- Objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General Techniques / Chapter 2.1.1 --- Plasmid Minipreparation --- p.26 / Chapter 2.1.2 --- Large Scale of Plasmid DNA Purification Using QIAGEN-tip500 --- p.28 / Chapter 2.1.3 --- Extraction of Human Blood Genomic DNA --- p.30 / Chapter 2.1.4 --- UV Spectroscopy for determining DNA/RNA Concentration --- p.31 / Chapter 2.1.5 --- Agarose Gel Electrophoresis of DNA --- p.31 / Chapter 2.1.6 --- Purification of DNA Fragment from Agarose Gel using GeneClean III ® (BIO 101 Inc.) Kit --- p.32 / Chapter 2.1.7 --- Restriction Digestion of DNA --- p.32 / Chapter 2.1.8 --- Southern Blot --- p.33 / Chapter 2.1.9 --- Probe Labelling --- p.33 / Chapter 2.1.10 --- Hybridization by Radio-labelling --- p.34 / Chapter 2.1.11 --- DNA Sequencing Reaction --- p.35 / Chapter 2.1.12 --- "Preparation of 6% Polyacrylamide, 8M Urea Denaturing Gel for DNA Sequencing Analysis" --- p.37 / Chapter 2.1.13 --- Preparation of Escherichia coli DH5α Competent Cells --- p.38 / Chapter 2.1.14 --- Modification of 5'Protruding end with T4DNA Polymerase --- p.39 / Chapter 2.1.15 --- Ligation and Transformation of Foregin DNA --- p.39 / Chapter 2.1.16 --- Rapid Screening for the Presence of Plasmid --- p.40 / Chapter 2.2 --- Expression of Glycogen Synthase Kinase-3 / Chapter 2.2.1 --- Preparation of Mammalian cells in Culture --- p.41 / Chapter 2.2.2 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.2.3 --- Western Blot Detection of Glycogen Synthase Kianse-3 --- p.43 / Chapter 2.3 --- Assay of Glycogen Synthase Kinase Promoter Activity / Chapter 2.3.1 --- Preparation of SHSY5Y in Culture --- p.45 / Chapter 2.3.2 --- Trypsinization for Removing Adherent Cells --- p.45 / Chapter 2.3.3 --- Transfection of Mammalian Cells by Calcium Phosphate Precipitation --- p.46 / Chapter 2.3.4 --- Stimulation of Transfection Cells by different Chemicals and Preparation of Cell Extract --- p.47 / Chapter 2.3.5 --- CAT-ELISA and β-Gal ELISA Assay --- p.47 / Chapter 2.4 --- Isolation of Glycogen Synthase Kinase-3α 5，Promoter Region / Chapter 2.4.1 --- 5'Rapid Amplification of cDNA End (5'RACE) --- p.48 / Chapter 2.4.2 --- PromoterFinder DNA Walking --- p.49 / Chapter 2.4.3 --- YAC Clone Genomic Construction --- p.50 / Chapter 2.5 --- Construction of Plasmid for Assay of Glycogen Synthase Kinase-3α Promoter Activity --- p.53 / Chapter 2.6 --- Genomic Organization of Glycogen Synthase Kinase-3α --- p.53 / Chapter 2.7 --- Primer Extension Assay / Chapter 2.7.1 --- Isolation of Total RNA by TRIZOL Reagent --- p.57 / Chapter 2.7.2 --- Primer Extension by SuperScript II --- p.57 / Chapter 2.8 --- Reagents and Buffers / Chapter 2.8.1 --- Nucleic Acid Electrophoresis Buffers --- p.59 / Chapter 2.8.2 --- Reagents for Preparation of Plasmid DNA --- p.59 / Chapter 2.8.3 --- Media for Bacterial Culture --- p.60 / Chapter 2.8.4 --- Reagents for Southern Blot --- p.60 / Chapter 2.8.5 --- Reagents for SDS-PAGE --- p.61 / Chapter 2.8.6 --- Reagents for Western Blot --- p.62 / Chapter 2.8.7 --- Reagents for DNA Sequencing --- p.62 / Chapter Chapter 3 --- Isolation of 5´ة Glycogen Synthase Kinase-3α Promoter Region / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results --- p.66 / Chapter 3.2.1 --- 5' Rapid Amplification of cDNA End (5'RACE) --- p.66 / Chapter 3.2.2 --- PromoterFinder DNA Walking --- p.68 / Chapter 3.2.3 --- YAC Clone Library Construction --- p.71 / Chapter 3.2.3.1 --- Southern Blotting --- p.71 / Chapter 3.2.3.2 --- Isolation of Sequence Upstream of Glycogen Synthase Kinase-3α region from YAC Clone Using PromoterFider DNA Walking --- p.71 / Chapter 3.2.3.3 --- Sequences of 5，Glycogen Synthase Kinase -3α Promoter --- p.73 / Chapter 3.2.4 --- Primer Extension Assay --- p.78 / Chapter 3.2.5 --- Assay of Glycogen Synthase Kinase-3α Promoter Activity using CAT-ELISA --- p.78 / Chapter 3.2.6 --- Genomic Structure of Glycogen Synthase Kinase-3α --- p.84 / Chapter 3.3 --- Discussion --- p.90 / Chapter 3.3.1 --- Glycogen Synthase Kinase-3a Promoter --- p.90 / Chapter 3.3.2 --- Glycogen Synthase Kianse-3a Promoter Activity --- p.92 / Chapter 3.3.3 --- Prospective and Future Studies --- p.94 / Chapter Chapter 4 --- Expression of Glycogen Synthase Kinase-3 / Chapter 4.1 --- Introduction --- p.96 / Chapter 4.2 --- Results Expression of GSK-3 under Stresses --- p.97 / Chapter 4.3 --- Discussion --- p.105 / Chapter 4.3.1 --- Post-translation regulation of Glycogen Synthase Kinase-3 --- p.105 / Chapter 4.3.2 --- Prospective and Future Studies --- p.107 / Chapter Chapter 5 --- Conclusion --- p.109 / Chapter 5.1 --- Promoter study --- p.110 / Chapter 5.2 --- Genomic organization study --- p.111 / Chapter 5.3 --- Expression study --- p.112 / Reference --- p.113 / Appendices / Appendix I G/C contents of GSK-3α Promoter Region --- p.128 / Appendix II Restriction sites of GSK-3α Promoter Region --- p.134 / Appendix III Primers designed on GSK-3α Promoter Region --- p.139 / Appendix IV Restriction sites of GSK-3α cDNA --- p.142 / Appendix V Vectors --- p.150 / Appendix VI Adaptors Sequences --- p.152 / Appendix VII Anti-GSK-3 Antibody --- p.153 / Appendix VIII Raw data of GSK-3α promoter activity assay --- p.154

"November 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 131-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Integrin recycling has been generally assumed to be important for cell migration but the trafficking pathways and the molecules regulating integrin trafficking remain poorly characterized. Furthermore, little is known about the activation status of endocytosed integrins and how it affects the recycling of these receptors. It is likely that FA-engaged integrins will follow different trafficking pathways than bulk integrins and here I sought to study the endocytic fate of this particular integrin pool using the MT-induced FA disassembly assay. I found that integrins previously resident at FAs travel through different Rab compartments after FA disassembly and that their return to the plasma membrane is Rab11- and Src-dependent. Strikingly, I unveiled new functions for FAK and Src family kinases in this process by showing that these kinases are critical to keep integrins active during endocytic trafficking. This finding is unprecedented since it was not known whether endocytosed integrins were kept active during their trafficking. Interestingly, reassembly of FAs from endocytosed integrin occurred preferentially at the leading edge of migrating cells suggesting that integrins are trafficked in a polarized fashion. Furthermore, the recycling of integrins from the Rab11-positive compartment to the plasma membrane is a long-range transport implying the existence of a MT motor committed to this task. Consistently, I identified that a kinesin-II motor, Kif3AC, is engaged in this process. My work establishes a FAK- and Src family kinases-based mechanism for integrin "adhesion memory" during endocytic trafficking and identifies a direct link between FA disassembly and reassembly through an endocytic recycling pathway involving Rab5 and Rab11 and a kinesin-II family member.

Regulatory protein phosphorylation is a well conserved mechanism of signal transduction in all biological systems. In bacteria, signal transduction by phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on serine (Ser) and/or threonine (Thr) residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such as E. coli, which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Bacterial Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C-type phosphatases are referred to as eukaryotic-like Ser/Thr phosphatases (eSTPs). eSTPs have been identified in a number of bacteria, but none have been reported in E. coli. The work presented in this thesis was aimed at identifying and biochemically characterizing a eukaryotic-like Ser/Thr phosphatase and its partner Ser/Thr kinase in E.coli. Chapter 3 describes the identification of a novel PP2C-like Ser/Thr phosphatase PphC encoded by an E. coli ORF, yegK, and characterization of its biochemical properties including kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. I investigated differences in the activity of this protein in closely related E. coli strains. Finally, I demonstrated that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon suggesting that they most likely function as a pair in regulating Ser/Thr phosphorylation. Chapter 4 describes the biochemical characterization of a Ser/Thr kinase YegI in E. coli. I show that YegI is an active kinase with significant structural homology to eukaryotic Ser/Thr kinases. The YegI kinase domain is tethered to a cytoplasmic C-terminal domain containing two non-specific DNA binding Helix-hairpin helix motifs. I have identified enolase and elongation factor-Tu (EF-Tu) as potential physiological substrates of YegI and have demonstrated that phosphorylation of EF-Tu by YegI inhibits protein translation in vitro.

SR蛋白在前體信使核糖核酸(pre-mRNA)的組成性剪接和選擇性剪接中扮演者重要的角色，在這個過程中它需要被SR蛋白激酶(SRPK) 燐酸化才能正常行使功能。經典的SR蛋白是由N端一到二個RNA識別基序(RRM) 以及C端一串精氨酸－絲氨酸(RS) 二肽所構成。SR蛋白的燐酸化調控它的亞細胞定位以及生理功能。此外，SR 蛋白激酶1(SRPK1) 和SR蛋白原型ASF/SF2的復合物結構顯示底物的結合需要第二個非標準的RRM結構域以及在N端可以被燐酸化的RS結構域，但是，第一個標準的RRM結構域對於SR 蛋白激酶1的結合卻是可以或缺的。 / 在這裡，我們展示了SR蛋白激酶2(SRPK2) 結合並且燐酸化SRp20的RS結構域，SRp20是另外一個只包含一個RNA識別基序(RRM) 的SR蛋白。與ASF/SF2相似的是，SRp20中的標準RNA識別基序對於SRPK2的結合並不是必要的。與此同時，我們發現錨定槽對於底物的識別作用在SRPK2中也是保守的，因為，錨定槽中四個關鍵氨基酸的突變會削弱它對SRp20的結合。 / 此外，現在認為SRPK2的功能已經不限於對前體信使核糖核酸(pre-mRNA) 的剪接調控。最近發現，SRPK2也可以燐酸化Tau蛋白並且介導阿爾茨海默疾病中的認知性缺陷。組成性的激活是SR蛋白激酶中的一個固有特性，然而人們對於它的調控機制還不是很清楚。因此, 為了更好的瞭解SRPK2，我們采用酵母雙雜交的方法並且最終發現一個新的SRPK2相互作用蛋白: ZNF187。 / ZNF187是一個可以結合血清反應元件(SRE) 的轉綠因子。我們的研究發現，它可以正向調控SRE的轉錄激活。然而，SRPK2在EGF的刺激下卻起着抑制的效果，其中EGF的刺激會促使SRPK2進入細胞核。進一步證實，通過RNAi干擾的方法敲掉SRPK2可以增加ZNF187誘導的SRE活性。在共轉染實驗中，SRPK2可以把ZNF187誘導的SRE活性逆轉到本底水平。對於可以和EGF刺激的SRPK2有着相似細胞定位的缺失或者突變研究發現，它們都可以產生相一致的抑制現象。於此相反，對於和SRPK2有着不同細胞定位的突變，它卻不能產生抑制效果。因此，我們認為在EGF的刺激下，SRPK2進入細胞核並且負向的調控ZNF187激活的SRE。令人驚訝的是，如果細胞在FBS的刺激下，SRPK2卻上調SRE活性，並且它可以協同增加ZNF187對於SRE的誘導。這些結果表明SRPK2對於ZNF187誘導的SRE轉綠調控是刺激物依賴的。 / SR proteins are critical players in regulating both constitutive and alternative pre-mRNA splicing, during which the phosphorylation by SR Protein Kinases (SRPKs) is required. Classical SR proteins contain one or two RNA Recognition Motifs (RRM) in their N terminus and a stretch of Arginine-Serine (RS) dipeptides in C terminus. Phosphorylation status of SR proteins regulates their subcellular localization as well as physiological function. In addition, complex structure of SRPK1 with ASF/SF2, a prototype of SR protein, shows that substrate binding requires non-canonicalRRM2 domain and RS domain, which can be extensivelyphosphorylated. However, the canonical RRM1 domain is dispensable for such interaction. / Here we show that SRPK2 binds and phosphorylates SRp20, a classical single RRM domain-containing SR protein, at its RS domain. Similarly with ASF/SF2, the canonical RRM domain of SRp20 is dispensable for interacting with SRPK2. Meanwhile, we also find that a docking groove that iscritical for substrate binding in SRPK1 is also conserved in SRPK2, since mutations on four key residues in docking groove impair its binding affinity with SRp20. / In addition, SRPK2 is now known to function more then regulating mRNA splicing, such as cell proliferation and cell apoptosis. Recently, SRPK2 is also shown to be a kinase phosphorylating Tau and mediate the cognitive defects in Alzheimer’s disease (AD). Besides, an intrinsic character of SRPKs lies in that they are constitutively active, but the regulation mechanism is not well understood. Therefore, in order to obtain a better recognition about SRPK2, we applied yeast two-hybrid assay and eventually anew interaction partner called ZNF187 was identified. / ZNF187 is a transcriptional factor that binds with Serum Response Element (SRE). Our studies showed that it isa positive regulator of SRE activity. However, SRPK2 showed inhibiting effect on SRE activation with the treatment of EGF, which could induce its nucleus entry, when co-transfected, it reversed the stimulating effect on SRE by ZNF187 to basal level. Furthermore, knockdown of SRPK2 by RNAi would enhance ZNF187-stimuated SRE activation. Studies on truncation and mutations that have the similar effect with EGF-induced subcellular localization of SRPK2 also generated the same inhibiting phenomenon. In contrast, mutant that has distinct localization with SRPK2 wild type failed to exert suppression. Therefore, we conclude that with the treatment of EGF, SRPK2 moves into nucleus and negatively regulates ZNF187-stimulated transactivation of SRE. Surprisingly, when cells were treated with FBS, SRPK2 showed stimulation on SRE activity and it synergized ZNF187-stimulated effect on SRE, indicating that transcriptional regulation of SRPK2 on ZNF187-stimulated SRE activity is stimuli-dependent. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shang, Yong. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 113-137). / Abstracts also in Chinese.

Parkinson’s disease remains a major neurodegenerative disease with prevalence that is second only to Alzheimer’s disease. Despite much advancement in the understanding of the pathogenesis of Parkinson’s disease, current therapeutic options are limited to those that are only symptomatic and are not disease-modifying. Furthermore, due to what seems to be increasingly complex underlying mechanisms of the disease, identifying a broadly applicable therapeutic strategy is difficult. There is much evidence surrounding the role of apoptosis and conversely, dysfunction of anti-apoptotic signaling in the progressive neurodegeneration that causes Parkinson’s disease. In particular, suppressed PI3K signaling has been implicated in the literature as a key event that occurs during neurodegeneration. Thus, regardless of the diverse upstream mechanisms that may lead to the disease, targeting a downstream effector of neuronal survival presents a therapeutic strategy that may be broadly effective against Parkinson’s disease. For this dissertation, we have developed a method to control the levels of active Akt, the main mediator of the PI3K signaling pathway, using an innovative protein destabilizing technique to create an inducible Akt, DD-Akt(E40K). This method permits the control of active Akt levels through a commonly used and blood-brain barrier permeable antibiotic, Trimethoprim. We have successfully established the inducibility of DD-Akt(E40K) across various cellular contexts, including neuronal cell types and conditions with suppressed PI3K signaling. This inducibility was found to be dose-responsive to Trimethoprim, reversible, and able to induce a known downstream substrate, FoxO4, that is an important regulator of cell survival. Importantly, DD-Akt(E40K) was found to inducibly protect neuronal PC12 cells against Parkinson’s disease mimetic toxins as well as growth-factor removal, indicating a proof of principle for the targeting of Akt activity as a protective strategy against Parkinson’s disease. The reported trophic effects of active Akt were also corroborated using our inducible DD-Akt(E40K) system in vivo, demonstrating significant increases in neuronal cell size within the substantia nigra of mice. Intriguingly, the inducibility of DD-Akt(E40K) was found to be dependent on the region of expression in the brain of mice such that the levels of this protective protein were not controllable by Trimethoprim in the substantia nigra but were controllable in the striatum. Taken together, the studies presented in this dissertation establish a new tool for the study of Akt signaling in various cellular and disease contexts and validate Akt as a promising therapeutic target in Parkinson’s disease. Our results also suggest an intriguing mechanism for the underlying pathology and selective degeneration observed in the disease.

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Li, Man Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-119). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VI / Abbreviations --- p.VIII / Abbreviations of Chemicals --- p.X / List of Tables --- p.XI / List of Figures --- p.XII / Table of Contents --- p.XIII / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- General amino acid control in yeast --- p.1 / Chapter 1.2 --- Mammalian eIF2α kinases --- p.7 / Chapter 1.2.1 --- Heme-regulated inhibitor kinase (EIF2AK1/HRI) --- p.7 / Chapter 1.2.2 --- Protein kinase dsRNA-dependent (EIF2AK2/PKR) --- p.8 / Chapter 1.2.3 --- PKR-like ER kinase (EIF2AK3/PERK) --- p.9 / Chapter 1.2.4 --- General control non-repressible 2 (EIF2AK4/GCN2) --- p.10 / Chapter 1.2.5 --- Activating transcription factor 4 (ATF4) --- p.11 / Chapter 1.3 --- Plant General Amino Acid Control --- p.12 / Chapter 1.3.1 --- Studies of the homolog of GCN2 in Arabidopsis thaliana --- p.12 / Chapter 1.3.2 --- Studies of the homolog of other eIF2a kinase in plant --- p.14 / Chapter 1.3.3 --- Studies of the homolog of other GAAC components --- p.14 / Chapter 1.4 --- Previous works in our lab --- p.15 / Chapter 1.5 --- Hypothesis and Objectives --- p.17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.18 / Chapter 2.1.1 --- "Bacterial cultures, plant materials and vectors" --- p.18 / Chapter 2.1.2 --- Primers --- p.21 / Chapter 2.1.3 --- Commercial kits --- p.25 / Chapter 2.1.4 --- "Buffer, solution, gel and medium" --- p.25 / Chapter 2.1.5 --- "Chemicals, reagents and consumables" --- p.25 / Chapter 2.1.6 --- Enzymes --- p.25 / Chapter 2.1.7 --- Antibodies --- p.25 / Chapter 2.1.8 --- Equipments and facilities --- p.25 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- Growth conditions of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.1 --- Surface sterilize of Arabidopsis thaliana seed --- p.26 / Chapter 2.2.1.2 --- Growing of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.3 --- Treatment of Arabidopsis seedling --- p.26 / Chapter 2.2.2 --- Basic molecular techniques --- p.27 / Chapter 2.2.2.1 --- Liquid culture of Escherichia coli --- p.27 / Chapter 2.2.2.2 --- Preparation of plasmid DNA --- p.27 / Chapter 2.2.2.3 --- Restriction digestion --- p.27 / Chapter 2.2.2.4 --- DNA purification --- p.28 / Chapter 2.2.2.5 --- DNA gel electrophoresis --- p.28 / Chapter 2.2.2.6 --- DNA ligation --- p.29 / Chapter 2.2.2.7 --- CaCl2 mediated E. coli transformation --- p.29 / Chapter 2.2.2.8 --- Preparation of DNA fragment for cloning --- p.29 / Chapter 2.2.2.9 --- PCR reaction for screening positive E. coli transformants --- p.30 / Chapter 2.2.2.10 --- DNA sequencing --- p.30 / Chapter 2.2.2.11 --- RNA extraction from plant tissue with tRNA --- p.31 / Chapter 2.2.2.12 --- Extraction of RNA without tRNA --- p.31 / Chapter 2.2.2.13 --- cDNA synthesis --- p.32 / Chapter 2.2.2.14 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.2.15 --- Western blotting --- p.33 / Chapter 2.2.3 --- Sub-cloning of AtGCN2 --- p.34 / Chapter 2.2.3.1 --- Sub-cloning full length AtGCN2 into pMAL-c2 --- p.36 / Chapter 2.2.3.2 --- Sub-cloning of the N-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.3.3 --- Sub-cloning of the C-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.4 --- Cloning of the eIF2α candidates for the in vitro assay --- p.41 / Chapter 2.2.4.1 --- Cloning of At2g40290 (putative eIF2α candidate) --- p.41 / Chapter 2.2.4.2 --- Cloning of At5g05470 (putative eIF2α candidate) into pBlueScript KS II + --- p.43 / Chapter 2.2.4.3 --- Sub-cloning of At5g05470 into pGEX-4T-1 --- p.43 / Chapter 2.2.4 --- Expression and purification of fusion proteins --- p.45 / Chapter 2.2.5 --- Expression of fusion proteins in E. coli --- p.45 / Chapter 2.2.5.2 --- Extraction of E. coli soluble proteins --- p.45 / Chapter 2.2.5.3 --- Purification of GST tagged fusion protein --- p.46 / Chapter 2.2.5.4 --- Purification of MBP tagged fusion protein --- p.46 / Chapter 2.2.5.5 --- Concentration of purified fusion proteins --- p.46 / Chapter 2.2.5.6 --- MS/MS verification of purified fusion proteins --- p.47 / Chapter 2.2.6 --- Gel mobility shift assay --- p.47 / Chapter 2.2.6.1 --- Synthesis of short biotinylated RNA --- p.47 / Chapter 2.2.6.2 --- Ligation of short biotinylated RNA with tRNA --- p.48 / Chapter 2.2.6.3 --- Gel mobility shift assay --- p.48 / Chapter 2.2.6.4 --- Blotting of the sample on to nitrocellulose membrane --- p.48 / Chapter 2.2.6.5 --- Detection of the tRNA on the membrane --- p.49 / Chapter 2.2.6.6 --- Detection of the MBP fusion proteins on the membrane --- p.49 / Chapter 2.2.7 --- In vitro kinase assay of AtGCN2 --- p.49 / Chapter 2.2.8 --- In vitro translation inhibition assay --- p.50 / Chapter 2.2.8.1 --- In vitro transcription of HA mRNA --- p.50 / Chapter 2.2.8.2 --- In vitro translation --- p.51 / Chapter 2.2.8.3 --- Detection of the protein dot blot --- p.51 / Chapter 2.2.9 --- Gene expression analysis by real time PCR --- p.52 / Chapter 2.2.10 --- Total seed nitrogen analysis --- p.53 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Blast search results suggested that AtGCN2 may be the sole eIF2α kinase in Arabidopsis thaliana --- p.54 / Chapter 3.2 --- Existence of two eIF2α candidates in Arabidopsis thaliana genome --- p.59 / Chapter 3.3 --- Fusion proteins were successfully expressed and purified --- p.63 / Chapter 3.4 --- C-terminal of AtGCN2 has a higher affinity toward tRNA than rRNA --- p.67 / Chapter 3.5 --- Both eIF2α candidates can be phosphorylated by full length AtGCN2 in vitro --- p.70 / Chapter 3.6 --- AtGCN2 can inhibit translation in vitro --- p.72 / Chapter 3.7 --- Overexpression of AtGCN2 did not affect expression of selected genes --- p.74 / Chapter 3.8 --- Overexpression of AtGCN2 did not affect seed nitrogen content and C:N ratio under normal growth conditions --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Existing evidence supported that AtGCN2 is the sole eIF2α kinase in Arabidopsis thaliana --- p.85 / Chapter 4.2 --- Kinase activities of AtGCN2 and its two substrates in Arabidopsis --- p.86 / Chapter 4.3 --- C-terminal binds tRNA in the gel mobility shift assay --- p.88 / Chapter 4.4 --- Overexpression of AtGCN2 did not affect gene expression of the transgenic lines under nitrogen starvation and azerserine treatment --- p.90 / Chapter 4.5 --- Overexpression of AtGCN2 did not alter the seed nitrogen content --- p.91 / Chapter 4.6 --- Existence of GCN4 and ATF4 in plant --- p.92 / Chapter 4.7 --- Alternative model without GCN4 and ATF4 homolog --- p.93 / Chapter 4.8 --- Possible application of the in vitro kinase assay --- p.94 / Chapter 4.9 --- Possible application of the in vitro translation inhibition analysis platform in future study --- p.95 / Chapter Chapter 5 --- Conclusion and Future Prospective --- p.97 / Appendices / Appendix I Commercial kits used in this project --- p.98 / "Appendix II Buffer, solution, gel and medium" --- p.99 / "Appendix III Chemicals, reagents and consumables" --- p.102 / Appendix IV Enzymes --- p.103 / Appendix V Antibodies --- p.104 / Appendix VI Equipments and facilities --- p.105 / Appendix VII Supplementary Data --- p.106 / Appendix VIII Amplification efficiency of real time primers --- p.108 / References --- p.109

Diseases such as epilepsy, pain, and neurodegenerative disorders are associated with changes in neuronal dysfunction due to an imbalance of excitation and inhibition. This work details a novel electroconvulsive seizure assay for C. elegans using the well characterized cholinergic and GABAergic excitation and inhibition of the body wall muscles and the resulting locomotion patterns to better understand neuronal excitability. The time to recover normal locomotion from an electroconvulsive seizure could be modulated by increasing and decreasing inhibition. GABAergic deficits and a chemical proconvulsant resulted in an increased recovery time while anti-epileptic drugs decreased seizure duration. Successful modulation of excitation and inhibition in the new assay led to the investigation of a cGMP-dependent protein kinase (PKG) which modulates potassium (K+) channels, affecting neuronal excitability, and determined that increasing PKG activity decreases the time to recovery from an electroconvulsive seizure. The new assay was used as a forward genetic screening tool using C. elegans and several potential genes that affect seizure susceptibility were found to take longer to recover from a seizure. A naturally occurring polymorphism for PKG in D. melanogaster confirmed that both genetic and pharmacological manipulation of PKG influences seizure duration. PKG has been implicated in stress tolerance, which can be affected by changes in neuronal excitability associated with aging, so stress tolerance and locomotor behavior in senescent flies was investigated. For the first time, PKG has been implicated in aging phenotypes with high levels of PKG resulting in reduced locomotion and lifespan in senescent flies. The results suggest a potential new role for PKG in seizure susceptibility and aging. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection