Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus of significant public health concern closely related to other highly pathogenic flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV). With the rise of ZIKV in Brazil in 2015, its potential link to microcephaly and other severe neurological birth defects prompted the World Health Organization to declare ZIKV a Public Health Emergency of International Concern. Since this time, numerous studies have provided ample evidence to establish ZIKV as the causative agent of microcephaly, yet the molecular mechanisms underlying these neurodevelopmental defects are not well understood. We therefore establish a tractable experimental model system to investigate the impact of ZIKV on human neural development. We demonstrate that ZIKV efficiently infects human cortical neural progenitor cells (hNPCs) derived from induced pluripotent stem cells, but less efficiently infects other cells along the neural differentiation pathway, including immature cortical neurons. Infected hNPCs further release infectious ZIKV particles. Importantly, ZIKV infection disrupts cell cycle progression and induces cell death in hNPCs contributing to their attenuated growth. Global transcriptome analyses of ZIKV-infected hNPCs reveal transcriptional dysregulation, notably a downregulation of cell-cycle-related genes, highlighting the potential involvement of cell cycle pathways in ZIKV biology. We then study the molecular mechanisms by which ZIKV manipulates the cell cycle in hNPCs and the functional consequences of cell-cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that host cell-cycle disruption is unique to ZIKV among the flaviviruses tested, including DENV and WNV, however similar among the two strains of ZIKV tested, including the prototype Uganda strain and a Puerto Rican strain. ZIKV, but not DENV, infection induces DNA double-strand breaks, triggering the DNA damage response through the ATM/Chk2 signaling pathway, while suppressing activation of the ATR/Chk1 signaling pathway in hNPCs. Furthermore, ZIKV infection impedes the progression of cells through S phase thereby preventing the completion of host DNA replication. Recapitulating the S-phase arrest state with S-phase inhibitors leads to an increase in ZIKV replication, but not of WNV or DENV replication. Together, our results identify hNPCs as a direct target of ZIKV and the damaging impact of ZIKV on the growth of hNPCs. Importantly, our data demonstrate ZIKV’s ability to induce host DNA damage and arrest cell cycle progression, which results in a cellular environment favorable for its replication. As hNPCs generate the cortical neurons during early fetal brain development, the ZIKV-mediated growth retardation likely contributes to the neurodevelopmental defects of the congenital Zika syndrome. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / January 23, 2018. / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Timothy Megraw, University Representative; Brian P. Chadwick, Committee Member; David M. Gilbert, Committee Member; Yan Li, Committee Member; Fanxiu Zhu, Committee Member.

The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding. Misfolded proteins are aggregation prone and protein aggregation are associated with many human diseases, such as neurodegenerative disorders and cancer. However, the molecular basis underlying the proteotoxicity and the mechanisms to combat this toxicity remain poorly understood. We used S. cerevisiae, budding yeast, as a model organism to address these questions as much of the protein homeostasis machinery is conserved form yeast to humans. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which is responsible for the development of Huntington's disease, a neurodegenerative disorder. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and degradation of misfolded proteins. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Our previous data indicate that the degradation of mutated Htt with a 103 polyQ expansion (Htt103QP) depends on both the ubiquitin proteasome system and autophagy in budding yeast. Extended induction of Htt103QP-GFP leads to the formation of a single inclusion body in wild-type yeast cells. We showed that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. In addition, mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Notably, we detected more aggregated forms of Htt103QP in sse1∆ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance but are less critical for proteasome-dependent degradation. These findings uncover the role of HSPs in the inclusion body formation and autophagy-mediated clearance of mutated Huntingtin. Using budding yeast as a model system, we further asked why misfolded proteins are toxic, and how eukaryotic cells combat this toxicity. Our results support the notion that ubiquitinated misfolded protein aggregates drain free ubiquitin and compromise ubiquitin-dependent protein degradation, but the AAA+ ATPase Cdc48 counteracts the toxicity by segregating these protein aggregates. Using Htt103QP as model misfolded protein, we found that Cdc48 and its two predominant cofactors, Npl4 and Ufd1, are required for the segregation and degradation of Htt103QP in yeast cells. We also identified the E3 ubiquitin ligase San1 that catalyzes Htt103QP ubiquitination and facilitates its proteasome-dependent degradation. Unexpectedly, deletion of San1 and another ubiquitin ligase, Ubr1, suppressed the growth defects and accumulation of ubiquitinated substrates in cells lacking functional Cdc48Ufd1/Npl4. We further show compromised ubiquitin-proteasome system in cdc48 mutants, as well as the suppression of these defects by san1∆ ubr1∆, indicating that ubiquitination of misfolded proteins contributes to the growth defect in cdc48 mutants. Importantly, we found that overexpression of ubiquitin partially rescued the growth defects in cdc48 mutants. Finally, we showed that blocking ubiquitination of misfolded proteins by san1∆ ubr1∆ increases the resistance of yeast cells to some proteotoxic stressors. Our results reveal the basis for the cytotoxicity of misfolded proteins and highlight the role of Cdc48 in alleviating this toxicity. Lastly, we showed the ubiquitin ligase Rsp5 is necessary for the inclusion body formation and autohagic degradation of Htt103QP. Also, cells with defective Rsp5 exhibit compromised K63-linked ubiquitination of Htt103QP indicating K63-linked ubiquitination could facilitate autophagic clearance of Htt103QP. Supporting this notion, cells expressing a mutant form of ubiquitin (K63R) that are unable to promote K63-linked ubiquitination exhibit Htt103QP IB defect. Rsp5 has also been implicated in ubiquitinating the autophagy adapter protein Cue5. We showed yeast cells lacking Cue5 exhibit Htt103QP autophagy defect which is consistent with published data that used a different mutated Huntingtin protein. Taken together, our research work identified several components in the cellular response to misfolded proteins. We identified a several factors required for mutated Huntingtin inclusion body formation and autophagic degradation. Also, we uncovered a mechanism that can explain why misfolded proteins are cytotoxic and how cells combat this toxicity. These findings may provide novel targets in developing strategies to combat protein misfolding diseases and cancer. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester 2018. / November 16, 2018. / Includes bibliographical references. / Yanchang Wang, Professor Directing Dissertation; Hong-Guo Yu, University Representative; Timothy Megraw, Committee Member; Yi Zhou, Committee Member; Akash Gunjan, Committee Member.

University of Macau / Institute of Chinese Medical Sciences

Both structural and immunogold EM studies have also been carried to identify the storage PVCs in developing tobacco seeds and mungbean cotyledons. Biochemically, storage PVCs in both developing tobacco seeds and mungbean cotyledons were labeled by VSRAt-1, S2 (globulin-like proteins), BiP and DIP antibodies. Structurally, storage PVCs in developing tobacco seeds, sized about 200 nm diameter, contain wavy limiting membrane with electron-dense core and periphery translucent outer layer with internal vesicles. In contrast, storage PVCs in mungbean cotyledon, sized about 400 nm diameter, contain electron-dense and translucent area located adjacent to each other. / Further drug treatments studies demonstrated that the lytic PVCs/MVBs in tobacco BY-2 cells were distinct from the storage PVCs in seed cells. BFA and wortmannin treatments respectively caused the lytic PVCs in tobacco BY-2 cells to become aggregate and vacuolated, whereas the storage PVCs in seed cells remained unchanged in response to treatments of these drugs. Therefore, the storage PVCs in developing seeds are biochemically distinct from the lytic PVCs in tobacco BY-2 cells. / Plant cells contain both lytic vacuole and protein storage vacuole. Prevacuolar compartments (PVCs) are membrane-bounded organelles mediating protein trafficking between the Golgi apparatus and vacuoles in the plant secretory pathways. Multivesicular bodies (MVBs) have recently identified as the lytic PVCs in tobacco BY-2 cells. However, little is known about the dynamics of the lytic PVCs. In addition, the existence and identity of PVCs for protein storage vacuole (termed storage PVCs in this study) remain unknown. / This thesis research addressed two important biological questions: the dynamics of the lytic PVCs and the identity of the storage PVCs in plant cells. Towards this goal, I have demonstrated that the Golgi apparatus and the lytic PVCs, marked by YFP fusion reporters in transgenic tobacco BY-2 cells, have different sensitivity to brefeldin A (BFA) treatments. BFA at high concentrations (50 to 100 microg/mL) caused both YFP-marked Golgi stacks and lytic PVCs to form aggregates in a dosage-dependent and time-dependent manner. Confocal immunofluorescence and immunogold EM studies with specific organelle antibody markers further demonstrated that BFA-induced aggregates derived from the lytic PVCs were distinct from but physically associated with the Golgi aggregates. Thus, the BFA effects on the secretory organelles have been extended to the lytic PVCs. / Tse, Yu Chung. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4521. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 156-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

种子发育是开花植物生命周期中的一个关键阶段。理解控制这一过程的机理既是植物生物学的重要基础研究课题，也是农业应用上的重要任务。因为种子是我们的主食，而种子质量和大小正是主要由那些控制种子发育过程的基因控制着。玉米作为一种典型的单子叶植物，是很好的研究单子叶植物种子发育的模式植物。 / 在开花植物中，RNA编辑是发生在线粒体和质体中的一种转录后机制，主要将特定的胞嘧啶转换为尿嘧啶。这一过程很多时候将改变基因组的遗传信息。这是一种非常重要的转录后调节机制，许多案例表明它对编码蛋白的功能十分关键。例如，RNA编辑能修复一个保守的氨基酸密码，创造一个起始或终止密码子，或移除一个终止密码子而编码出一个更大的功能蛋白。因此，RNA编辑不能正常进行可能损害或完全失去编码蛋白的功能，对植物的生长和发育造成严重后果。 / 本研究中，我们报道了玉米种子突变体emp5(empty pericarp 5)的分子特征研究。Emp5基因的无效突变导致玉米种子胚和胚乳发育在早期发育阶段停止。Emp5基因编码一个定位在线粒体的PPR-DYW蛋白。对emp5突变体线粒体转录组的分析表明，EMP5蛋白功能失效阻碍了rpl16-458（野生型中100%被编辑）这一位点的C-to-U RNA编辑，降低了nad9, cox3和rps12这三个转录本中总共9个位点的C-to-U RNA编辑水平。令人意外的是，同时也增加了atp6, nad1, cob 和 rpl16 4个转录本中共5个位点的RNA编辑水平。EMP5蛋白缺失E+和DYW结构域仍然保留了底物的特异性和RNA编辑功能，只是编辑效率有所降低。这表明EMP5蛋白的E+和DYW结构域对其编辑功能不关键，但对编辑效率是必需的。对EMP5在水稻中的同源蛋白的分析表明，OsEMP5在水稻线粒体rpl16-458位点的编辑功能是保守的。OsEMP5的基因沉默表达导致水稻植株生长缓慢及种子缺陷。这些结果表明Emp5基因编码的这一PPR-DYW蛋白对多个线粒体转录本的RNA编辑是必需的。尤其是rpl16-458这一位点的编辑对线粒体的功能十分重要，因而对玉米种子发育非常关键。 / Seed development is a critical stage in the life cycle of flowering plants. Understanding the mechanism governing this process is both a fundamental question in plant biology and also an important task in agriculture application as seeds are staple food and seed quality and size are controlled by the genes governing seed development process. Maize as a typical monocot plant, is also an excellent model system for monocot seed development research. / In flowering plants, RNA editing is a post-transcriptional mechanism that converts specific cytidines to uridines in both mitochondrial and plastidial transcripts, altering the genetic information encoded by these genes. It is important for posttranscriptional regulation and in some cases critical to the functions of the encoded proteins. For example, editing can restore a conserved amino acid codon, create an initiation or stop codon, or remove a stop codon that leads to a functional larger protein. Therefore, deficiency in editing may result in a compromised or complete loss of function for the encoded proteins, leading to a severe consequence in plant growth and development. / In this study, we report the molecular characterization of the empty pericarp 5 (emp5) mutant in maize (Zea mays). Null mutation of Emp5 results in abortion of embryo and endosperm development at early stages. Emp5 encodes a mitochondrion targeted DYW-subgroup PPR protein. Analysis of the mitochondrial transcripts reveals that loss of the EMP5 function abolishes the C-to-U editing of rpl16-458 (100% edited in the wildtype), decreases the editing at nine sites in nad9, cox3 and rps12, and surprisingly increases the editing at five sites of atp6, nad1, cob and rpl16. EMP5 lacking the E+ and DYW domain still retains the substrate specificity and editing function, only at reduced efficiency. This suggests that the E+ and DYW domains of EMP5 are not essential to the EMP5 editing function, but necessary for efficiency. Analysis of the ortholog in rice indicates that OsEMP5 has a conserved function in C-to-U editing of the rice mitochondrial rpl16-458 site. Knock-down expression of OsEmp5 results in slow growth seedlings and defective seeds. These results demonstrate that EMP5 encodes a PPR-DYW protein that is required for the editing of multiple transcripts in mitochondria and the editing events, particularly the C-to-U editing at the rpl16-458 site, are critical to the mitochondrial functions and hence to seed development in maize. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Yujun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 78-86). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Thesis/Assessment Committee --- p.i / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.6 / Chapter 2.1. --- Seed development in flowering plants --- p.7 / Chapter 2.1.1 --- Seed morphogenesis of flowering plants --- p.7 / Chapter 2.1.2 --- Molecular mechanisms of seed development --- p.8 / Chapter 2.2 --- PPR protein family --- p.11 / Chapter 2.2.1 --- Definition of PPR protein --- p.11 / Chapter 2.2.2 --- Subgroups of PPR protein --- p.12 / Chapter 2.2.3 --- PPR protein distribution and evolution --- p.13 / Chapter 2.2.4 --- Functions of PPR proteins in plants --- p.14 / Chapter 2.3 --- PPR proteins involving in seed development --- p.15 / Chapter 2.4 --- PPR proteins studied in maize --- p.17 / Chapter 2.5 --- RNA processing in plant mitochondria --- p.19 / Chapter 2.5.1 --- RNA splicing --- p.20 / Chapter 2.5.2 --- 5’ and 3’ processing of plant mitochondrial transcrips --- p.23 / Chapter 2.5.3 --- RNA stabilization --- p.24 / Chapter 2.6 --- RNA editing --- p.25 / Chapter 2.7 --- Status of C to U RNA editing mechanism --- p.28 / Chapter Chapter 3. --- Materials and methods --- p.31 / Chapter 3.1 --- Plant Materials --- p.32 / Chapter 3.2 --- Light Microscopy of Cytological Sections --- p.32 / Chapter 3.3 --- Immunohistochemistry Analysis Materials --- p.32 / Chapter 3.4 --- Isolation of genomic DNA Materials --- p.33 / Chapter 3.5 --- Southern Analysis Materials --- p.34 / Chapter 3.6 --- Inverse PCR cloning Materials --- p.35 / Chapter 3.7 --- RNA Extraction and RT-PCR Materials --- p.35 / Chapter 3.8 --- Subcellular Localization of EMP5 Protein Materials --- p.36 / Chapter 3.9 --- Analysis of Mitochondrial RNA Editing Materials --- p.37 / Chapter 3.10 --- Rice Transformation Materials --- p.37 / Chapter 3.10.1 --- OsEmp5 RNAi vector construction Materials --- p.37 / Chapter 3.10.2 --- Callus induction from mature rice seeds Materials --- p.38 / Chapter 3.10.3 --- Callus subculture Materials --- p.38 / Chapter 3.10.4 --- Preparation of Agrobacterium tumefaciens Materials --- p.38 / Chapter 3.10.5 --- Co-cultivation Materials --- p.39 / Chapter 3.10.6 --- Callus washing and selection Materials --- p.39 / Chapter 3.10.7 --- Regeneration Materials --- p.40 / Chapter 3.10.8 --- Screening of transgenic plants Materials --- p.40 / Chapter Chapter 4. --- Results --- p.42 / Chapter 4.1 --- Phenotypic and Genetic Characterization of emp5-1 --- p.43 / Chapter 4.2 --- Cloning of Emp5 --- p.45 / Chapter 4.3 --- Emp5 Encodes a Mitochondrion-Targeted PPR-DYW Subclass Protein --- p.48 / Chapter 4.4 --- Expression of Emp5 --- p.51 / Chapter 4.5 --- EMP5 is Required for Mitochondrial RNA Editing --- p.52 / Chapter 4.6 --- Molecular Characterization of emp5-4 Allele --- p.54 / Chapter 4.7 --- Functional Analysis of the Rice OsEmp5 Gene --- p.58 / Chapter Chapter 5. --- Discussion --- p.62 / Chapter 5.1 --- Abortion of emp5-1 Mutant Seed Development is Caused by Defective Mitochondrial RNA Editing --- p.63 / Chapter 5.2 --- Increased Editing in the emp5 Mutant --- p.65 / Chapter 5.3 --- Substrate Specifying Sequence of EMP5 Are Not Conserved --- p.66 / Chapter 5.4 --- The E+ and DYW Motif of EMP5 Is not Essential for This Protein Function --- p.67 / Chapter Chapter 6. --- Conclusion --- p.75 / References --- p.78

<p> Plants produce a variety of small molecules, including those essential for survival in all conditions (primary metabolites) or for more ecologically specific conditions (secondary metabolites). While primary metabolic pathways are broadly shared among plants, secondary metabolism is under constant selective pressure towards chemical innovation, given the continual fluctuation of the environment. Thus, plant secondary metabolism - whose constituents number in the hundreds of thousands - is lineage-specific, highly structurally diverse, and ultimately of high value to medicine, agriculture, and industry. Efforts to optimize the production of specific metabolites or to discover new compounds remain difficult primarily due to inadequate understandings of the metabolic genes involved and how these genes are regulated. This work first examines co-regulation, a major form of organization by which plant secondary metabolic genes are organized. In response to the bacterial crop pathogen <i>Pseudomonas syringae, Arabidopsis thaliana</i> and its relatives in the mustard family produce numerous secondary metabolites from the amino acid tryptophan, including the antimicrobial compound camalexin. However, hundreds of biosynthetic genes of unknown function are also simultaneously upregulated. Using metabolic profiling and co-expression analysis, I helped to identify the complete biosynthetic pathway to the indole-3-carbonylnitriles (ICNs), a previously unknown class of compounds. When the cytochrome P450 gene <i>CYP82C2</i> is mutated, biosynthesis of the compound 4-hydroxy-ICN (4OH-ICN) is abolished, and plant defense against <i>P. syringae</i> is impaired. Conversely, addition of 4OH-ICN to plants is sufficient to suppress bacterial growth. Next, this work examines the evolution of camalexin and 4OH-ICN metabolism. Cytochrome P450-directed secondary metabolism has been shown almost without exception to be evolutionarily derived from changes to enzymes with broad substrate specificity. By contrast, I observe through genetics, enzyme phylogenetic analysis, and transient expression assays that the ICN and camalexin biosynthetic pathways evolved from a common chemical substrate. In particular, changes to camalexin catalysis by the newly duplicated gene <i>CYP71A12</i> led to the formation of ICN metabolism in several mustard species, although both compounds are directly derived from indole cyanohydrin. Furthermore, 40H-ICN is an extremely recently evolved metabolite, derived from a flurry of genic, epigenetic and transposon-mediated rearrangements of a yet-more recent gene duplicate (<i>CYP82C2</i>). These regulatory changes to <i>CYP82C2</i> lead to its pathogen-inducibility solely in the species <i>A. thaliana</i>. I additionally identify WRKY33 and MYB51 as two sets of defense regulators that carefully fine-tune 40H-ICN metabolism by direct biosynthetic gene regulation. WRKY33 transcription factor, which is involved in the species-specific regulation of <i>CYP82C2</i>, is conserved throughout flowering plants, indicating that transcriptional recruitment is an important feature in the expansion of secondary metabolism. Finally, this work probes possible molecular functions of 40H-ICN and camalexin by exploring the molecular mechanisms underlying their secretion from roots and regulation of cell death processes. This study ultimately reveals that the proliferation of diverse chemical arsenals in plants is greatly aided by the regulatory capture of new and rapidly evolving genes by evolutionarily more stable transcription factors. Future emphases on transcriptional regulators of secondary metabolism may thus aid in the discovery of new secondary metabolic pathways on a more rapid scale.</p><p>

<p> The Glucagon-like peptide 1 receptor (GLP-1R) belongs to the pharmaceutically important Class B family of G-protein coupled receptors (GPCRs) and its incretin peptide ligand GLP-1 analogs are adopted drugs for the treatment of type 2 diabetes (T2D). Despite remarkable anti-diabetic effects, Glucagon Like Peptide-1 (GLP-1) peptide-based drugs are limited by the need of injection or high cost oral formulation. On the other hand, developing non-peptide small molecule drugs targeting GLP-1R remains elusive likely due to the large nature of the orthosteric binding site on GLP-1R. A promising approach is to develop small molecule agonistic positive allosteric modulators (ago-PAMs) or positive allosteric modulators (PAMs) of GLP-1R by targeting the potential allosteric sites in the transmembrane (TM) domain of human GLP-1R. </p><p> As the first step of taking this approach, we constructed a three-dimensional structure model of the TM domain of human GLP-1R using homology modeling and conformational sampling techniques. Next, a potential allosteric binding site on the TM domain was predicted computationally. <i>In silico</i> screening of drug-like compounds against this predicted allosteric site has identified nine compounds as potential GLP-1R agonists. The independent agonistic activity of two compounds was subsequently confirmed using cyclic adenosine monophosphate (cAMP) response element (CRE)-based luciferase reporting system. One compound was also shown to stimulate insulin secretion through <i> in vitro</i> assay. In addition, this compound synergized with GLP-1 to activate human GLP-1R. </p><p> In 2017, the crystal structures of GLP-1R in its active state (PDB ID: 5VAI) became available. Hence, we have performed another round of <i> in silico</i> screening employing this structure. First, the potential ligand binding sites in 5VAI were identified using computational tools and <i> in silico</i> screening procedure as described above was carried out again. A new small 8 molecule with low molecular weight and logP was identified. <i> In vitro</i> studies of this compound confirmed that it acts as the ago-Positive Allosteric Modulator (PAM) of GLP-1R that improves GLP-1's affinity and efficacy towards GLP-1R. When used in combination with GLP-1, this compound improves insulin secretion than using GLP-1 alone. Site specific mutagenesis studies confirmed its binding site as predicted in the TM domain of GLP-1R. </p><p> Finally, this ago-PAM molecule was further optimized to improve its potency and specificity towards GLP-1R using structure-based optimization strategy and medicinal synthesis. The newly designed compound, whose molecular weight was less than the parental compound, was found to act as the PAM of GLP-1R and showed improvement in the specificity than the parental compound. Thus, this new compound could be further exploited in the drug development for T2D treatment. </p><p> These results demonstrated that allosteric regulation exists in GLP-1R and can be exploited for developing small molecule agonists. The success of this work will help pave the way for small molecule drug discovery targeting other Class B GPCRs through allosteric regulations.</p><p>

by Jingou Tong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 152-166). / 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.

This thesis presents two separate pieces of work pertaining to pre-mRNA splicing in mammalian cells. The first part examines the regulation of the alternative splicing of the PKM gene in cancer cells, while the second part investigates the physical connections between the transcriptional apparatus and splicing factors. Cancer cells uniformly alter key aspects of their metabolism, including their glucose usage. In contrast to quiescent cells, which use most of their glucose for oxidative phosphorylation when oxygen is present, under the same conditions, most of the glucose consumed by cancer cells is converted to lactate. This phenomenon is known as aerobic glycolysis, and is critical for cancer cell growth. The pyruvate kinase isoform expressed by the cell is a key determinant of glucose usage. Pyruvate kinase in most tissues is produced from the PKM gene, which is alternatively spliced to produce to produce the PKM1 or PKM2 isoforms, which contain exons 9 or 10 respectively. Adult tissues express predominantly the PKM1 isoform, which is universally reverted to the embryonic PKM2 isoform in cancer cells. PKM2 expression promotes aerobic glycolysis. In Chapter 3, I describe a mechanism by which cancer cells promote switching to PKM2. We show that PKM exon 9 is flanked by binding sites for the RNA-binding proteins hnRNP A1/A2 and PTB. These proteins bind to exon 9 and repress its inclusion in the mRNA, resulting in PKM2 production. Additionally, we show that hnRNP A1/A2 and PTB are all overexpressed in cancers in a way that precisely correlates with the expression of PKM2. Finally, we show that the oncogenic transcription factor c-Myc promotes PKM2 expression by transcriptionally upregulating the genes encoding hnRNP A1/A2 and PTB. In the second part of my work, presented in Chapter 5, I examine the coupling of transcription and splicing. The RNA polymerase II C-terminal domain (CTD) plays an important role in ensuring that pre-mRNA transcripts are efficiently spliced, most likely through interactions between splicing factors and the CTD. We have established a biochemical complementation system that has facilitated the identification of a splicing factor that binds to the CTD. Surprisingly, purification of the factor revealed it to be a complex containing U2AF65 and the Prp19 complex, two central splicing factors that had not previously been shown to interact. This complex is functional: I present evidence that the two factors can only activate splicing of the IgMA3 pre-mRNA when they are engaged in a complex. I go on to show that U2AF65 binds directly to the CTD, and this interaction stimulates the RNA binding of U2AF65.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder characterized by a progressive loss of cognition. Histopathologically, AD is defined by the presence of two lesions, senile plaques (SP) and neurofibrillary tangles (NFT), which result from the accumulation and deposition of the amyloid-β peptide (Aβ) and the aggregation of hyperphosphorylated tau protein, respectively. Aβ is formed upon sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases and is secreted extracellularly. The accumulation of extracellular Aβ is thought to initiate a pathogenic cascade resulting in synaptic dysfunction in neurons, followed by the their eventual demise through apoptosis. However, while Aβ has been shown to be increased in AD patients' brains, little is known about how the cleavage of APP and the subsequent generation of Aβ is influenced or if the cleavage process changes over time. Moreover, while the effects of Aβ on neurons are known, the exact mechanism remains unclear. Many have postulated that Aβ exerts its effects by binding a putative receptor, but the search for an Aβ receptor has so far remained inconclusive. Interestingly, one of the proposed potential receptor for Aβ is APP itself. In this model, soluble oligomeric Aβ binds cell-surface APP, inducing its dimerization leading to all the downstream effects of Aβ in cells -- e.g. cell death and/or synaptic dysfunction. Moreover, it has been proposed that Aβ can promote its own production in neurons, thereby initiating a pathogenic loop. However, isolating Aβ-induced APP signaling has remained challenging due to the promiscuous nature of Aβ binding. To work around this problem, we used an antibody-mediated approach to artificially trigger the dimerization of cell-surface APP in cells. We found that dimerization of APP could recapitulate all of the effects of oligomeric Aβ in hippocampal neurons, triggering neuronal death at high concentrations and interfering with normal synaptic functions low concentrations. We also found that dimerization of APP is sufficient to promote the amyloidogenic pathway, by increasing levels of the β-secretase BACE1, resulting in increased Aβ production. Finally, we found that dimerization of APP triggered caspase-dependent cleavage of APP and the formation of a second neurotoxic fragment, termed C31, which also mimics the effects of Aβ in hippocampal neurons. Taken together, our data provides support for the occurrence of a positive pathogenic feedback loop involving Aβ, APP and C31 in neurons.