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Yao, Yao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 131-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Developmental Dyslexia (DD) refers to a reading disorder affecting individuals that possess otherwise normal intelligence. Having demonstrated by familial and twin studies, genetic factors are found to be of major significance to DD development. A strong dyslexia susceptibility gene KIAA0319 (K), of which crucial role in DD had been revealed by various linkage and association studies, was found to have 40% reduction in expression in the DD risk haplotype. Besides, both up- and down-regulation of K would result in impaired neuronal migration in rat. Despite the undoubtedly strong linkage of K to DD, biological and molecular knowledge of K is still lacking. Consequently, how K plays its role in DD remains unclear. To address this question, investigations of human K protein and its interactions in molecular level were performed. K protein is a large transmembrane protein which consists of four main parts, including the N-terminus of K which has a MANSC domain downstream of the signal peptide, a large cluster of five PKD domains in the middle of the protein sequence, a Cysteine -rich C6 region together with a transmembrane domain which had been demonstrated to be critical for forming K protein homodimer, and the only cytoplasmic C-terminus of K. Having shown that no gross effect on gene expression at both mRNA and protein level was found with overexpressing K by DNA microarray and two-dimensional gel electrophoresis, protein interactions involving K were targeted for investigation. Towards this goal, a monoclonal antibody against K was raised, which is capable for recognizing native full-length K proteins in immunoblotting, indirect immunofluorescence staining, as well as in immunoprecipitation. A novel K interaction partner protein KIAA0319-Like (KL), which is a homologous protein of K with high sequence similarity (59%), has been found and confirmed by co-immunoprecipitation. No interaction was shown for truncation mutants of Cysteine-rich C6 region in either K or KL proteins, cuing that the interaction of K and KL at C6 region is a mimic of K homodimer, and led to a hypothesis that the function of K is regulated by KL, which serves as a molecular control of neuronal migration by regulating the formation of K dimer. Another known interaction partner of K protein, the mu---subunit of Adaptor protein 2 complex (AP2M1) which binds to cytoplasmic C-terminus of K (55% similarity to that of KL), was found to have similar binding behaviour towards K as well as KL by co-immunoprecipitation and molecular docking. In addition to AP2M1, two adaptor proteins FEM and SH2 were also confirmed to be interacting with cytoplasmic C-terminus of K, suggested that cytoplasmic region of K is responsible for interactions of downstream cellular pathways. Interaction of K with adaptor proteins also suggested that K might be a membrane receptor that mediates signalling via various adapter proteins. The N-terminus of K protein which has the least sequence similarity to KL (31%) is hence thought to confer to the specificity of the receptor and is critical to the function of K in DD. / Chan, Hoi Ling. / Adviser: Mary M. Y. Waye. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 164-170). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Chen, Shuyan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 91-98). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLEGGMENTS --- p.v / LIST OF FIGURES --- p.vi / LIST OF TABLES --- p.vii / ABBREVIATIONS --- p.viii / CONTENTS --- p.ix / Chapter Chapter I --- Introduction / Chapter 1.1 --- Introduction of BRE / Chapter 1.1.1 --- Discovery of BRE --- p.1 / Chapter 1.1.2 --- Isoforms of BRE --- p.2 / Chapter 1.1.3 --- Homology and orthologs of BRE --- p.3 / Chapter 1.1.4 --- Expression studies of BRE mRNA --- p.4 / Chapter 1.1.5 --- Expression and cellular localization of BRE protein --- p.5 / Chapter 1.1.6 --- Interaction between BRE and death receptor --- p.6 / Chapter 1.1.7 --- Anti-apoptotic effect of BRE in cell line studies --- p.9 / Chapter 1.1.8 --- Anti-apoptotic effect of BRE in vivo --- p.11 / Chapter 1.1.9 --- BRE's role in DNA repair and ubiquitination --- p.12 / Chapter 1.1.10 --- BRE's role in regulation of Prohibitin and p53 expression --- p.13 / Chapter 1.2 --- Hepatocellular carcinoma / Chapter 1.2.1 --- Carcinogenesis --- p.15 / Chapter 1.2.2 --- Diethylnitrosamine -induced HCC --- p.15 / Chapter 1.2.3 --- Mouse model for HCC studies --- p.17 / Chapter 1.2.4 --- BRE in human HCC --- p.18 / Chapter 1.3 --- Green Fluorescent Protein / Chapter 1.3.1 --- Application of GFP in biological research --- p.19 / Chapter 1.3.2 --- Advantage of GFP applied in protein localization --- p.19 / Chapter Chapter II --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Primer used for cloning --- p.20 / Chapter 2.1.2 --- DNA clones used in the studies --- p.21 / Chapter 2.1.3 --- Materials for DNA manipulation --- p.24 / Chapter 2.1.4 --- Materials for protein manipulation --- p.24 / Chapter 2.1.5 --- Antibodies --- p.25 / Chapter 2.1.6 --- Chemicals --- p.25 / Chapter 2.1.7 --- Kits --- p.26 / Chapter 2.1.8 --- Culture media and reagents --- p.26 / Chapter 2.1.9 --- Bacterial strain used for transformation and cloning --- p.26 / Chapter 2.1.10 --- Instrumentation --- p.27 / Chapter 2.1.11 --- Animals --- p.27 / Chapter 2.1.12 --- Slides --- p.27 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Construction of Plasmids / Chapter 2.2.1.1 --- Polymerase chain reaction (PCR) --- p.28 / Chapter 2.2.1.2 --- Enzyme Digestion and Ligation --- p.29 / Chapter 2.2.1.3 --- Transformaion / Chapter 2.2.1.3.1 --- Preparation of competent cells --- p.29 / Chapter 2.2.1.3.2 --- Heat-shock Transformation --- p.29 / Chapter 2.2.1.4 --- Midi Prep of plasmids --- p.30 / Chapter 2.2.2 --- Cell Culture --- p.30 / Chapter 2.2.3 --- Transfection --- p.30 / Chapter 2.2.4 --- MG-132 treatment --- p.31 / Chapter 2.2.5 --- Flow Cytometry --- p.32 / Chapter 2.2.6 --- Western blotting / Chapter 2.2.6.1 --- SDS-PAGE --- p.32 / Chapter 2.2.6.2 --- Immunoblotting --- p.32 / Chapter 2.2.7 --- Production of Monoclonal Antibody --- p.33 / Chapter 2.2.8 --- Mice --- p.34 / Chapter 2.2.9 --- Tissue Processing --- p.35 / Chapter 2.2.10 --- Tissue Section --- p.35 / Chapter 2.2.11 --- Immunostaining --- p.36 / Chapter 2.2.12 --- H&E staining --- p.36 / Chapter 2.2.13 --- Picture Capture --- p.37 / Chapter 2.2.14 --- Confocal imaging --- p.37 / Chapter 2.2.14 --- Statistical Analysis --- p.37 / Chapter Chapter III --- BRE promotes growth of chemically-induced hepatocellular carcinoma / Chapter 3.1 --- DEN induced HCC in male mice --- p.38 / Chapter 3.2 --- BRE facilitates HCC in female mice --- p.44 / Chapter 3.3 --- Over-expression of BRE in tumor portion --- p.45 / Chapter 3.4 --- Direct effect of DEN on BRE expression --- p.47 / Chapter 3.5 --- Contribution of infiltrating cells in up-regulation of BRE --- p.50 / Chapter Chaper IV --- Subcellular localization of BRE / Chapter 4.1 --- GFP-BRE fusion constructs --- p.55 / Chapter 4.1.1 --- Transfection of GFP-BRE fusions --- p.58 / Chapter 4.1.2 --- Flow cytometry analysis of GFP-BRE fusions --- p.59 / Chapter 4.1.3 --- Western blot analysis of GFP-BRE fusions --- p.62 / Chapter 4.1.4 --- Stabilities of GFP-BRE fusions --- p.64 / Chapter 4.2 --- Fusions between GFP and the deletion mutants of BRE --- p.66 / Chapter 4.2.1 --- Transfection of mutants --- p.68 / Chapter 4.2.2 --- Low expression of mutants --- p.69 / Chapter 4.3 --- MG-132 treatments / Chapter 4.3.1 --- Increased expression of fusion proteins --- p.74 / Chapter 4.3.2 --- Subcellular localization of GFP-BRE fusions --- p.77 / Chapter Chapter V --- Discussion / Chapter 5.1 --- Functional role of BRE in HCC / Chapter 5.1.1 --- Stage model of carcinogenesis --- p.81 / Chapter 5.1.2 --- Anti-apoptotic genes in cancer --- p.84 / Chapter 5.1.3 --- Limitation of the study --- p.85 / Chapter 5.1.4 --- Conclusion --- p.85 / Chapter 5.2 --- Subcellular localization of BRE / Chapter 5.2.1 --- Low expression of GFP-BRE fusions --- p.86 / Chapter 5.2.2 --- Additional study --- p.90 / Chapter 5.2.3 --- Conclusion --- p.90 / Reference --- p.91 / Appendix --- p.99

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 90-106).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 88-103).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 63-82).

Agrin triggers the subsynaptic aggregation of acetylcholine receptor (AChR) via activation of the receptor tyrosine kinase MuSK (muscle-specific kinase). At present, the intracellular mechanisms utilized by MuSK to initiate such a complex process remain unknown. In the present study, I first tested if H-ras was involved in the process of synaptogenesis induced by agrin. The data presented suggest that ras could have a role in this process because a dominant inhibitory ras mutant (ras-N17) partially blocked the inductive effects of agrin while two activated ras mutants (ras-V12 and ras-V12-D38) induced agrin-independent AChR clusters. These effects were not due to major alterations in the levels of AChR, though more experiments are required to confirm these preliminary findings. / Second, I investigated whether the adaptor protein Shc was a downstream effector of activated MuSK. MuSK and Shc could be co-immunoprecipitated, but this association was not consistently observed nor was it modulated by agrin at all times. Generally, no alteration in Shc phosphotyrosine content was observed in response to agrin, and when an increase was detected, it was modest. Finally, agrin did not modulate the interaction between Shc and Grb2. Based on these results, I conclude that Shc interaction with MuSK is not regulated by agrin.

Thesis (Ph. D. in Neuroscience)--Vanderbilt University, May 2008. / Title from title screen. Includes bibliographical references.

Clearance of molecularly damaged and misfolded synaptic vesicle (SV) proteins is vital for the maintenance of healthy, functional synapses. However, this process poses significant trafficking challenges for neurons, as the majority of degradative organelles and machinery are localized in the somatodendritic compartment, far from SV pools in presynaptic terminals. Our previous work showed that SV protein degradation is mediated by the endosomal sorting complex required for transport (ESCRT) pathway in an activity-dependent manner. Moreover, we found that neuronal activity increased ESCRT protein recruitment to axons and SV pools, suggesting a novel mechanism for regulating the trafficking of this critical degradative machinery, whose localization and transport in neurons has been unexplored. Here, we characterize the axonal transport of ESCRT-0 proteins Hrs and STAM1, the first components of the ESCRT pathway, which are critical for initiating SV protein degradation. We find that Hrs- and STAM1-positive transport vesicles exhibit increased anterograde and bidirectional motility in response to neuronal activity, as well as frequent contact with SV pools. ESCRT-0 vesicles typically colocalize with early endosome marker Rab5, but their transport dynamics do not mirror those of the total Rab5 vesicle pool. Moreover, other ESCRT pathway components and effectors do not show activity-dependent changes to motility, indicating that neuronal firing specifically regulates the motility of the ESCRT-0+ subset of Rab5+ structures in axons. Finally, we identify kinesin-3 motor protein KIF13A as essential for the activity-dependent transport of ESCRT-0 vesicles as well as the degradation of SV membrane proteins. Altogether, these studies demonstrate a novel activity-dependent mechanism for mobilizing the axonal transport of a newly characterized endosomal subtype carrying ESCRT machinery. This activity-induced transport is necessary for ESCRT-mediated degradation of synaptic vesicle proteins.