Zhang, Bin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 154-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Novel pairs of Bet/Exo recombineering proteins were identified in the beta-proteobacterium Laribacter hongkongensis (LHK) and in the SXT genetic element isolated from Vibrio cholerae. In this research, these new recombineering proteins were functionally characterized using a variety of in vivo and in vitro techniques. The SXT-Exo and LHK-Exo proteins were both found to be alkaline exonucleases, with activities similar to those of Lambda-Exo. Both the SXT-Bet/Exo and LHK-Bet/Exo protein pairs had dsDNA recombination activity within E. coli. / Recombineering is a powerful tool used to manipulate or engineer DNA in vivo, which enables chromosomes and plasmids to be modified precisely and efficiently. It is of critical importance for research into genome and proteome function, and greatly facilitates metabolic engineering applications. The Lambda-Red (Bet and Exo) and RecET proteins constitute the most efficient bacterial recombineering systems characterized to date. However, they work only in E. coli or closely-related bacteria (e.g. Salmonella spp.), which limits their widespread application. / The Lambda-Red and RecET recombineering systems can use PCR products (double stranded DNA, dsDNA) or single stranded DNA (ssDNA, oligonucleotides) to create precise point mutations (substitutions), gene deletions and insertions in chromosomal or episomal DNA. The Exo/RecE exonuclease proteins digest dsDNA and produce long 3'-ssDNA tails. The Bet/RecT ssDNA annealing proteins bind to these 3'-ssDNA tails and promote their homologous recombination with complementary ssDNA regions on the chromosome or episome. / The results described in this thesis will be very useful in assisting the future development of novel recombineering systems that can be used for genetic engineering applications across a wide range of bacterial organisms. Such tools will greatly promote functional genomic and proteomic studies within these organisms, and may also be used for microbial engineering and biotechnological applications. / The ssDNA recombination activities of five different Bet/RecT recombinases were directly compared using an E. coli reporter system. The comparison revealed that Bet protein from LHK had a higher efficiency than Lambda-Bet or RecT. Based on their predicted secondary structure, a set of rationally-designed lambda-Bet protein truncations were prepared and their biological activity was examined, to investigate structure-function relationships within this recombinase. / Chen, Wenyang. / Adviser: Ho W.S. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 113-128). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract 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.

Yun Jing-ping. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 185-226). / 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.

Shan Sze Wan. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 125-139) / 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.

Or Man Wai. / "January 2002." / "glued" in title is superscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references. / 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.

by Wai-Man Tong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 155-174). / Abstracts in English and Chinese. / ABSTRACT --- p.I-IV / Chapter 1. --- INTRODUCTION / Chapter 1.1. --- General / Chapter 1.1.1. --- Ribosome Inactivating Protein --- p.1 / Chapter 1.1.1.1. --- Ricin --- p.2 / Chapter 1.1.1.2. --- Trichosanthin --- p.5 / Chapter 1.1.2. --- In Vitro Study of RIP --- p.6 / Chapter 1.2. --- Uptake of Ribosome Inactivating Proteins / Chapter 1.2.1. --- Suicide Transport --- p.7 / Chapter 1.2.1.1 . --- Endocytic Uptake of Ricin --- p.8 / Chapter 1.2.1.2. --- Endocytic Uptake of Trichosanthin --- p.11 / Chapter 1.2.2. --- Pervious Studies in This Laboratory --- p.11 / Chapter 1.3. --- Apoptosis And Ribosome Inactivation / Chapter 1.3.1. --- Apoptosis / Chapter 1.3.1.1. --- Morphological Feature of Apoptosis --- p.14 / Chapter 1.3.1.2. --- Molecular Changes of Apoptosis --- p.15 / Chapter 1.3.2. --- Toxicity of Ribosome Inactivating Protein / Chapter 1.3.2.1. --- Toxicity of Ricin --- p.20 / Chapter 1.3.2.2. --- Toxicity of Trichosanthin --- p.21 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1. --- GENERAL / Chapter 2.1.1. --- Cell Culture / Chapter 2.1.1.1 . --- Schwann Cell Culture --- p.23 / Chapter 2.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.24 / Chapter 2.1.1.3. --- Identification of Schwann Cell and Dorsal Root Ganglion Neuron --- p.25 / Chapter 2.1.2. --- Labeling of Toxins --- p.26 / Chapter 2.1.3. --- Administration of Toxin --- p.27 / Chapter 2.2. --- UPTAKE OF RIBOSOME INACTIVATING PROTEINS / Chapter 2.2.1. --- Real-Time Observation of Toxin Uptake by Neurons --- p.27 / Chapter 2.3. --- APOPTOSIS STUDY OF RIBOSOME INACTIVATING PROTEINS' TOXICITY / Chapter 2.3.1. --- TUNEL Staining --- p.28 / Chapter 2.3.2. --- Annexin V Staining --- p.30 / Chapter 2.4. --- MOLECULAR STUDY OF THE DEATH MECHANISM OF RIBOSOME INACTIVATING PROTEINS / Chapter 2.4.1. --- NIH/3T3 Cell Line Culture --- p.33 / Chapter 2.4.2. --- Differential Display / Chapter 2.4.2.1. --- Differential Display --- p.34 / Chapter 2.4.2.2. --- Cloning and Sequencing --- p.38 / Chapter 2.4.2.3. --- RT-PCR --- p.42 / Chapter 2.4.3. --- Two Dimension Gel Electrophoresis --- p.43 / Chapter 2.4.4. --- Ribosomal RNA Analysis --- p.48 / Chapter 3. --- RESULTS / Chapter 3.1. --- General / Chapter 3.1.1. --- Cell Culture / Chapter 3.1.1.1 . --- Schwann Cell Culture --- p.50 / Chapter 3.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.51 / Chapter 3.1.1.3. --- Identification of Schwann Cell and Dorsal Root Ganglion Neuron --- p.51 / Chapter 3.1.2. --- RIPs Labeling --- p.52 / Chapter 3.2. --- Uptake of Ribosome Inactivating Protein / Chapter 3.2.1. --- Real-Time Observation of Toxin Uptake --- p.53 / Chapter 3.3. --- Apoptosis Study of Ribosome Inactivating Proteins' Toxicity / Chapter 3.3.1. --- TUNEL Staining --- p.55 / Chapter 3.3.2. --- Annexin V Assay / Chapter 3.3.2.1. --- Schwann Cell Culture --- p.57 / Chapter 3.3.2.2. --- Dorsal Root Ganglion Neuron Culture --- p.58 / Chapter 3.3.2.3. --- Unique Observable Pattern --- p.60 / Chapter 3.4. --- Molecular Study of the Death Mechanism of Ribosome Inactivating Proteins / Chapter 3.4.1. --- NIH/3T3 Cell Line Culture --- p.60 / Chapter 3.4.1.1. --- TUNEL Staining --- p.61 / Chapter 3.4.1.2. --- Annexin V Staining --- p.61 / Chapter 3.4.2. --- Differential Display / Chapter 3.4.2.1. --- Observation --- p.61 / Chapter 3.4.2.2. --- Primer Combination --- p.62 / Chapter 3.4.2.3. --- Differential Display --- p.62 / Chapter 3.4.3. --- Two-Dimensional Gel Electrophoresis / Chapter 3.4.3.1. --- Observation --- p.63 / Chapter 3.4.3.2. --- Comparison of Gels --- p.63 / Chapter 3.4.4. --- Ribosomal RNA Analysis --- p.63 / Chapter 4. --- DISCUSSION / Chapter 4.1. --- General / Chapter 4.1.1. --- The Selection of In Vitro Model / Chapter 4.1.1.1. --- Schwann Cell Culture --- p.65 / Chapter 4.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.66 / Chapter 4.1.2. --- Labeling of Toxins with Fluorochromes --- p.67 / Chapter 4.1.3. --- Dosage Used in In Vitro Study --- p.68 / Chapter 4.2. --- Uptake of Ribosome Inactivating Proteins / Chapter 4.2.1. --- Real-Time Examination of Toxin Uptake --- p.69 / Chapter 4.3. --- Involvement of Apoptosis in Ribosome Inactivating Proteins' Intoxication / Chapter 4.3.1. --- TUNEL Staining --- p.75 / Chapter 4.3.2. --- Annexin V and Propidium Iodide Staining --- p.77 / Chapter 4.3.3. --- Special Pattern of Fluorescence Signal in Neuronal Cell Bodies --- p.82 / Chapter 4.4. --- Molecular Study of Death Mechanism of Ribosome Inactivating Proteins / Chapter 4.4.1. --- NIH/3T3 Cell Line Culture --- p.84 / Chapter 4.4.2. --- Differential Display --- p.84 / Chapter 4.4.3. --- Two Dimensional Polyacrylamide Gel Electrophoresis --- p.86 / Chapter 4.4.4. --- Ribosomal RNA Alternation --- p.88 / Chapter 5. --- CONCLUSIONS --- p.89 / Chapter 6. --- "FIGURES, GRAPHS AND LEGENDS" --- p.91 / Chapter 7. --- REFERENCES --- p.155 / APPENDIX / Appendix A Materials --- p.175 / Appendix B Source of Chemicals and Equipments --- p.184 / ACKNOWLEDGEMENTS --- p.186

Macrophages are active participants in many important biological processes, including antimicrobial activity, tumour surveillance, apoptotic cell clearance, homeostasis and wound healing. The activity of all cells is under the direct influence of their genetic makeup and macrophages are no exception. Natural resistance-associated macrophage protein 1 (Nramp1, also known as SLC11A1) is a macrophage-restricted gene that confers resistance to intracellular pathogens in mice. Mitogen activated protein kinase activated protein kinase 2 (MAPKAPK-2 or MK2), a substrate of p38 MAPK, is known to influence the activation of macrophages in response to stressors, including the Toll-like receptor (TLR)-4 ligand LPS. Like NRAMP1, MK2 has also been shown to influence the efficiency of the antibacterial response. The present study evaluates the role of NRAMP1 and MK2 in TLR-mediated cytokine induction and their role in cutaneous wound healing. Mice lacking NRAMP1 are severely impaired in their rate of cutaneous wound healing. Nramp1 gene ablation has been associated with lower levels of SLPI, a protein previously demonstrated to influence the rate of wound healing in a non-redundant fashion. Macrophages derived from Nramp1-null mice are less efficient in activating p38 MAPK signaling, which results in lower levels of MK2 phosphorylation. The reduced level of p38 MAPK and MK2 activation in Nramp1-null macrophages also correlates with decreased cytokine induction in response to TLR7 ligand stimulation of these cells. Using p38 MAPK inhibitor and MK2-deficient macrophages, we demonstrate that TLR7- and TLR9-mediated cytokine induction is directly under the control of this signaling pathway. Furthermore, cytokine induction is regulated by MK2 at the post-transcriptional level. Macrophage-induced cytokines play an important role in cutaneous wound healing. Since MK2-deficient macrophages are severely impaired in their ability to induce cytokines following activation, we next evaluated the role of MK2 in cutaneous wound healing. Our results demonstrate that the rate of wound healing is significantly delayed in the absence of MK2. The level of cytokine expression in the wounds is impaired and macrophages are major players in cutaneous wound healing. Our data also show that intradermal transfer of macrophages with intact MK2 significantly improved wound healing kinetics. Overall, the studies presented in this dissertation demonstrate the importance of NRAMP1 and MK2 in the modulation of macrophage gene expression, and their important role in the control of cutaneous wound healing.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease which results from the degeneration of upper and lower motor neurons in the brainstem, spinal cord and motor cortex. Tragically there is no treatment to prevent ALS. The drug Riluzole acts to delay progression, but only by a month or so in this disease that has a survival length of three to five years. The identification of genes that are mutated in patients with ALS would help devise novel therapeutic strategies as much remains to be discovered about the genetics of ALS. Familial forms of the disease account for only 5-10% of patients. Among these familial cases, about 15-20% are caused by mutations in the zinc/copper superoxide dismutase gene, but the genetic basis of the remaining familial cases and the many sporadic cases continues to be largely unknown. / Altogether, the results presented in this thesis came from the use of several strategies to establish the genetic cause of ALS and the related motor neuron disorders like hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS). A concerted and collaborative effort was put forth to identify the gene causative for ALS3 on chromosome 18. In addition, a recently reported locus has been confirmed on chromosome 9p for patients that present both ALS and frontotemporal dementia. The major finding involves the discovery of eight mutations in the TARDBP gene in nine patients with sporadic and familial ALS. Furthermore, a large association study evaluated the role of common polymorphisms in the paraoxonase gene cluster in susceptibility to the development of ALS. In the analysis of upper motor neuron diseases, mutations in a novel gene, KIAA0196, were identified for the HSP locus SPG8 on chromosome 8. Finally, the first locus for PLS was discovered on the p-arm of chromosome 4 following genome scan analysis of a large Quebec family with PLS. / These genetic discoveries all contributed novel advances to the field of motor neuron disorders. As more is elucidated regarding the biochemical function of these the proteins encoded by these genes, a more comprehensive picture of ALS and other motor neuron disorders will hopefully emerge.

Diss. (sammanfattning Stockholm : Karol. inst. / Härtill 4 uppsatser.